**2. Bacterial inoculants**

Ensiling is the most common method used to preserve a great variety of forages for use during those seasons when the crop is unavailable and/or is decreasing in nutritive value. Ensiling is based on the conversion of simple plant sugars, such as glucose and fructose, to lactic acid by lactic acid bacteria (LAB) under anaerobic conditions [6, 7]. Epiphytic LAB are essential microflora for spontaneous silage fermentation; however, the number and genera of bacteria varies widely in forages [8]. Thus, bacterial inoculants (specifically homofermentative LABhoLAB) have been used in order (1) to inhibit the growth of aerobic and undesirable anaerobic microorganisms, (2) promote a rapid decline in the pH of forage after ensiling in order to avoid greater activity of proteases and deaminases derived from its own plant tissues and/or microorganisms, and (3) increase DM recovery [9].

The international literature is rich with data describing the eventual benefits of inoculation. However, no conclusion has been reached about the effect of bacterial inoculants on silage quality and animal performance in Brazil (see [5]) considering previously summarized studies carried out from 1999 to 2009. After 2009, 85 new Brazilian studies (scientific articles published in national and international journals) evaluating the effect of bacterial inoculants for silage production were published (**Figure 1**). Thus, analyzing real life scenarios are important to understand how bacterial inoculants alter silage quality and how they affect the performance of animals consuming inoculated silages.

Survey About the Use of Bacterial Inoculants in Brazil: Effects on Silage Quality and Animal Performance http://dx.doi.org/10.5772/64472 5

**1. Introduction**

4 Advances in Silage Production and Utilization

quality and animal performance.

**2. Bacterial inoculants**

microorganisms, and (3) increase DM recovery [9].

of animals consuming inoculated silages.

Silage is the feedstuff produced by the fermentation of a crop, forage, or agricultural byproduct, usually at greater than 50% moisture content [1]. In Brazil, silage is the principal source of energy and fiber in the diets of dairy cattle [2] and is frequently used in feedlots for the production of beef cattle [3]. However, descriptions of silage production practices and utilization in Brazilian literature are poor [4]. Furthermore, there is a lack of extension programs in Brazil that disseminate and enhance the knowledge of farmers regarding silage management, which has contributed to the production of low-quality products in many cases. As a strategy to alter this scenario, several farmers have chosen to use bacterial inoculants in order to improve silage quality and reduce production costs by decreasing the loss of dry matter (DM). Nevertheless, in Brazil, there are few reviews and surveys concerning the impact of bacterial inoculants on ensiling practices. In addition, the most complete review of this topic (see [5]) indicated that the low number of studies conducted in Brazil at that time did not produce a definitive conclusion about the magnitude of the effect of additives on silage

Therefore, our objective was to conduct a survey on the use of bacterial inoculants in Brazil and understand how they affect ensiling processes and animal performance. Here, we highlight that the present survey had an exploratory focus and, because of this, we conducted only a descriptive analysis of the data found in the accessed studies throughout of this text.

Ensiling is the most common method used to preserve a great variety of forages for use during those seasons when the crop is unavailable and/or is decreasing in nutritive value. Ensiling is based on the conversion of simple plant sugars, such as glucose and fructose, to lactic acid by lactic acid bacteria (LAB) under anaerobic conditions [6, 7]. Epiphytic LAB are essential microflora for spontaneous silage fermentation; however, the number and genera of bacteria varies widely in forages [8]. Thus, bacterial inoculants (specifically homofermentative LABhoLAB) have been used in order (1) to inhibit the growth of aerobic and undesirable anaerobic microorganisms, (2) promote a rapid decline in the pH of forage after ensiling in order to avoid greater activity of proteases and deaminases derived from its own plant tissues and/or

The international literature is rich with data describing the eventual benefits of inoculation. However, no conclusion has been reached about the effect of bacterial inoculants on silage quality and animal performance in Brazil (see [5]) considering previously summarized studies carried out from 1999 to 2009. After 2009, 85 new Brazilian studies (scientific articles published in national and international journals) evaluating the effect of bacterial inoculants for silage production were published (**Figure 1**). Thus, analyzing real life scenarios are important to understand how bacterial inoculants alter silage quality and how they affect the performance

**Figure 1.** Number of Brazilian articles published concerning bacterial inoculant utilization from the last 26 years (total number of articles accessed = 178).

