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Article

A Meta-Analysis of the Effects of a Chemical Additive on the Fermentation and Aerobic Stability of Whole-Plant Maize Silage

by
Érica Benjamim da Silva
and
Limin Kung, Jr.
*
Department of Animal and Food Sciences, University of Delaware, 531 S. College Ave., Newark, DE 19716, USA
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(2), 132; https://doi.org/10.3390/agriculture12020132
Submission received: 10 December 2021 / Revised: 12 January 2022 / Accepted: 13 January 2022 / Published: 19 January 2022
(This article belongs to the Special Issue Forage Quality, Conservation and Evaluation in Ruminant Production Systems)
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Abstract

:
Our objective was to conduct a meta-analysis on the effects of a chemical additive containing sodium benzoate, potassium sorbate, and sodium nitrite on the fermentation and aerobic stability of whole-plant maize silage. We used data from 28 experiments, with 56 untreated to treated comparisons, carried out over a 9-year period. The influence of dry matter (DM) content and length of ensiling on the additive effect were assessed by meta-regression and subgroup analysis. Treatment with the additive reduced the pH and concentrations of ammonia-N, lactic acid, and ethanol, but increased the concentration of residual reducing sugars, DM recovery, and aerobic stability (by about 5 days). As the forage DM decreased, there was a greater reduction in ethanol by the additive treatment. The additive reduced the number of yeasts in silages made with forage harvested at >32% DM. The improvement in aerobic stability by the additive increased as ensiling time progressed. Overall, these findings show that the chemical additive has the ability to consistently and markedly improve the aerobic stability of maize silage under a wide range of conditions.

1. Introduction

Chemical additives are often used to inhibit the fermentation of undesirable bacteria, reducing losses and preventing the development of yeasts that initiate aerobic deterioration in silages [1]. Auerbach and Nadeau [2] reported that the use of chemical additives with a single compound is uncommon, and mixtures of compounds are preferred as they might promote a reduction in costs, increase the range of inhibition to multiple microorganisms, and may have synergistic effects. In the initial experiments evaluating an additive containing sodium benzoate, potassium sorbate, and sodium nitrite (ADT), Knicky and Sporndly [3] and da Silva et al. [4] reported that it markedly improved the aerobic stability of various silages.
Sodium benzoate and potassium sorbate are antifungal compounds used in the human food and animal feed industries, and in silage production, they aim to inhibit yeasts that commonly initiate aerobic deterioration [5]. Sodium nitrite, however, is mainly applied to silages to inhibit bacteria such as clostridia and enterobacteria, which cause undesirable fermentations [1]. In most cases, undesirable fermentation by bacteria are not as concerning in whole-plant maize silage compared to grass or legume silages because the former has a lower buffering capacity and adequate content of water-soluble carbohydrates, which allows for a rapid decrease in pH and an inhibition of these bacteria [6]. However, the addition of sodium nitrite to mixtures of inhibitors of aerobic spoilage might still be beneficial to maize silage, as it could induce synergistic actions with antifungal compounds. For example, Stanojevic et al. [7], in an in vitro trial, observed synergy between sodium benzoate and sodium nitrite against Escherichia coli, Staphylococcus aureus, Bacillus mucoides, and Candida albicans, and between potassium sorbate and sodium nitrite against B. mucoides, Pseudomonas aeruginosa, and E. coli.
Microbial inoculants can also effectively improve the aerobic stability of maize silage, and often have a lower initial cost than chemical additives [1]. Still, chemical additives can lead to greater dry matter (DM) recovery [2], which might make up for their higher costs. Research has focused on the development of inoculants that can improve aerobic stability after short periods of ensiling (e.g., 30 days or less). Such inoculants contain bacteria capable of producing moderate amounts of acetic acid, which have a good antifungal activity against yeasts that often initiate aerobic spoilage in silages. However, achieving early improvements in aerobic stability with inoculants has been inconsistent [8,9]. In their favor, chemical additives have the probability of acting quickly in a silo, as their action is direct and not dependent on a microorganism to outcompete epiphytic microorganisms and to produce antifungal end products. For example, Kung et al. [10] and da Silva et al. [11] observed that ADT improved the aerobic stability of maize silage after as early as 5 d and 1 d of ensiling, respectively.
In addition to experiments conducted to purposely evaluate the ability of ADT to improve the aerobic stability of silages [10,11,12,13,14], our laboratory used it as a positive control in experiments with maize silage, creating a unique database that documents the effectiveness of ADT over a broad range of conditions over several years. Therefore, our objective was to perform a meta-analysis of the effects of ADT on the fermentation and aerobic stability of whole-plant maize silage, as well as a meta-regression analysis of the influence of the DM content and length of ensiling on the magnitude of the additive effect. We hypothesized that the treatment with ADT would reduce the number of yeasts and the ethanol production, increase DM recovery and aerobic stability, and that a more significant effect would be observed in drier silages and silages ensiled for more extended periods.

