A database was developed from several types of literature that reported the effect of wilting on silage. The search of the literature was conducted using Scopus with the keywords used being ‘wilting’ and ‘silage/ensiling’. The database was made in August 2023 from the Scopus research database. The selection criteria were: i) English-language articles; ii) direct comparison between wilting and un-wilting silage; iii) comparison of chemical content, bacteria silage population, and DM digestibility and; iv) replication and variance were reported (standard deviation [SD] or standard error or means). These criteria followed the preferred reporting item for systematic reviews and meta-analysis protocol.

All relevant literature titles are collected along with other information. All literature is collected and a database is formed using a data aggregation process. Data aggregation is the arrangement of data from the literature to facilitate the analysis calculations used.

The selection process is shown in Figure 1. The initial search resulted in 452 articles. A total of 346 articles were excluded for several reasons (non-related titles, review articles, or conference proceedings). Hence, the full-text evaluation resulted in 106 articles while 70 articles were excluded due to lack of comparison (n = 30), irrelevant contents or variables (n = 20), and insufficient data (n = 20). The final articles (n = 36) after assessment were considered a database in the meta-analysis shown in Table 1.

Data were analyzed using the random-effects meta-analysis method as described by Risyahadi et al [15]. The mathematical modeling of one-way random effects follows:

In this equation, y_i represents the effect size (Hedge’s d) for the i-th observation, θ is the general parameter for the combined effect size, v_i represents the actual variation in the effect size, and ɛ_i is the error for the i-th observation.

In brief, the effect size (d) was calculated based on Hedges’ standardized mean difference, with the formula [16]:

Where the mean of the experimental or wilting process is (X¯^E), the control group or without wilting process is (X¯^C), and the pooled standard deviation is (S) S defined as:

and J is the correction factor for the small sample size, i.e.:

where: N^E, sample size of the experimental group; N^c, sample size of the control group; S^E, standard deviation of the experimental group; S^C, standard deviation of the control group. The variance of Hedges’d (V_d) is described as follows:

The cumulative effect size (d₊₊) was formulated as follows:

where: W_i, the inverse of the sampling variance: W_i = 1/vd. The precision of the effect size was described using a 95% confidence interval (CI), i.e. d±(1.96×SD). All the above equations were derived from the study of Sánchez-Meca and Marín-Martínez [17]. Statistical significance was established by verifying that the CI did not encompass a null effect size. Significance was set at a p-value of 0.05. A fail-safe number (Nfs) was calculated to identify publication bias caused by non-significant studies, which were not included in the analysis. Nfs > 5N+10 was considered to provide evidence of a robust meta-analysis model. Nfs was calculated using the method of Rosenthal [18]. The smallest sample size from individual studies was applied as N. Cohen’s benchmarks were used as standard judgment borders for effect size assessment. These benchmarks were: 0.2 for small, 0.5 for medium, and 0.8 for large effect size.

Between-study variance (τ²) was estimated using DerSimonian and Laird’s method [19]

Where Q is the weighted sum square, df is the degrees of freedom, and C is the value. The meta-analysis was conducted using OpenMEE for performance variables. To address potential publication bias from omitted studies, a fail-safe number (Nfs) was computed [18], with Nfs > 5N+10 indicating robustness, applying the smallest sample size from individual studies as N.

Moisture content stands out as a crucial factor influencing silage quality. Excessively wet silage may result in poor fermentation and spoilage, while overly dry silage can lead to inadequate packing and diminished nutritional value. The optimal moisture content for silage falls within the range of 60% to 65% [20].

Dry matter content serves as a more precise indicator of forage moisture levels and is the recommended metric to ensure the correct moisture level for effective silage fermentation and preservation. Digestible energy intake is estimated from DM intake by ruminants, and energy digestibility is obtained from ruminants fed at maintenance levels. Wilting brings about proportional increases in silage DM. Meta-analysis findings indicate that wilting significantly (p<0.05) influences forage silage by elevating DM content and reducing DM loss. This outcome is consistent with numerous experimental studies [21,22]. The increase in DM value results from a lesser decrease in DM losses within the silage. According to Borreani et al [1], the wilting process significantly reduces DM loss, especially in leaves, and is directly associated with the initial DM content of the forage during treatment and the severity of its condition.

The ensiling process is initiated by LAB during fermentation, utilizing water-soluble WSC as energy and carbon sources. Therefore, WSC is crucial for achieving well-preserved silages, with a recommended concentration level of 60 to 70 g/kg of DM [23]. Increasing WSC concentration can enhance fermentation efficiency, promoting faster forage preservation with minimal acid or inoculant use, which can lead to cost savings in silage production.

The meta-analysis reveals that the wilting process significantly increases (p<0.05) WSC content in silage. This finding aligns with the experimental study by Zhang et al [23]. However, it is essential to note that certain researchers have reported no significant impact of wilting on WSC levels [24]. The rise in WSC content can be attributed to the higher concentration of DM. During wilting, the concentration of carbohydrates, including WSC, increases as the forage loses moisture. This elevated concentration results in a higher quantity of WSC relative to the overall DM content of the silage. This finding corresponds with the research by Yahaya et al [25], which indicates that silage with higher DM content contains higher WSC compared to silage with medium and lower DM. Additionally, wilting slows down the respiration of plant cells, leading to reduced carbohydrate consumption. By decreasing respiration, more carbohydrates, including WSC, are preserved in the forage, contributing to higher WSC levels in the silage [1].

The pH value serves as a crucial indicator of silage quality [26]. During the ensiling process, the pH of forage is reduced to a level that inhibits the proliferation of undesirable bacteria, including clostridia, enterobacteria, yeasts, and molds. Conversely, wilting involves elevating the initial DM content of silage to a level that effectively hinders the growth of harmful bacteria, such as clostridia [20].