Initially, the small interest on the topic in the last century in Brazil likely reflected questions about the cost of those inoculants and their effectiveness as in other countries [10], although these are questions that are debated very often. The inconsistent results obtained from early studies carried out in Europe and North America due to low rates of inoculation and questionable viabilities of the bacteria [9], also likely contributed to the initial small interest. Conversely, advances in molecular biology associated with positive responses found across the world may have moved the crescent interest from Brazilian researchers to study bacterial inoculants for silage production. Moreover, the increasing number of techniques used to produce more viable and stable bacteria, and the additional tools developed to access the effects of silage inoculants, may also be part of the reason for the increased interest. Indeed, poor silage management has led to the production of silages of low nutritional value and undesirable sanitary aspects under tropical conditions. Surely, sugarcane and tropical grass silages are still the crops most susceptible to problems that occur during fermentation due to the action of undesirable microorganisms. Thus, these crops comprised 58.1% of all studies evaluated regarding the use of bacterial inoculants (**Figure 2**).

**Figure 2.** Number of Brazilian studies published regarding the utilization of silage inoculants by crop. \*HMC, highmoisture corn; HMS, high-moisture sorghum.


follows: (1) obligate homofermentative → unable to ferment pentoses because the lack enzyme phosphoketolase; (2) facultative heterofermentative → ferment hexoses similarly to the obligate homofermentative but they are able to ferment pentoses; and (3) obligate heterofermentative → ferment hexoses to a range of products. Overall, under most silage conditions where substrate is not lacking, facultative heterofermentative LAB primarily make only lactic acid [9]. Thus, for the sake of simplicity, facultative heterofermentative LAB will be considered

Survey About the Use of Bacterial Inoculants in Brazil: Effects on Silage Quality and Animal Performance

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7

In Brazil, several homofermentative (*Lactobacillus plantarum*, *L. curvatus*, *L. acidophilus*, *L. paracasei*, *Lactococcus lactis*, *Leuconostoc mesenteroides*, *Pediococcus pentosaceus*, *P. acidilactici*, *Streptococcus faecium*, *S. faecalis*, and *S. bovis*) and heterofermentative LAB (*L. buchneri*, *L. hilgardii*, *L. kefiri*, *L. salivarius*, and *L. brevis*) have been used as silage inoculants, leading to different combinations for each crop (**Table 1**). Other microorganisms have also been tested, such as *Propionibacterium acidipropionici*, *Bacillus subtilis*, and *Saccharomyces* spp., but less

As described earlier, hoLAB and heterofermentative LAB (heLAB) comprised first and second generation of silage inoculants, respectively. The hoLAB gained popularity in the late 1970s and early 1980s because it must quickly grow to dominate silage fermentation reducing DM and nutritive losses [9]. Conversely, homofermentative-inoculated silages often have lower stability during the feed-out phase, because of the greater concentration of lactic acid and residual water-soluble carbohydrates (WSC) [13]. Lactic acid and WSCs are utilized as substrates for the growth of aerobic microorganisms, notably yeasts [13]. Thus, *L. buchneri* was developed as a second generation inoculant to produce acetic acid and improve the aerobic stability of silage by inhibiting the growth of spoilage microorganisms [14]. Nowadays, some commercial silage inoculants contain multiple strains of hoLAB and often one strain of heLAB, because of the potential synergistic actions among bacterial strains. For example, previous studies showed that the association between *L. plantarum* and *L. buchneri* accelerated the initial rate of lactic acid fermentation, reducing the pH and causing lower protein degradation, in

addition to enhancing the aerobic stability of corn and sorghum silages [13, 15].

but articles evaluating hoLAB and heLAB combined started to be published earlier.

In Brazil, hoLAB were primarily investigated and used as commercial silage inoculants to ensure suitable fermentation (**Figure 3**). Around the year 2000, Brazilian researchers turned their attention and curiosity to investigate the effects of heLAB on the ensiling of tropical crops, but articles on this topic only started to be published in 2006. Moreover, studies combining hoLAB and heLAB started at the same time that second generation silage inoculants were used,

Despite the type of silage inoculant used for the six main crops used for ensiling in Brazil, hoLAB composed the only silage inoculant assessed for alfalfa and sorghum silages (**Figure 4**). Moreover, hoLAB still composed the majority (>69%) of the treatments for corn, HMC, and grass silages. Sugarcane was the only crop in which heLAB composed the majority (57%) of the treatments assessed. This scenario is not a surprise, since hoLAB were primarily investigated and used as commercial silage inoculants in the worldwide, and likely this reflected in a greater number of studies assessing hoLAB in Brazil. Alfalfa and grass silages often have low WSC content and high buffer capacity, and then pH declines more slowly after the crop is ensiled

part of homofermentative LAB in this review for furthers comparison.

frequently.