2. Materials and Methods

We performed a meta-analysis with data from 28 experiments conducted between 2012 and 2020, which included 56 different comparisons between maize silage treated with ADT vs. untreated silage (Table S1). All of the experiments were conducted by the Dairy Nutrition and Silage Fermentation Laboratory at the University of Delaware, Newark, DE, USA. In several experiments, more than one length of ensiling was evaluated, and data collected at each length of ensiling were used as a different comparison.
In most of the experiments, whole maize plant was harvested from the University of Delaware Farm (Newark, DE, USA) and was chopped to a theoretical length of 19 mm using a pull-type chopper (3975, John Deere, Moline, IL, USA) equipped with a kernel processer (roller gap set to 1.4 mm). The exception was in comparisons 25 and 26 harvested from the University of Pennsylvania Dairy (New Bolton, PA, USA), and 29 and 30 from the Chesapeake Gold Dairy (Chesapeake City, MD, USA). Following harvest, forages were untreated (CTR) or treated with an additive (ADT) containing 20% sodium benzoate, 10% potassium sorbate, and 5% sodium nitrite (Safesil, Salinity, Göteborg, Sweden) at 2 L/t of fresh forage weight. The forages were packed into 7.5-L laboratory silos using a custom-made gas-driven hydraulic press, modified from a log splitter in all of the experiments. Densities ranged from 200 to 240 kg DM/m3. Silos were sealed with plastic lids with O-ring seals, ensiled for varying times, and stored in laboratory conditions between 18 to 24 °C. The number of replicates per treatment, the DM content of the fresh forage prior to ensiling, and the length of ensiling for each experiment are shown in Table S1.
The variables analyzed in the meta-analysis were the concentrations of residual reducing sugars, ammonia-N, lactic acid, acetic acid, ethanol, the pH, and the numbers of LAB and yeasts, DM recovery, and aerobic stability. The DM recovery was calculated using the weight and DM content of the fresh forage packed in the silo and of the silage after the ensiling period. The methods of chemical and microbial analyses were the same as described in Kung et al. [10]. An exception was the enumeration of yeasts in Comparisons 17 to 24, which were determined on 3M Yeast and Mold Petrifilm (3M Corporation, 3M, St. Paul, MN, USA) and incubated aerobically at 32 °C for 48 h. The lowest dilution for LAB and yeast detection was 2 log10 cfu/g of fresh weight. Aerobic stability was defined as the number of hours before the silage mass temperature increased 2 °C above the baseline temperature of each silo after exposure to air at 18 to 24 °C. The aerobic stability of the silage was measured for different lengths of time in each comparison, and to standardize, we established an upper limit of 240 h of aerobic exposure. Therefore, in comparisons where silage did not spoil for more than 240 h, this value was used as the input. Six comparisons (16, 25, 27, 31, 35, and 36), in which the CTR or ADT silages did not spoil (i.e., silages temperatures did not increase 2 °C above the baseline) and the aerobic stability measurement ended before 240 h, were not used for the meta-analysis of aerobic stability.
The means and standard deviations of the CTR and ADT treatments were extracted for each comparison using JMP Pro 15 (SAS Institute Inc., Cary, NC, USA), and together with the sample sizes of the treatments, were used as the input for the meta-analysis. The meta-analysis was performed using a random-effects maximum likelihood model with the raw means differences of ADT and CTR on Jamovi Version 1.6.23.0 (available at https://www.jamovi.org, accessed on 9 August 2021) [15]. Significance was declared at p ≤ 0.05 and tendency at p > 0.05 and ≤0.10. Studentized residuals and Cook’s distances were used to determine if comparisons were outliers [16]. Comparisons were considered outliers when they had a studentized residual larger than the 100 × (1 − 0.05/(2 × k))th percentile of a standard normal distribution (using a Bonferroni correction with two-sided alpha = 0.05 for k comparisons included in the meta-analysis) and a Cook’s distance larger than the median plus six times the interquartile range of the Cook’s distances. The amount of heterogeneity (τ2) was estimated using the restricted maximum-likelihood estimator [17]. In addition, the percentage of total variation due to heterogeneity (I2) and the Q-test for heterogeneity [18] were also reported. Finally, to check for bias, we performed the rank correlation test [19] and the Egger’s regression test [20], using the standard error of the observed outcomes as the predictor, as well as the Fail-Safe N test using the Rosenthal method [20]. The Egger’s test is based on a linear regression of the intervention effect estimates on their standard errors, weighting by their inverse variance, and the Fail-Safe N test is based on the number of missing studies needed to nullify the effect (i.e., change the meta-analysis results) [20]. A Fail-Safe N test with a p-value greater than 0.05 indicates that few studies are needed to change the meta-analysis results. Rank correlation and Egger’s regression tests with a p-value lower than 0.05 indicate a high risk of bias.
A forest plot showing the effect of additive treatment on the aerobic stability of maize silage was made using JMP Pro 15 (SAS Institute Inc., Cary, NC, USA). A meta-regression analysis was performed to detect the influence of the covariates’ DM content of the forage before ensiling and the length of ensiling on the additive effect on Jamovi Version 1.6.23.0 [15]. The comparisons were separated into three categories according to the forage DM content and the length of ensiling. For the DM content, groups 1, 2, and 3 comprised forages with ≤32, 32.01 to 37.99, and ≥38% DM, respectively. Comparisons were categorized in groups 1, 2, or 3 if the ensiling occurred for ≤20, 21 to 69, or ≥70 d, respectively. The meta-regression analysis was conducted using a random-effects maximum likelihood model with the raw means differences. Subgroup analysis using a random-effects maximum likelihood model with the raw means differences was performed on Jamovi Version 1.6.23.0 [15] if a covariate effect was detected.

