Effects of monensin supplementation on rumen fermentation, methane emissions, nitrogen balance, and metabolic responses of dairy cows: a systematic review and dose–response meta-analysis

To investigate the effects of supplemental monensin administration on the metabolic responses of dairy cows, a systematic review and dose-response meta-analysis was conducted. Initially, 604 studies were identified through comprehensive database searches, including Google Scholar, Scopus, Science Direct, and PubMed, using keywords related to dairy cows, monensin, and metabolic outcomes. After a 2-stage screening process, 51 articles with a total of 60 experiments were selected for meta-analysis based on criteria such as the study implementation date between 2001 and 2022, the presence of a control group that did not receive monensin supplementation, the reporting of at least one outcome variable, and the presentation of means and corresponding errors. The meta-analysis used the one-stage random effects method, and sensitivity analyzes were performed to assess the robustness of the results. The results showed that the administration of monensin at a dosage of 19–26 ppm was inversely related to methane emissions and that the administration of monensin at a dosage of 18–50 ppm resulted in a significant decrease in dry matter intake (DMI). Administration of monensin at doses of 13–28 and 15–24 ppm also resulted in a significant decrease in ruminal acetate proportion and an increase in propionate proportion, respectively, with no effects on ruminal butyrate, NH3, or pH levels. There were no effects on blood parameters or nitrogen retention, but a significant negative correlation was observed between monensin supplementation and fecal nitrogen excretion. Based on the analysis of all variables evaluated, the optimal dose range of monensin was estimated to be 19–24 ppm.


INTRODUCTION
Monensin, an ionophore antibiotic, affects the ion balance of gastrointestinal microorganisms by altering their membranes, leading to the promotion of certain bacterial species and resistance to antimicrobial agents (McGuffey et al., 2001;Vendramini et al., 2016).Monensin is widely used to improve feed efficiency by enhancing the efficiency of rumen fermentation (Grainger et al., 2010).In addition, it can reduce methane emissions in ruminants leading to a reduced carbon footprint of ruminant-derived foods such as beef and dairy products (Haque 2018).The latter has received increased attention in recent years as ruminants are thought to be responsible for 11.6% of global methane production and 43% of greenhouse gas emissions in the agricultural sector (Ripple et al., 2014;Herrero et al., 2016).
However, reports of the effects of monensin on methane production have been inconsistent.Some studies show suppression (Odongo et al., 2007;Junior et al., 2017), while others find no effect (Grainger et al., 2008;Grainger et al., 2010;Benchaar, 2016;Benchaar, 2020).Research has examined the effects of monensin on rumen fermentation, blood metabolites, and nitrogen balance, but results, also remain inconsistent (Do Prado et al., 2015;de Jesus et al., 2016;Benchaar, 2016;Schären et al., 2017;Santos et al., 2019;Silva et al., 2021;Grigoletto et al., 2021;Silva et al., 2022).These inconsistencies may be due to a variety of factors, such as differences in monensin dose, basal diet, and more, thus a meta-analysis is needed to reach a definitive conclusion.A recent dose-response meta-analysis examined the relationship between monensin supplementation and its effect on dairy cow performance and milk composition (Rezaei Ahvanooei et al., 2023).The results showed that monensin supplementation of up to 23 ppm increases milk production, with the optimal dose being 12.6 ppm.Monensin supplementation also significantly decreased DMI, milk protein, milk fat content, and milk fat yield at doses of 22 to 96 ppm, 12 to 36 ppm, and below 58 ppm and 35 ppm, respectively; however, results varied depending on days in milk.Overall, the optimal dose of monensin was found to be about 16 ppm (Rezaei Ahvanooei et al., 2023).
Meta-analysis is a useful technique in scientific fields, provides a comprehensive conclusion by estimating treatment effects, considers sources of heterogeneity, and identifies study limitations (St-Pierre, 2001).The traditional meta-analysis framework is limited in its ability to account for correlations between doses tested and between outcome variables.A dose-response metaanalysis that accounts for these correlations and can estimate optimal levels of independent variables in the case of nonlinear relationships, would be more appropriate for analyzing the effect of monensin supplementation in dairy cows (Crippa et al., 2019).The presence of one or more influential studies in the analysis process is one of the most important factors affecting the robustness and validity of the meta-analysis results.Therefore, without conducting a sensitivity analysis and identifying the possible existence of such studies and excluding them from the analysis process in case of their detection, the results of a meta-analysis study are not considered valid.Previous meta-analyses, such as those by Duffield et al. (2008) and Appuhamy et al. (2013), did not perform sensitivity analyses and therefore may have led to biased results.Similarly, de Moura et al. ( 2021) performed a traditional meta-analysis without considering parity and year of publication as possible sources of heterogeneity.Conversely, the onestage approach, as proposed by Crippa et al. (2019), can find curved dose-response relationships and provide more precise information on the sources of heterogeneity.
In the present study, a one-stage dose-response metaanalysis was conducted to investigate the effects of monensin supplementation on feed intake, rumen fermentation pattern, methane emissions, blood metabolite levels, and nitrogen balance parameters in dairy cows due to the limitations of earlier meta-analyses and the necessity to estimate the optimal dose of monensin supplementation.It was hypothesized that monensin supplementation would have a significant effect on rumen fermentation and metabolic responses of dairy cows and that the optimal dose could be estimated by a one-stage dose-response meta-analysis.