**Table 1.** Bacterial species applied in the six main crops used to produce silage in Brazil (number of treatments).

As mentioned earlier, the crescent development in molecular biology techniques has led to a wide range of microbial additives to aid in crop preservation. The LAB (genera *Lactobacillus*, *Pediococcus*, *Lactococcus*, *Enterococcus, Streptococcus*, and *Leuconostoc*) are the main group of bacteria used as silage inoculants, because they all produce lactic acid as a principal product from sugar fermentation [6]. Commonly, the LAB are classified into two groups based on the products of fermenting glucose, as follow: (1) homofermentative (first generation of silage inoculants) → produce two moles of lactic acid from one mole of glucose; and (2) heterofermentative (second generation of silage inoculants) → produce one mole of lactic acid, one mole of carbon dioxide (CO2), and either one mole of ethanol or one mole of acetic acid from one mole of glucose [11]. However, actually three groups of LAB have been considered [12], as follows: (1) obligate homofermentative → unable to ferment pentoses because the lack enzyme phosphoketolase; (2) facultative heterofermentative → ferment hexoses similarly to the obligate homofermentative but they are able to ferment pentoses; and (3) obligate heterofermentative → ferment hexoses to a range of products. Overall, under most silage conditions where substrate is not lacking, facultative heterofermentative LAB primarily make only lactic acid [9]. Thus, for the sake of simplicity, facultative heterofermentative LAB will be considered part of homofermentative LAB in this review for furthers comparison.

**Item Alfalfa Corn Grass HMC1 Sorghum Sugarcane**

*Bacillus subtilis* – 4– – – – *Lactobacillus brevis* – –– – – 27 *Lactobacillus buchneri* – 16 8 8 – 62 *Lactobacillus hilgardii* – –– – – 10 *Lactobacillus kefiri* – –– – – 1 *Lactobacillus paracasei* – –– – – 2 *Lactobacillus plantarum* – 8 18 9 – 59 *Leuconostoc mesenteroides* – 1– – – – *Streptococcus bovis* – – 14 – – – *Streptococcus faecium* – –3 – – –

*L. buchneri + L. kefiri* – –– – – 1 *L. buchneri + Propionibacterium acidipropionici* – –– 1 – – *Lactobacillus casei + Streptococcus faecalis* – –– 2 – – *L. plantarum + B. subtilis* – 1– – – – *L. plantarum + L. buchneri* – 4– – – 1 *L. plantarum + Pediococcus acidilactici* – 6 17 – – 1 *L. plantarum + Pediococcus pentosaceus* 5 16 3 – – 7 *L. plantarum + P. acidipropionici* – 22 – – 4 *L. plantarum + S. faecium* 6 14 12 – 26 3 Combo2 4 46 42 16 16 3

**Table 1.** Bacterial species applied in the six main crops used to produce silage in Brazil (number of treatments).

As mentioned earlier, the crescent development in molecular biology techniques has led to a wide range of microbial additives to aid in crop preservation. The LAB (genera *Lactobacillus*, *Pediococcus*, *Lactococcus*, *Enterococcus, Streptococcus*, and *Leuconostoc*) are the main group of bacteria used as silage inoculants, because they all produce lactic acid as a principal product from sugar fermentation [6]. Commonly, the LAB are classified into two groups based on the products of fermenting glucose, as follow: (1) homofermentative (first generation of silage inoculants) → produce two moles of lactic acid from one mole of glucose; and (2) heterofermentative (second generation of silage inoculants) → produce one mole of lactic acid, one mole of carbon dioxide (CO2), and either one mole of ethanol or one mole of acetic acid from one mole of glucose [11]. However, actually three groups of LAB have been considered [12], as

One specie

6 Advances in Silage Production and Utilization

Two species

1

2

HMC, high-moisture corn.

Combination of three or more bacteria.

In Brazil, several homofermentative (*Lactobacillus plantarum*, *L. curvatus*, *L. acidophilus*, *L. paracasei*, *Lactococcus lactis*, *Leuconostoc mesenteroides*, *Pediococcus pentosaceus*, *P. acidilactici*, *Streptococcus faecium*, *S. faecalis*, and *S. bovis*) and heterofermentative LAB (*L. buchneri*, *L. hilgardii*, *L. kefiri*, *L. salivarius*, and *L. brevis*) have been used as silage inoculants, leading to different combinations for each crop (**Table 1**). Other microorganisms have also been tested, such as *Propionibacterium acidipropionici*, *Bacillus subtilis*, and *Saccharomyces* spp., but less frequently.