3. Results and Discussion

3.1. Effect of Additive Treatment on Maize Silage Characteristics

Table 1 shows the effects of additive treatment on maize silage characteristics. Treatment with ADT did not affect the concentration of acetic acid (p = 0.52) and the numbers of lactic acid bacteria (LAB; p = 0.98). However, it increased (p < 0.001) the concentration of residual reducing sugars, DM recovery, and aerobic stability, and decreased (p < 0.001) the concentrations of ammonia-N, lactic acid, and ethanol, pH, and the number of yeasts.
Sodium benzoate and potassium sorbate are mostly utilized in silage production as inhibitors of yeasts and molds, but they can also inhibit some bacteria, such as Bacillus sp. [1,21]. Conversely, sodium nitrite is mainly applied to inhibit undesirable bacteria [22,23]. Therefore, a small inhibition of the fermentative processes in the silo by the additive was not unexpected. Even though the additive effects on pH, lactic acid, and ammonia-N were significant, they were small and possibly of limited biological relevance. Still, these results facilitated the interpretation of the additive effects on the fermentation. Treatment did not affect the numbers of LAB and only slightly reduced (−0.21% of DM) the lactic acid concentration (Table 1). In comparison, Zhang et al. [24] observed a greater reduction (−1.76% of DM) in the lactic acid concentration in maize silage treated with a mixture of 40% potassium sorbate and 60% sodium benzoate at 2 g/kg of fresh matter, after 42 d of ensiling. Silages treated with the ADT had 6.94% less ammonia-N than CTR (Table 1), which indicates that the activity of the proteolytic microorganisms might have been slightly inhibited by the additive treatment. This reduction in ammonia-N content was detected even though the application of sodium nitrite can directly increase the ammonia concentration in the silo. Therefore, the actual reduction in ammonia-N content by the inhibition of proteolytic organisms by additive treatment is possibly even greater than what was estimated, as suggested by Auerbach and Nadeau [2]. The additive appeared not only to have inhibited the development of proteolytic microorganisms, but also of sugar fermenters (e.g., LAB, undesirable bacteria, or yeasts), as ADT silages had 15.91% more residual reducing sugars than CTR silages (Table 1). In agreement with our findings, when evaluating 20 comparisons between high-moisture maize treated with various chemical additives and untreated maize, Morais et al. [25] observed that treated silages had 35.5% more residual water-soluble carbohydrates than the untreated ones. Similarly, Knicky and Sporndly [26] evaluated a high dose (5 L/t of fresh forage) of the same combination of salts of acids tested in the present study on the fermentation of a mixture of red clover and grasses inoculated with spores of Clostridium tyrobutyricum. They observed that treated silage had 90% more residual water-soluble carbohydrates after 119 d of ensiling than the untreated silage.
Another indication of the inhibition of fermentative processes by ADT is that those silages had 45% less ethanol than the CTR silages (Table 1). Comparably, Weiss et al. [27] observed that maize silage treated with 2 L/t of an additive containing 250 g/L of sodium benzoate and 150 g/L of potassium sorbate and ensiled for 90 d had 58% less ethanol and 41% fewer yeasts than the untreated silage. Several microorganisms can produce ethanol during silage fermentation, such as heterofermentative LAB, enterobacteria, and yeasts [28]. The overall inhibition of fermentation and the reduction of 18% in the numbers of yeasts (Table 1) might explain the reduction in the ethanol concentration in ADT silages.