The meta-analysis results indicated that wilting and non-wilting treatments had no significant impact on pH reduction. This finding aligns with the report by Hao et al [27], who observed that neither wilting nor additive addition to silage affected pH value. The pH value in silage is influenced by microbial heterogeneity, which can alter the lactic acid to acetic acid ratio [28]. Other organic acids, such as propionic acid and butyric acid, also influence pH value. Microbial heterogeneity in silage is further influenced by the type and maturity of the forage, as well as the temperature at ensiling. This variation in silage material and environmental conditions affects microbial populations, which in turn impacts the fermentation process and the resulting pH value [29].

Wilting contributes to the enhancement of silage fermentation quality, particularly by impacting the lactic acid content, which can undergo degradation into other components [30]. This aligns with the findings of the meta-analysis, revealing a decrease in lactic acid levels, accompanied by reductions in acetic-propionic and butyric acids. The wilting process diminishes the overall activity of microorganisms in the silage [31], with a notable decline in clostridium bacteria, as evidenced by a significant reduction in butyric acid levels. This observation is in line with the report by Cole Diepersloot et al [21], which emphasizes that non-wilted forage fermentation yields low butyric acid levels, further emphasizing how wilting can effectively decrease butyric acid content. Low butyric acid levels indicate that silage can be considered high-quality even if lactic acid levels are relatively low and the pH is relatively high, as this suggests effective nutrient preservation [32].

While this meta-analysis revealed a slight decrease in CP content in wilted silage compared to non-wilted silage, the difference was not statistically significant (p>0.05). This inconsistency in findings across studies could be attributed to varying conclusions in the research. For example, Kim et al [5] reported lower CP content in wilted silage, while Yahaya et al [25] found higher CP content. Additionally, studies by Hao et al [27] and Wang et al [33] observed both increased and decreased CP levels in treated silages. The potential reduction in CP content due to wilting could be linked to the continued activity of respiration enzymes in surviving plant cells after harvesting, albeit at a reduced level, as evidenced by the nonsignificant decrease in CP observed with wilting.

These findings support the meta-analysis result showing lower ammonia content (p<0.05) in wilted silage compared to unwilted silage, indicating reduced proteolysis activity. The main purpose of wilting before ensiling is to increase DM content, thereby reducing respiration activity and preserving nutrients like CP, which can then be used by animals during feeding. The positive effect of wilting on reducing proteolysis activity has been well-documented by many researchers including [6,23,34,35].

Based on the results of the meta-analysis, wilting in silage leads to a significant increase (p<0.05) in NDF and ADF contents. This finding aligns with the observations made by Reppeto et al [36], who noted a substantial rise in NDF and ADF contents after 8 hours and 10 days of ensiling forage. The significance of this meta-analysis study lies in the variations reported by different researchers. Contrary to these results, Cole Diepersloot et al [21] found no effects on NDF after 30 days of storage, but observed lower levels of NDF for wilting silage after 90 days of storage. Moreover, some studies, such as Zhang et al [23] and Herrmann et al [37], reported decreases in NDF during fermentation, possibly due to solubilization by acid components affecting certain NDF fractions, and this may be linked to the degradation occurring in the wilting process [23]. Despite these differences, it is generally accepted that storage has minimal effects on NDF [38].

Silage digestibility stands as a critical parameter in determining overall silage quality. The meta-analysis results suggest that wilting treatment does not have a significant impact on IVDMD of silage. This observation aligns with the primary objective of ensiling, which aims to minimize nutrient loss and preserve forage digestibility [39]. The wilting process involves a gradual reduction of moisture, with the goal being not to alter the nutritional content but rather to preserve it as effectively as possible, maintaining the digestible material. This approach differs from rapid drying methods which can lead to diminished nutrient levels and reduced digestibility across various components [40].

The present meta-analysis indicated no significant influence on microbial populations in silage, including LAB, yeast, and aerobic bacteria (p>0.05). Nonetheless, the expectation for wilting silage was to have a positive effect on bacterial populations, especially in reducing unwanted clostridial populations. This anticipation is due to the diminished oxygen availability resulting from reduced water content in wilted silage, which should inhibit their growth [41]. Different studies have reported varied impacts on the LAB population in wilted silages. For instance, Wan et al [8] observed an increase, whereas Tao et al [4] noted a decrease, and Liu et al [3] recorded both increases and decreases in their respective studies. The unaltered LAB population compared to non-wilted silage might be attributed to the absence of added inoculants. Augmenting the LAB population in silage can be accomplished by including LAB inoculants [33]. According to Kung et al [32], reducing the risk of undesirable bacteria growth can be achieved by ensiling forages at a DM content above 30% to 35%. This is due to the higher DM content, which diminishes the available moisture for bacterial growth and fosters an acidic environment less favorable for harmful bacteria. Additionally, wilting before ensiling further reduces forage moisture, making the environment less conducive to bacterial proliferation [5]. The suppression of bacterial growth due to the wilting process might be indicated by the decrease in lactic acid levels, accompanied by reductions in acetic-propionic and butyric acids, as shown in the current meta-analysis results.

The wilting treatment process positively influences the DM content of forage intended for silage, as demonstrated by the regression equation: DM = 4.24+0.009×Wilting (Figure 2). A more extended wilting duration correlates with a higher DM value. It is crucial to achieve fast wilting with a shorter duration in the field to minimize DM loss [1]. Nevertheless, extended exposure of harvested forage to sunlight may negatively impact the quality of silage as it promotes the growth of undesirable microorganisms. Prolonged wilting durations could also undermine the aerobic stability and nutritional value of silages [42,43].