MATERIALS AND METHODS
This meta-analysis was conducted according to the principles outlined by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Moher et al., 2009; https: / / www .prisma-statement .org).

Data Sources
The framework of PICO was used to establish study inclusion criteria: population (dairy cows), intervention (monensin supplementation), comparison (control group without monensin), and outcome (listed in Table 1).Databases searched included Google Scholar, Scopus, Science Direct, and PubMed.Keywords used in the search were "dairy cow," "dairy cattle," "monensin," "ionophore," "methane," "fermentation," "blood metabolites," and "nitrogen balance" (Figure 1).We hand-searched all reference lists of eligible research and review articles to find relevant publications.Our data source was limited to peer-reviewed articles.A total of 604 articles were initially identified through database searches, of which 265 were considered for further screening, excluding articles conducted in goats, lambs, or sheep, as well as buffalo and duplicate articles.There were 89 articles reviewed after the second screening process.This meta-analysis was based on a rigorous set of criteria: (1) studies between 2001 and 2022 were included; (2) studies without a control group that did not receive monensin supplementation were excluded; (3) studies that reported at least one outcome variable listed in Table 1 were included; (4) studies had to report mean values and corresponding errors; (5) only articles in English were included; and (6) all studies were in vivo studies conducted in dairy cows.The collected literature was imported into the Endnote program for analysis (EndNote X8).Duplicate articles were detected and excluded by the EndNote software.Selection of appropriate articles was performed by MRRA.Questions regarding the selection of appropriate articles were resolved by collective consultation.Data were extracted from relevant studies by 2 independent reviewers (MRRA and AHP) and entered using a standard template in Excel, Microsoft Office.A total of 51 articles with 60 studies were able to meet the criteria and were included in the study.Table 1 summarizes the details of the articles used in this meta-analysis.Our meta-analysis examined DMI, methane production, rumen fermentation parameters, blood metabolites, and nitrogen balance.

Risk of bias assessment
Study quality and risk of bias were assessed using the Systematic Review Center for Laboratory Animal Experimentation tool (SYRCLE's RoB tool; Hooijmans et al., 2014).The RoB tool from SYRCLE is an  (Higgins et al., 2011) for use in animal studies.This tool contains 10 entries related to selection, performance, detection, attrition, reporting bias, and other biases to assess the risk of bias in animal studies.Two reviewers (MRRA and AHP) independently assessed the quality of the included studies based on the 10 entries listed.