Several microorganisms can contribute to the losses of DM during the ensiling process, such as heterofermentative LAB, enterobacteria, clostridia, and yeasts [5]. The development of clostridia and enterobacteria can be controlled by fast acidification of the silage [29], which is often observed in maize silage. Therefore, in maize silage, heterofermentative LAB and yeasts are most likely responsible for the majority of the DM losses, at least when air is excluded from the system. Borreani et al. [5] pointed out that the total elimination of fermentation losses was not possible, but that the use of additives could help to minimize these losses. Morais et al. [25], when evaluating ten comparisons of high-moisture maize treated with various chemical additives and untreated high-moisture maize, observed that chemical additive treatments increased the DM recovery by 0.49 percentage points. In the present meta-analysis, ADT increased the DM recovery by 1.24 percentage points (Table 1). Similarly, Bernardes et al. [30] observed that the difference in weight losses between untreated maize silage and maize silage treated with potassium sorbate at 2 g/kg of fresh matter or with sodium benzoate at the same dose was 1.18 and 0.93 percentage points, respectively, after 88 d of ensiling.
Overall, treatment with ADT increased (p < 0.001) the aerobic stability of silage by 117 h compared to CTR (Table 1). Figure 1 displays the forest plot showing the effect of ADT treatment on the aerobic stability of maize silage overall and for each comparison individually. From the 46 comparisons analyzed, only one (Comparison 55) had a negative raw means difference, and two others (Comparisons 8 and 44) had a negative 95% confidence interval lower limit. In Comparison 8, the CTR silage was stable for an average of 235 h, and none of the ADT silages spoiled until 240 h when the measurements ended. Therefore, the fact that CTR silage was stable for a more extended period than usual and the aerobic stability measurements ended before all silages spoiled might have influenced the lack of significant improvement in aerobic stability by additive treatment. In Comparison 44, a similar situation was observed, in which two replicates of CTR and all three replicates of ADT did not spoil in 240 h. It is uncommon for untreated silages to have a high aerobic stability after short-term ensiling, as observed in Comparison 8 after 56 d of ensiling and in Comparison 44 after 30 d of ensiling. In Comparison 55, the CTR silage was stable for 130 h and ADT silage for 104 h, and the reasoning previously elucidated does not apply. Interestingly, Comparison 56, which belonged to the same study of Comparison 55, but had silages ensiled for 103 d instead of 60 d, showed improved aerobic stability in response to additive treatment. In this case, the reason for the lack of additive effects in Comparison 55 is unknown.
In agreement with our findings, Bernardes et al. [30] observed that treatment with potassium sorbate or sodium benzoate at 2 g/kg fresh forage weight increased maize silage aerobic stability by 194 and 196 h, respectively, after 88 d of ensiling. Likewise, da Silva et al. [31] observed that maize silage treated with sodium benzoate at 2 g/kg of fresh forage weight and ensiled for 103 d was 120 h more stable than the untreated silage. Auerbach and Nadeau [32], when testing the same additive evaluated in the present study, also at 2 L/t of fresh forage weight, observed that it increased the aerobic stability of maize silage by 5 d compared to the untreated silage, after 89 d of ensiling. Even though the improvement in aerobic stability observed by Auerbach and Nadeau [32] was only numerical and not statistical, it was very similar to the one observed in the present study (5 vs. 117 h or 4.9 d). The 67%-increase in aerobic stability by additive treatment is possibly explained by the inhibition of undesirable fermentation and a reduction in the number of yeasts.