As described earlier, hoLAB and heterofermentative LAB (heLAB) comprised first and second generation of silage inoculants, respectively. The hoLAB gained popularity in the late 1970s and early 1980s because it must quickly grow to dominate silage fermentation reducing DM and nutritive losses [9]. Conversely, homofermentative-inoculated silages often have lower stability during the feed-out phase, because of the greater concentration of lactic acid and residual water-soluble carbohydrates (WSC) [13]. Lactic acid and WSCs are utilized as substrates for the growth of aerobic microorganisms, notably yeasts [13]. Thus, *L. buchneri* was developed as a second generation inoculant to produce acetic acid and improve the aerobic stability of silage by inhibiting the growth of spoilage microorganisms [14]. Nowadays, some commercial silage inoculants contain multiple strains of hoLAB and often one strain of heLAB, because of the potential synergistic actions among bacterial strains. For example, previous studies showed that the association between *L. plantarum* and *L. buchneri* accelerated the initial rate of lactic acid fermentation, reducing the pH and causing lower protein degradation, in addition to enhancing the aerobic stability of corn and sorghum silages [13, 15].

In Brazil, hoLAB were primarily investigated and used as commercial silage inoculants to ensure suitable fermentation (**Figure 3**). Around the year 2000, Brazilian researchers turned their attention and curiosity to investigate the effects of heLAB on the ensiling of tropical crops, but articles on this topic only started to be published in 2006. Moreover, studies combining hoLAB and heLAB started at the same time that second generation silage inoculants were used, but articles evaluating hoLAB and heLAB combined started to be published earlier.

Despite the type of silage inoculant used for the six main crops used for ensiling in Brazil, hoLAB composed the only silage inoculant assessed for alfalfa and sorghum silages (**Figure 4**). Moreover, hoLAB still composed the majority (>69%) of the treatments for corn, HMC, and grass silages. Sugarcane was the only crop in which heLAB composed the majority (57%) of the treatments assessed. This scenario is not a surprise, since hoLAB were primarily investigated and used as commercial silage inoculants in the worldwide, and likely this reflected in a greater number of studies assessing hoLAB in Brazil. Alfalfa and grass silages often have low WSC content and high buffer capacity, and then pH declines more slowly after the crop is ensiled

[6]. Therefore, is comprehensive why only hoLAB were assessed for alfalfa and why hoLAB composed the majority of the treatments for grass. However, considering that corn and sorghum silages that are most susceptible to aerobic deterioration under tropical conditions [16] would be expected a greater number of studies concerning heLAB or combining hoLAB and heLAB to reduce this trouble.

time of cutting when the highest nutritive value of the grass is achieved and at high buffering

Survey About the Use of Bacterial Inoculants in Brazil: Effects on Silage Quality and Animal Performance

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9

Epiphytic LAB utilize carbohydrates as energy and carbon sources for growth, and these microorganisms are only able to convert nonstructural carbohydrates (notably WSCs—monoand disaccharides) into organic acids, because they lack the enzymatic complex required to metabolize complex polysaccharides [7]. Thus, enzyme-bacterial inoculants may become useful to improve the fermentation patterns and nutritive value mainly of ensiled crops having low WSC content. Bacterial inoculants ensure that LAB will dominate in silage fermentation, whereas the enzymes (i.e., fibrolytic enzymes) contained in those inoculants act on the cell wall, releasing a greater amount of fermentable sugars and increasing substrate availability, thereby improving silage digestibility [18]. Amylolytic and proteolytic enzymes are also commonly used in silage inoculants, and they are particularly useful for cereal silages, reducing the negative effect of the starch-protein matrix on starch digestion in ruminants [19, 20]. Therefore, it is easy to understand why enzyme-bacterial inoculants are used primarily in high-moisture corn (HMC) silages (>55%), followed by grass, corn, sorghum, alfalfa, and sugarcane silages (**Figure 5**). Obviously, the little interest in evaluating enzyme-bacterial inoculants for sugarcane silage is related to the great amount of WSC in this crop, particularly

**Figure 5.** Enzyme utilization in silage inoculants by crop in Brazil (% related to the number of treatments containing

The use of bacterial inoculants as additives to improve silage fermentation has a long and diverse history. As described earlier, although silage inoculant utilization occurred later in Brazil than Europe and North America, many types and formulations of bacteria are currently sold commercially for this purpose. However, the compatibility between the plant and

**2.1. Fermentation patterns, nutritive value, and aerobic stability of silages**

capacity, results in poor fermentation and low silage digestibility [17].

sucrose [21].

bacterial inoculants). \*HMC, high-moisture corn.