Statistical analysis
In the present study, the standardized mean difference (SMD) was used as the effect size.There are 2 approaches for dose-response meta-analysis: the one-stage approach and the 2-stage approach (Piray and Foroutanifar, 2022).The 2-stage approach has been used in the majority of previous studies (Piray and Foroutanifar, 2021).Any study eligible for a 2-stage dose-response meta-analysis must include at least 3 doses of the independent variable.The one-stage dose-response metaanalysis, on the other hand, consists of eligible studies that tested only at least 2 doses of the treatment (Piray and Foroutanifar, 2022).The one-stage approach may also provide more precise insight into the causes of heterogeneity among studies (Crippa et al., 2019).To find truly curvilinear dose-response relationships, Crippa et al. (2019) suggested that the one-stage method can take the place of the conventional 2-stage method.By including all appropriate studies, a one-stage doseresponse meta-analysis allows the complexity of the research question to be examined (Crippa et al., 2019).We used the one-stage random-effects meta-analysis method to evaluate the possible nonlinear associations between monensin supplements and outcome measures because it was preferable to the 2-stage method.For this purpose, a restricted cubic spline with 3 knots at the fixed percentiles (10th, 50th, and 90th) was implemented.The overall P-value was calculated by testing whether the 2 regression coefficients were both equal to zero.The test of whether the second spline coefficient was equal to zero was used to determine the nonlinearity P-value.The variance partition coefficient was used to calculate heterogeneity between studies (Crippa et al., 2019).Several rules were followed in conducting subgroup analyzes: (1) any subgroup analysis must be based on sound scientific evidence, (2) analyzes were pre-defined, (3) the overall effect of the independent variable was significant, and (4) subgroup analysis was performed for data subgroups of the meta-analysis with heterogeneity of >50% and ≥10 studies (Piray and Foroutanifar, 2022).Potential factors of heterogeneity that could influence dairy cow response to supplemental monensin were parity (multiparous, multiparous and primiparous and primiparous), decade of article publication (2001-2011 and 2012-2022), method of mo-nensin application (total mixed ration (TMR), TMR top-dressed, controlled-release capsule (CRC) and others), type of diet (TMR, Pasture, and others), protein and NDF content of the diet (divided into 2 categories based on median), number of cows per trial (sample size), length of the trial period (divided into 2 categories based on median), number of DIM at the beginning of the trial (day-28 to 0 (calving), 0 to 60, 60 to 150, and >150), and breed of cows (Holstein, Holstein × Friesian, and others) were considered in this study.Subgroup analysis was not performed because this meta-analysis did not reveal significant heterogeneity for any of the outcome variables.Publication bias can compromise the reliability and accuracy of the results of a meta-analysis.It means that studies with statistically significant results are more likely to be published than those with null or non-significant results (Piray and Foroutanifar, 2022).The Begg's funnel plot and Egger regression asymmetry were used to assess publication bias in this meta-analysis.In cases where publication bias was detected, the nonparametric trim-and-fill method was used to re-estimate an effect size corrected for publication bias (Piray and Foroutanifar, 2021).
As part of the systematic review, decisions must be made regarding data collection and quantitative analysis.A key step in this process is sensitivity analysis, which examines how different data and modeling decisions affect the final results.Results can be considered robust if they hold up in different re-analyzes; otherwise, the results of the primary analysis should be interpreted with great caution.One of the most common methods for conducting sensitivity analyzes for a doseresponse meta-analysis is to exclude one trial at a time and then analyze how the exclusion affects the overall treatment effect.Evaluating the impact of knot location selection and excluding high treatment doses from the analysis are 2 other methods of conducting sensitivity analyzes for a dose-response meta-analysis (Piray and Foroutanifar, 2022).The robustness of dose-response meta-analysis results may be affected by the presence of influential studies, excessive monensin doses, and the position of the knots.To investigate the robustness of the results, a sensitivity analysis was performed using the leave-one-out technique, examining the position of the nodes and excluding monensin doses above 24 ppm.For determining the optimal knot position, we considered various alternative knot locations, including different combinations of the 10th, 25th, 50th, 75th, and 90th percentiles of the total dose distributions.The results showed that the estimated curves were not affected by the position of the knots.Statistical analyses were performed using the R software packages version 4.1.3(https: / / www .r-project .org;access date: 2022-03-10) dosresmeta (version 2.0.1.)and metaphor ('metafor'

Simplified pre-analysis review
General information on the effects of monensin supplementation on outcome parameters was provided in Figure 2 and Supplemental Table S1.The eligible studies used in this meta-analysis examined different monensin supplementation doses ranging from 0 to 48 ppm.The figure also shows that the majority of monensin doses tested did not affect the outcome variables.DMI, methane emissions, and acetate and propionate appeared to be the outcomes most affected by monensin administration, while other outcomes were less affected.

Risk of bias assessment
The results of the risk of bias assessment for all included studies are visually presented in Figure 3 and Supplementary Table S2.Using the SYRCLE RoB tool, the collective assessment shows an overall low risk of bias, comprising 72.16% of the analyzed studies.Of note, 20.58% of the included studies had unclear risk of bias, while cases with high risk of bias accounted for a smaller proportion at 7.26%.Despite the shortcomings in reporting experimental details in animal studies highlighted by Avey et al. (2016), the results on risk of bias derived from our meta-analysis predominantly confirm the acceptable quality of the studies reviewed.