3.2. Influence of DM Content of the Forage on Additive Effects

Meta-regression showed that the DM content of the forage prior to ensiling influenced (p = 0.03) the effect of additive treatment on the ethanol concentration and tended (p = 0.10) to influence its effect on the yeast numbers (Table 2).
The lower the DM content of the forage prior to ensiling, the greater the reduction in ethanol concentration by treatment with the additive (reduction of 30.82%, p < 0.001, at ≥38% DM; 57.53%, p < 0.001, at 32.01−37.99% DM; and 69.86%, p = 0.02, at ≤32% DM; Figure 2). Such a finding might be explained by the fact that, considering only untreated silages, the ones made from wetter forages (<38% DM) had a greater concentration of ethanol than those made from drier forages (≥38% DM) (1.74 vs. 1.22% DM). Because the wetter silages had a greater initial ethanol concentration, in those silages, the additive effect could be more pronounced than in the drier silages. Less mature maize plants have a higher water activity and available sugars than more mature maize plants [33], which might facilitate the growth and activity of the microorganisms that cause undesirable fermentations and produce ethanol, such as enterobacteria, heterofermentative LAB, and yeasts [28]. However, such a hypothesis could not be confirmed in the present study as the numbers of yeasts and LAB were not greater in the wetter silage and the numbers of enterobacteria were not measured. Bal et al. [34] observed that silage made with whole maize plant at the early dent stage (30.1% DM) had a greater concentration of ethanol than silage made from forages at the quarter milk line, two-thirds milk line, and black layer stages (0.87 vs. average of 0.18% DM). In contrast, Hu et al. [35] reported that untreated maize silage made from forage with 33.1% DM had a lower concentration of ethanol than that made with forage at 40.6% DM. However, in agreement with our findings, Hu et al. [35] observed that treatment with Lactobacillus buchneri did not reduce the ethanol content compared to untreated silage when the initial forage had 40.6% DM, but in silage made from forage with 33.1% DM it reduced the ethanol concentration by approximately 57%.
The additive treatment did not affect (p = 0.95) the yeast numbers in silages made of forages that had ≤32% DM. However, treatment with ADT reduced (p < 0.001) the number of yeasts by 16.46% and 21.07% in silages made of forages with 32.01 to 37.99% and ≥38% DM, respectively (Figure 3). Silages made of forages with a DM content between 32.01 to 37.99% had 4.15 log cfu/g of fresh weight yeasts, and silages made of forages with ≥38% DM had 4.45 log cfu/g of fresh weight yeasts. However, in silages made of forages with ≤32% DM, the numbers of yeasts were relatively low (3.01 log fu/g of fresh weight). Therefore, the inhibition of yeasts by additive treatment was not statistically significant at ≤32% DM, possibly due to the already low numbers of yeasts in those silages. Another possible explanation for the lack of additive effect at ≤32% DM is that the detection limit was 2 log cfu/g of fresh weight. Therefore, if the additive reduced the yeast numbers below 2 log cfu/g of fresh weight, an effect could not be detected. Additionally, in the present study, CTR silages made of forages with ≤32% DM had concentrations of lactic and acetic acids of 6.06 and 1.67% of DM, respectively, whereas silages made of forages with >32% DM had on average 4.98 and 1.27% of DM of lactic and acetic acids, respectively. The higher levels of acids, especially acetic acid with antifungal properties [36], in silages with a lower DM content (≤32%) might have caused their yeast numbers to be relatively low.