Effect on DMI
Fifty eligible articles, including 59 studies, were included in the analysis to evaluate the effect of monensin supplementation on DMI.Monensin supplementation was found to have a significant nonlinear relationship with DMI (P < 0.01).Dry matter intake was significantly reduced when the monensin dose was increased from 18 to 50 ppm (Figure 4A).Leave-one-out analysis did not reveal any influential studies that altered the results of the model.In addition, the overall trend of the association remained unchanged when monensin doses above 24 ppm were excluded.These tests of sensitivity analysis indicate that the results are statistically robust.There was no substantial heterogeneity for this outcome measure (heterogeneity <50%).The test for publication bias became significant (P = 0.03, Figure 5A).However, the trim and fill test confirmed the reliability of the results.

Effect on fermentation parameters
Methane emissions.Eight eligible articles, including 13 studies, examined the effects of monensin supplementation on methane emissions.The results showed a significant effect of monensin supplementation on this variable (P < 0.01).Methane emission was significantly decreased (8.12-33.31g/d per cow) by monensin addition at dosages from 19 to 26, and outside this range, the effect of monensin was not significant (Figure 4B).Leave-one-out analysis showed that studies No. 2 (Grainger et al., 2008), No. 4 (Grainger et al., 2010), No. 5 (Grainger et al., 2010), No. 6 (Van Vugt, 2005), No. 9 (Van Vugt, 2005), and No. 12 (Junior et al., 2017) were influential studies.In addition, removing monensin doses above 24 ppm changed the results of the model and therefore, the above results are after removing these studies from the analysis.The heterogeneity  The result of quality and risk of bias assessment of all included studies.For each of the 10 entries, signaling questions were answered to facilitate judgment: "yes" indicates low risk of bias (green color), "no" indicates high risk of bias (red color), and "unclear" indicates an unclear risk of bias (yellow color).For example, if one of the relevant signaling questions is answered with "no," this indicates high risk of bias for that specific entry.index was low (heterogeneity <50%), and there was no significant publication bias for this measurement result (P > 0.1, Figure 5B).
Rumen pH.Twenty-one articles with 23 studies were included in the meta-analysis for rumen pH.This analysis revealed no association between monensin supplementation and rumen pH (Figure 4C).In the leave-one-out analysis, we determined that study No. 3 (Santos et al., 2019) was influential and was therefore removed from the analysis.Excluding data points above (heterogeneity <50%) and publication bias (P > 0.1) were observed for rumen pH (Figure 5C).
Rumen NH 3 .Twenty eligible articles with 21 studies were included in the analysis of the effects of monensin addition on NH3-N in the rumen.The results showed that the addition of monensin to the rations of dairy cows had no significant effect on rumen NH 3 -N (Figure 4D).The lack of association was also maintained in the tests of sensitivity analysis (leave-one-out analysis and exclusion of doses above 24 ppm).The extent of heterogeneity was low (heterogeneity <50%), and no publication bias was detected in this analysis (P > 0.1, Figure 5D).
Acetate.Twenty-two articles with 24 studies were included in the meta-analysis to evaluate the effects of monensin addition on rumen acetate concentration.A significant nonlinear relationship was found between monensin supplementation and the molar proportion of acetate in the rumen (P = 0.001).The addition of monensin at doses ranging from 13 to 28 ppm decreased acetate concentration, whereas the other doses had no effect (Figure 4E).Based on the results of the leaveone-out analysis, study No.3 (Santos et al., 2019) was identified as an influential study and was therefore removed from the analysis.In addition, the pooled association did not change when monensin doses above 24 ppm were removed from the analysis.No significant heterogeneity (<50%) and publication bias (P > 0.1) were observed for this result (Figure 5E).
Propionate.This analysis was performed using 22 articles with 24 eligible studies to determine the effects of monensin supplementation on the molar proportion of propionate in the rumen.There was a significant nonlinear relationship between monensin supplementation and rumen propionate proportion (P = 0.001).Monensin supplementation at doses between 15 and 24 ppm resulted in a significant increase in the molar proportion of propionate in the rumen.Outside this range, however, the effect was not significant.Based on the model results, the optimal monensin dose was estimated to be 13.29 ppm.(Figure 4F).In the leaveone-out analysis, studies No. 3 (Santos et al., 2019), No. 8 (Benchaar et al., 2006), No. 10 (Mathew et al., 2011), No. 14 (Ruiz et al., 2001), and No. 15 (Mutsvangwa et al., 2002) were found to be influential.In addition, the exclusion of study No. 21 (Drong et al., 2016) with a monensin dose greater than 24 ppm from the analysis changed the model results.Therefore, the above results are after removing these studies from the analysis process.In this analysis, there was no evidence of substantial heterogeneity (heterogeneity <50%).Although an obvious publication bias was detected by the funnel plot and Egger's test (P = 0.001, Figure 5F), while the trim and fill test confirmed the stability of the results.
Butyrate.Twenty-two articles with 24 studies were included in the meta-analysis for butyrate.The results showed that monensin administration had no significant effect on the molar proportion of butyrate in the rumen (Figure 4G).Leave-one-out analysis failed to find any influential studies.The monensin was used at a dose greater than 24 ppm in study No. 21 (Drong et al., 2016).When this dose was removed from the analysis, the overall P value of the model increased from 0.0054 to 0.1348.The heterogeneity index for this result was low (heterogeneity <50%), and there was no significant publication bias (P > 0.1, Figure 5G).

Effect on blood metabolites.
Glucose Seventeen articles with 19 eligible studies were included in this meta-analysis.This analysis found no association between supplemental intake of monensin and blood glucose concentration (Figure 4H).The robustness of the results was demonstrated by the tests of sensitivity analysis, which included leave-oneout analysis and removal of high doses of monensin.No significant heterogeneity was observed for this outcome measure (heterogeneity <50%).In addition, there was no evidence of obvious publication bias.(P > 0.1, Figure 5H).
Urea Thirteen articles with 15 studies were reviewed in this meta-analysis.In the present meta-analysis, no association was found between monensin supplementation and blood urea concentration (Figure 4I).The tests of sensitivity analysis showed that the results were stable.In addition, no significant heterogeneity (heterogeneity <50%) and publication bias (P > 0.1) were detected for blood urea concentration (Figure 5I).BHB This meta-analysis included 12 studies from 10 articles.The results showed that the addition of monensin to the diets of dairy cows did not affect blood BHB levels (Figure 4J).Studies No.2 (Schären et al., 2017), No. 5 (Azarfar et al., 2016), No. 7 (Karcher et al., 2007), No. 8 (Hausmann et al., 2018), and No. 11 (Drong et al., 2016) were identified as highly influential studies by leave-one-out analysis.When these studies were excluded from the analysis, the overall P-value of the model changed from 0.01 to 0.46 (Figure 4J).Sensitivity analysis was not performed to determine the effects of high doses of monensin because there was no study with a dose greater than 24 ppm.Low heterogeneity was observed for this result (heterogeneity <50%).The funnel plot and Egger's regression test indicated significant publication bias (P = 0.004), however, the trim and fill test confirmed the results (Figure 5J).
NEFA Fourteen studies were included in the metaanalysis on the effect of monensin on blood NEFA levels.This analysis showed no association between the supplemental intake of monensin and blood NEFA levels (Figure 4K).The validity of the results was confirmed by sensitivity analysis tests.No significant heterogeneity (heterogeneity <50%) and publication bias (P > 0.1) were detected (Figure 5K).