3.3. Influence of Length of Ensiling on Additive Effects

The length of ensiling influenced the effect of treatment with ADT on the concentration of residual reducing sugars (p = 0.03) and tended (p = 0.052) to influence its effects on aerobic stability (Table 2).
Treatment with ADT increased the concentration of residual reducing sugars (by 39.77%, p < 0.001) in forage ensiled for ≤ 20 d, tended to increase the concentration by 12.50% (p = 0.07) forage ensiled for 21 to 69 d, but did not affect (p = 0.11) the concentration of sugars in forage ensiled for ≥ 70 d (Figure 4). We interpret these findings to suggest that treatment with ADT moderately inhibited fermentation, especially in the early stages of ensiling, but that with longer ensiling times, the use of reducing sugars as a substrate reached the same level as observed in the CTR silages.
Treatment with ADT increased (p < 0.001) the aerobic stability of the silages at all of the three lengths of ensiling evaluated. The increase in aerobic stability was 39%, 57%, and 100% in silages ensiled for ≤20 d, 21 to 69 d, and ≥70 d, respectively (Figure 5). In a review by Auerbach and Nadeau [2], greater improvements in aerobic stability were reported with an extended length of ensiling in the maize silage treated with chemical additives. In particular, the authors reported data from an unpublished study by Auerbach et al., where the aerobic stability of maize silage treated with a chemical additive (257 g/L sodium benzoate, 134 g/L potassium sorbate, and 57 g/L ammonium propionate) at 2 L/t of fresh forage weight increased from 7 to 34 d of ensiling from 92 to 297 h. Such findings might be because yeasts are usually the main microorganisms responsible for initiating aerobic deterioration of silages [29], and that the inhibitory effect of organic acids with antifungal properties on their metabolism can be dependent on the length of exposure to the acid [37]. Although ADT improved the aerobic stability of the maize silages ensiled for ≤20 d, caution should be taken to allow enough time for sodium nitrite to be degraded in the silo before feeding, as it can be toxic to cattle [38].

4. Conclusions

The meta-analysis showed that over several years and in a multitude of experiments, treatment with an additive containing sodium benzoate, potassium sorbate, and sodium nitrite consistently altered the fermentation of maize silages. The main additive effects included a reduction in the numbers of yeasts when the DM was greater than 32%, a lower concentration of ethanol, a greater DM recovery, and improved aerobic stability. The additive improved the aerobic stability even in silages ensiled for less than 20 d, and the improvements further increased with extended times of ensiling.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture12020132/s1, Table S1: Meta-analysis dataset.