Effect on Nitrogen balance
Nitrogen intake The effect of monensin supplementation on nitrogen intake was meta-analyzed using data Ahvanooei et al.: Effects of monensin… from 16 articles with 17 eligible studies.This analysis revealed no association between supplemental monensin and nitrogen intake (Figure 4L).The results of the model were not affected by the sensitivity analysis and excluded high doses of monensin (>24 ppm).We found no significant heterogeneity in this meta-analysis (heterogeneity <50%) and publication bias (P > 0.1, Figure 5L).
Milk nitrogen Fifteen articles with 16 studies were used for this meta-analysis.The results showed that the addition of monensin had no significant effect on milk nitrogen (Figure 4M).Based on the results of the leave-one-out analysis, study No. 3 (Santos et al., 2019) was identified as an influential study, and its exclusion from the analysis increased the overall P value of the model from 0.004 to 0.30.Removal of doses above 24 ppm did not affect the results of the model.There was no substantial heterogeneity in this meta-analysis (heterogeneity <50%).However, publication bias (P = 0.07) was significant for milk nitrogen (Figure 5M), and the trim and fill test showed the reliability of results.
Urine nitrogen Data from 12 relevant studies that investigated the effects of monensin on urinary nitrogen status were used.The results showed that the addition of monensin to the feed of dairy cows did not affect urinary nitrogen levels (Figure 4N).In the leave-oneout analysis, studies No. 3 (Santos et al., 2019) andNo. 10 (Gandra et al., 2012) were considered influential and therefore excluded from the analysis.Their exclusion changed the association between monensin supplementation and urinary nitrogen levels (the P value of the model changed from 0.02 to 0.28).Sensitivity analysis for high doses of monensin (>24 ppm) was not performed because there were no studies with monensin levels above 24 ppm.No significant heterogeneity (heterogeneity <50%) and no significant publication bias (P > 0.1) were detected for this outcome variable (Figure 5N).
Fecal nitrogen This meta-analysis included 13 articles with 14 studies.Monensin supplementation and fecal nitrogen excretion were found to be associated in a nonlinear manner (P = 0.01).Addition of monensin to the diet at doses of 14-22 markedly reduced fecal nitrogen output and had no effect outside this range (Figure 4O).Studies No.3 (Santos et al., 2019) and No.12 (Gandra et al., 2012) were identified as influential studies based on the results of the leave-one-out analysis.When these studies were excluded from the analysis, the overall p-value of the model increased from 0.001 to 0.013 (Figure 4O).No study examined the effect of a high dose of monensin (>24 ppm) on fecal nitrogen excretion.The heterogeneity index for this outcome measure was low (heterogeneity <50%), and there was no significant publication bias (P > 0.1, Figure 5O).
Nitrogen retention Nine studies that examined the effects of monensin on nitrogen retention provided the data for this meta-analysis.The results showed that monensin supplementation had no significant effect on nitrogen retention in dairy cows (Figure 4P).The sensitivity analysis tests omitting the high dose of monensin (>24 ppm) did not change the model results.In addition, heterogeneity for nitrogen retention was low (heterogeneity <50%).The funnel plot and Egger's regression test indicated significant publication bias (P = 0.05).The trim and fill test revealed that publication bias may not be a problem in this analysis (Figure 5P).

DISCUSSION
The current meta-analysis compiled the results of published articles on the effects of monensin supplementation in lactating dairy cows on ruminal fermentation criteria, blood parameters, and nitrogen balance from 2001 to 2022.The results showed that monensin supplementation had a significant nonlinear relationship with DMI, with a significant decrease observed when the monensin dose was increased from 18 to 50 ppm.In addition, sensitivity analysis confirmed the statistical robustness of the results, and there was no substantial heterogeneity for this outcome measure in the current study.Different studies have reported different effects of monensin on DMI, resulting in either no effect or a decrease.For example, McGuffey et al. (2001) reported that supplemental monensin decreased DMI by 0.5 kg/day, whereas Symanowski et al. (1999) found that DMI decreased in the experimental groups supplemented with 16 and 24 ppm monensin.Similar to this study, Ipharraguerre and Clark (2003) reported a 1.5% decrease in DMI in 14 experiments with ionophoric compounds, and Santos et al. (2019) found an 18% decrease in DMI (3.58 kg d −1 ) in lactating dairy cows supplemented with high levels of monensin (48 mg/ kg DM).Conversely, the addition of monensin to the ration of early lactating dairy cows increased DMI by 5.8% (McCarthy et al., 2015).According to Benchaar et al. (2006) and Ipharraguerre and Clark (2003), the effect of monensin supplementation on DMI depends on the stage of lactation, the status of energy balances, the level of monensin administration, and the number of animals used in the study.
This meta-analysis found no relationship between monensin supplementation and rumen pH or rumen NH3 concentration.However, monensin supplementation was associated with methane emissions.This meta-analysis revealed low heterogeneity indices for the rumen fermentation parameters studied.Previous Ahvanooei et al.: Effects of monensin… studies reported inconsistent effects of monensin supplementation on rumen fermentation measurements.The DMI and nutrient composition of the experimental diets (type of carbohydrate and fat content of the diet), monensin dose, and duration of monensin treatment could explain the different results obtained in previous studies (Guan et al., 2006;Beauchemin et al., 2008;Ellis et al., 2012).
The addition of monensin to dairy cow rations at a dosage of 19-26 ppm significantly reduced (8.12-33.31g/d per cow) methane emissions.According to Beauchemin et al. (2009), monensin has a dose-dependent effect on CH4 production.It was observed that low doses (less than 20 mg/kg DMI) of monensin had no significant effect on CH4 production, whereas higher doses (24-35 mg/kg DMI) of monensin resulted in a decrease in CH4 production (by 4-10% on a g/day scale; Beauchemin et al., 2009).The decrease in CH4 production at higher doses of monensin could be due to an increase in the molar proportion of propionate to acetate and a decrease in protozoa in the rumen (Beauchemin et al., 2009).It has been suggested that each one-unit (mg/kg DMI) increase in monensin dose could increase the CH4 mitigation effect of monensin by 1.1 g/d in dairy cows and beef steers (Appuhamy et al., 2013).In addition, Odongo et al. (2007) observed a 7% reduction in CH4 production after monensin administration to dairy cows at a dose of 24 mg/kg DM.Despite the above reports, the results regarding the lack of effect of monensin on reducing methane production at low doses are contradictory.For example, Junior et al. (2017) found that a dose of 300 mg/day of monensin (17 mg/kg DM) could result in a 10.7% reduction in CH4 emissions.In contrast, Benchaar (2016) indicated that in foragebased diets, inclusion of monensin at doses of ≤24 mg/ kg DMI may not represent a CH4 reduction strategy in dairy cows.Grainger et al. (2008) also found that administration of monensin in the form of controlledrelease capsules containing 240 mg monensin/d (13 mg monensin/kg DM) did not affect enteric methane emission in Holstein cows.Some of the mentioned contradictions may be due to different conditions when conducting the experiment, such as the type of feed consumed (TMR or pasture), the method of using monensin (top-dressed, CRC or TMR), DIM, breed, etc.The reducing effect of monensin on DMI may also be a plausible reason to explain the reduction in methane emissions in this study.As reported by Appuhamy et al. (2013) and Beauchemin et al. (2009), the reduction in CH4 production when feeding monensin could be partially explained by the reduced DMI.
This meta-analysis found that monensin administration at doses of 15 to 24 ppm resulted in a significant decrease in acetate and an increase in propionate mole fraction in the rumen, but did not affect butyrate.Previous in vitro and in vivo studies (Mbanzamihigo et al., 1996;García et al., 2000) showed that monensin-fed cows had a higher molar fraction of propionate, whereas the butyrate fraction and acetate-propionate ratio were lower.According to Krause and Russell (1996), this increase in rumen propionate molar content is due to the inhibition of gram-positive bacteria, which interfere with acetate, butyrate, and ammonia production in the rumen and favor the growth of gram-negative bacteria in addition to propionate production.
According to the present meta-analysis, supplemental monensin had no significant effects on blood metabolites including glucose, urea, BHB, and NEFA.This result was surprising considering that a previous meta-analysis of 24 studies found that monensin can lower NEFA while increasing blood glucose concentrations (Duffield et al., 2008).The divergence between the results of our study and the meta-analysis performed by Duffield et al. (2008) may be attributed to differences in methodology.Specifically, our study included more recent data published through 2021 and used a random-effects model to account for heterogeneity.These methodologic differences and inclusion criteria may have led to the observed differences in the results.It is important to note that unlike our study, the previous meta-analysis by Duffield et al. (2008) was significantly limited by the lack of a sensitivity analysis, which may have influenced the results.Consistent with our results, as shown in Figure 2, most individual studies did not report a significant effect of monensin on blood metabolites.However, in contrast to our results, Petersson-Wolfe et al. (2007) showed that monensin could reduce BHB in dairy cows.According to them, this reduction could be related to the reduction of butyrate, which is converted to BHB in the rumen epithelium.Therefore, based on our results regarding the lack of effect of monensin on rumen butyrate, the results regarding BHB are also confirmed.
In contrast to the results of Markantonatos and Varga (2017) regarding the improvement of energy status in transition cows by monensin, the results of this study showed no significant effect of monensin on plasma NEFA concentration.The increase in NEFA concentration in dairy cows occurs following a negative energy balance in early lactation caused by a decrease in DMI and high milk production (McCarthy et al., 2015).On the other hand, Karcher et al. (2007) indicated that a decrease in NEFA and BHB concentration was observed during monensin feeding, but reported a lack of residual effect of prepartum monensin on NEFA and BHB in plasma postpartum.Plasma urea concentration is positively related to urea recycling, which in turn is negatively related to rumen NH3-N concentra-  (Kennedy et al., 1980).Therefore, according to our results, that monensin has no effect on NH3-N in the rumen, the result of ineffectiveness of monensin on blood urea is also reinforced.In contrast to our results, Santos et al. (2019) showed that the use of monensin at a high dose (48 mg/kg DM) can lead to a significant increase in blood urea concentration and attributed this to the effect of monensin on rumen microorganisms, especially those that promote proteolysis and deamination of amino acids.In a study by Arieli et al. (2001), glucose disposal rates were reduced but the plasma glucose pool increased, suggesting an improvement in the energy status of transition cows receiving monensin.Similarly, Markantonatos and Varga (2017) suggested that monensin could increase the efficiency of gluconeogenesis and contribute to the glucose pool.However, our results for blood glucose rely on the noneffect of most of the individual studies in Figure 2.
The results of this meta-analysis showed that the addition of monensin did not influence nitrogen retention in dairy cows.According to Plaizier et al. (2000), monensin administration improved N retention, whereas Benchaar et al. (2006) observed no effect of feeding monensin (16 mg/kg DM).The inherent error in N balance studies (Spanghero and Kowalski, 1997) likely overestimates actual nitrogen retention, as evidenced by the modest change in body weight (Benchaar et al., 2006).Assuming that body tissues contain approximately 20% protein (NRC, 2021), retention of 27.3 g N/d (i.e., 170.6 g protein/d) in the Benchaar et al. (2006) study should have resulted in a weight gain of 0.85 kg/d, whereas in that study, feeding monensin (16 mg/kg DM) resulted in a weight gain of only 70 g per d.Therefore, the effect of monensin on nitrogen retention in dairy cows needs further investigation.