Author Contributions

É.B.d.S.: conceptualization, methodology, formal analysis, investigation, data curation, writing—original draft preparation, review, and editing. L.K.J.: conceptualization, methodology, resources, writing—review and editing, supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially funded by Salinity AB, Gothenburg, Sweden.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the Supplementary Material.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Forest plot showing the effect of additive treatment on the aerobic stability (h) of maize silage (p < 0.001). The weight, raw mean difference (MD), and the lower and higher confidence limit for each comparison and across studies are shown in the right column. The x-axis shows the MD between maize silage treated with additive (ADT) and untreated maize silage (CTR). The circle represents the MD of each comparison and the star represents the MD across all comparisons. The length of the horizontal lines represents the 95% confidence interval for the effect size. The vertical dashed line represents an MD of zero and lack of effect. Points to the left of the line indicate a decrease in aerobic stability by additive treatment, and points to the right of the line indicates an increase in aerobic stability by additive treatment. PP—partially published. Kung et al. (2018) [10]; da Silva et al. (2017b) [13]; Savage et al. (2016) [14]; da Silva et al. (2017a) [11]; da Silva et al. (2020) [12].
Figure 1. Forest plot showing the effect of additive treatment on the aerobic stability (h) of maize silage (p < 0.001). The weight, raw mean difference (MD), and the lower and higher confidence limit for each comparison and across studies are shown in the right column. The x-axis shows the MD between maize silage treated with additive (ADT) and untreated maize silage (CTR). The circle represents the MD of each comparison and the star represents the MD across all comparisons. The length of the horizontal lines represents the 95% confidence interval for the effect size. The vertical dashed line represents an MD of zero and lack of effect. Points to the left of the line indicate a decrease in aerobic stability by additive treatment, and points to the right of the line indicates an increase in aerobic stability by additive treatment. PP—partially published. Kung et al. (2018) [10]; da Silva et al. (2017b) [13]; Savage et al. (2016) [14]; da Silva et al. (2017a) [11]; da Silva et al. (2020) [12].
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Figure 2. Influence of the DM content of the forage prior to ensiling on the effect of additive treatment on the concentration of ethanol in maize silage. Error bars show the 95% confidence interval. n—number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
Figure 2. Influence of the DM content of the forage prior to ensiling on the effect of additive treatment on the concentration of ethanol in maize silage. Error bars show the 95% confidence interval. n—number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
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Figure 3. Influence of the DM content of the forage prior to ensiling on the effect of additive treatment on the number of yeasts in maize silage. Error bars show the 95% confidence interval. n = number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
Figure 3. Influence of the DM content of the forage prior to ensiling on the effect of additive treatment on the number of yeasts in maize silage. Error bars show the 95% confidence interval. n = number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
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Figure 4. Influence of the length of ensiling on the effect of additive treatment on the concentration of reducing sugars in maize silage. Error bars show the 95% confidence interval. n—number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
Figure 4. Influence of the length of ensiling on the effect of additive treatment on the concentration of reducing sugars in maize silage. Error bars show the 95% confidence interval. n—number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
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Figure 5. Influence of the length of ensiling on the effect of additive treatment on the aerobic stability of maize silage. Error bars show the 95% confidence interval. n—number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
Figure 5. Influence of the length of ensiling on the effect of additive treatment on the aerobic stability of maize silage. Error bars show the 95% confidence interval. n—number of comparisons; MD—raw means difference between silage treated with additive and untreated; ADT—silage treated with additive; CTR—untreated silage.
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Table
Table 1. Additive effect on the chemical characteristics (% of DM), fermentation products (% of DM), microbial numbers (log10 cfu/g of wet silage weight), dry matter recovery (%), and aerobic stability (h) of maize silage.
Table 1. Additive effect on the chemical characteristics (% of DM), fermentation products (% of DM), microbial numbers (log10 cfu/g of wet silage weight), dry matter recovery (%), and aerobic stability (h) of maize silage.
ItemnCTR Mean
(Standard Deviation)		Means Difference (95% CI)HeterogeneityBias Assessment (p-Values)
Effect Sizep-Valueτ2I2p-ValueFail-Safe N *Rank
Correlation		Egger’s Regression *
Reducing sugars340.88 (0.71)0.14 (0.06, 0.21)<0.0010.04093.28<0.001<0.0010.9060.859
Ammonia-N380.072 (0.020)−0.005 (−0.007, −0.003)<0.0010.00576.86<0.001<0.0010.5490.192
pH563.77 (0.12)−0.03 (−0.04, −0.01)<0.0010.00395.57<0.001<0.0010.5270.346
Lactic acid504.95 (1.16)−0.21 (−0.33, −0.08)<0.0010.09056.04<0.001<0.0010.5980.969
Acetic acid501.30 (0.40)−0.02 (−0.08, 0.04)0.520.02474.47<0.0010.0700.9470.619
Ethanol501.46 (0.99)−0.66 (−0.89, −0.44)<0.0010.63698.77<0.001<0.0010.2710.914
LAB527.18 (1.17)0.001 (−0.07, 0.07)0.980.03489.83<0.0010.0330.0040.147
Yeasts564.13 (1.09)−0.73 (−0.95, −0.52)<0.0010.54699.84<0.001<0.0010.6160.002
DM recovery5696.09 (3.27)1.24 (0.56, 1.91)<0.0014.66383.03<0.001<0.0010.7730.883
Aerobic stability4670 (47)117 (99, 136)<0.001364399.82<0.001<0.0010.5960.224
n—number of comparisons; CTR—untreated silage; τ2—between cluster variance; I2—percentage of total variation that is due to heterogeneity; LAB—lactic acid bacteria; DM—dry matter. * Fail-Safe N test with p > 0.05 and Rank correlation and Egger’s regression tests with p < 0.05 indicate high bias risk.
Table
Table 2. Meta-regression of the effects of the three levels of DM content of the forage prior to ensiling and of the three categories of length of ensiling on the chemical characteristics (% of DM), fermentation products (% of DM), microbial numbers (log10 cfu/g of fresh forage weight), dry matter recovery (%), and aerobic stability (h) for the mean differences between maize silage treated with additive and untreated.
Table 2. Meta-regression of the effects of the three levels of DM content of the forage prior to ensiling and of the three categories of length of ensiling on the chemical characteristics (% of DM), fermentation products (% of DM), microbial numbers (log10 cfu/g of fresh forage weight), dry matter recovery (%), and aerobic stability (h) for the mean differences between maize silage treated with additive and untreated.
Dependent
Variables		nCovariates
Forage DM Content *Length of Ensiling **
Intercept
(p-Value)		Moderator
(p-Value)		τ2Heterogeneity p-ValueIntercept
(p-Value)		Moderator
(p-Value)		τ2Heterogeneity p-Value
Reducing sugars340.004 (0.97)0.059 (0.25)0.040<0.0010.40 (0.002)−0.12 (0.03)0.035<0.001
Ammonia-N38−0.005 (0.19)0.0001 (0.95)0.000<0.001−0.006 (0.12)0.0006 (0.75)0.000<0.001
pH56−0.07 (0.01)0.02 (0.11)0.003<0.001−0.05 (0.03)0.01 (0.28)0.003<0.001
Lactic acid500.09 (0.69)−0.12 (0.18)0.088<0.0010.01 (0.95)−0.11 (0.14)0.084<0.001
Acetic acid50−0.03 (0.78)0.01 (0.91)0.025<0.0010.01 (0.90)−0.02 (0.69)0.025<0.001
Ethanol50−1.41 (<0.001)0.31 (0.03)0.577<0.001−0.60 (0.07)−0.03 (0.83)0.642<0.001
LAB52−0.02 (0.86)0.01 (0.85)0.035<0.0010.01 (0.93)−0.004 (0.94)0.035<0.001
Yeasts56−0.17 (0.64)−0.24 (0.10)0.519<0.001−0.80 (0.01)0.03 (0.82)0.557<0.001
DM recovery562.00 (0.08)−0.32 (0.46)4.703<0.0012.16 (0.02)−0.46 (0.30)4.602<0.001
Aerobic stability46118 (<0.001)−0.08 (1.00)3728<0.00172 (0.004)22 (0.052)3395<0.001
n—number of comparisons; τ2—between cluster variance; LAB—lactic acid bacteria; DM—dry matter. * ≤32, 32.01 to 37.99, and ≥38% DM. ** 20, 21 to 69, or ≥70 d.
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Benjamim da Silva, É.; Kung, L., Jr. A Meta-Analysis of the Effects of a Chemical Additive on the Fermentation and Aerobic Stability of Whole-Plant Maize Silage. Agriculture 2022, 12, 132. https://doi.org/10.3390/agriculture12020132

AMA Style

Benjamim da Silva É, Kung L Jr. A Meta-Analysis of the Effects of a Chemical Additive on the Fermentation and Aerobic Stability of Whole-Plant Maize Silage. Agriculture. 2022; 12(2):132. https://doi.org/10.3390/agriculture12020132

Chicago/Turabian Style

Benjamim da Silva, Érica, and Limin Kung, Jr. 2022. "A Meta-Analysis of the Effects of a Chemical Additive on the Fermentation and Aerobic Stability of Whole-Plant Maize Silage" Agriculture 12, no. 2: 132. https://doi.org/10.3390/agriculture12020132

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