Study Limitations
In conducting our systematic review, one of the PRIS-MA guidelines was deemed 'not applicable' reflecting our unique study structure.This included the results of individual studies (forest plot).Another limitation was that we used only English-language articles.In addition, our data source was limited to peer-reviewed articles, reflecting our commitment to rigor and reliability.Despite these considerations, we are confident in the soundness of our methodology and the authenticity of our findings.

CONCLUSION
Our results suggest that supplementation with monensin at a dosage of 19-26 ppm was inversely related to methane emissions.DMI was significantly decreased by increasing the monensin dose from 18 to 50 ppm.In addition, supplementation with monensin at doses ranging from 15 to 24 ppm resulted in a significant decrease in acetate production and an increase in propionate production.However, no significant effects of feeding different doses of monensin on blood parameters and nitrogen retention were observed.Overall, based on the metabolic and rumen fermentation parameters covered in the present meta-analysis, the recommended optimal dose range of monensin would be 19-24 ppm.Although the use of monensin in dairy cow diets has been extensively studied, more research is needed to determine the optimal use rate and potential environmental, animal health, and performance benefits of this feed additive.

Figure 1 .
Figure 1.Flowchart of the literature search, identification, and screening process for selecting suitable studies (search conducted from April 2021 to February 2022).

Figure 2 .
Figure2.Percent effectiveness of "examined monensin doses" on outcomes in dairy cows.The numbers in parentheses before the results indicate the number of doses studied in the studies that fall within the above dose ranges, separated by the slash '/'.For example, for DMI (56/5/4), a total of 65 different doses were evaluated, of which 56, 5, and 4 doses fell within the ranges of 10-24, 24-35, and 35-48 ppm, respectively.The effect was considered significant if the overall P-value of the model was less than 0.05.Uncolored columns indicate the absence of studies in the target dose range and intended outcome.

Figure 3 .
Figure3.The result of quality and risk of bias assessment of all included studies.For each of the 10 entries, signaling questions were answered to facilitate judgment: "yes" indicates low risk of bias (green color), "no" indicates high risk of bias (red color), and "unclear" indicates an unclear risk of bias (yellow color).For example, if one of the relevant signaling questions is answered with "no," this indicates high risk of bias for that specific entry.
Ahvanooei et al.: Effects of monensin… adapted version of the Cochrane RoB tool

Table 1 .
Ahvanooei et al.:Effects of monensin… Summary of studies used in the meta-analysis on the effect of monensin supplementation in dairy cows Ahvanooei et al.: Effects of monensin… tion