**Figure 3.** Evolution of the utilization of homofermentative and heterofermentative LAB, either alone or combined (mixed) in Brazil (% related to the number of treatments).

**Figure 4.** Assessment of homofermentative and heterofermentative LAB, either alone or combined (mixed) by crop in Brazil (% related to the number of treatments containing bacterial inoculants). \*HMC, high-moisture corn.

The use of bacterial inoculants has also claimed to improve the nutritive value of silages by reflecting alterations in fermentation patterns, which may be important for tropical silages in particular. The use of tropical forages often results in silages with lower nutritive value than those produced under temperate conditions [16]. Unfavorable aspects of some crops (especially grasses), such as low WSC and DM content (both needed for proper fermentation) at the time of cutting when the highest nutritive value of the grass is achieved and at high buffering capacity, results in poor fermentation and low silage digestibility [17].

[6]. Therefore, is comprehensive why only hoLAB were assessed for alfalfa and why hoLAB composed the majority of the treatments for grass. However, considering that corn and sorghum silages that are most susceptible to aerobic deterioration under tropical conditions [16] would be expected a greater number of studies concerning heLAB or combining hoLAB and

**Figure 3.** Evolution of the utilization of homofermentative and heterofermentative LAB, either alone or combined

**Figure 4.** Assessment of homofermentative and heterofermentative LAB, either alone or combined (mixed) by crop in

The use of bacterial inoculants has also claimed to improve the nutritive value of silages by reflecting alterations in fermentation patterns, which may be important for tropical silages in particular. The use of tropical forages often results in silages with lower nutritive value than those produced under temperate conditions [16]. Unfavorable aspects of some crops (especially grasses), such as low WSC and DM content (both needed for proper fermentation) at the

Brazil (% related to the number of treatments containing bacterial inoculants). \*HMC, high-moisture corn.

heLAB to reduce this trouble.

8 Advances in Silage Production and Utilization

(mixed) in Brazil (% related to the number of treatments).

Epiphytic LAB utilize carbohydrates as energy and carbon sources for growth, and these microorganisms are only able to convert nonstructural carbohydrates (notably WSCs—monoand disaccharides) into organic acids, because they lack the enzymatic complex required to metabolize complex polysaccharides [7]. Thus, enzyme-bacterial inoculants may become useful to improve the fermentation patterns and nutritive value mainly of ensiled crops having low WSC content. Bacterial inoculants ensure that LAB will dominate in silage fermentation, whereas the enzymes (i.e., fibrolytic enzymes) contained in those inoculants act on the cell wall, releasing a greater amount of fermentable sugars and increasing substrate availability, thereby improving silage digestibility [18]. Amylolytic and proteolytic enzymes are also commonly used in silage inoculants, and they are particularly useful for cereal silages, reducing the negative effect of the starch-protein matrix on starch digestion in ruminants [19, 20]. Therefore, it is easy to understand why enzyme-bacterial inoculants are used primarily in high-moisture corn (HMC) silages (>55%), followed by grass, corn, sorghum, alfalfa, and sugarcane silages (**Figure 5**). Obviously, the little interest in evaluating enzyme-bacterial inoculants for sugarcane silage is related to the great amount of WSC in this crop, particularly sucrose [21].

**Figure 5.** Enzyme utilization in silage inoculants by crop in Brazil (% related to the number of treatments containing bacterial inoculants). \*HMC, high-moisture corn.

#### **2.1. Fermentation patterns, nutritive value, and aerobic stability of silages**

The use of bacterial inoculants as additives to improve silage fermentation has a long and diverse history. As described earlier, although silage inoculant utilization occurred later in Brazil than Europe and North America, many types and formulations of bacteria are currently sold commercially for this purpose. However, the compatibility between the plant and microorganisms used will determine the success of the application of bacterial inoculants in silages [22]. When that compatibility is better understood, positive responses from inoculation occur more often.

In order to understand the extent to which each type of bacterial inoculant affects silage quality, we summarized data from corn, grass, sugarcane, alfalfa, sorghum, and HMC silages produced in Brazil. All comparisons in this survey were made from studies (at least two studies for each variable) that used a negative treatment (untreated forage—control) against one or more treatments containing bacterial inoculants. Some calculations were made when data were lacking from these publications as follows: hemicellulose content was calculated as neutral detergent fiber (NDF) minus acid detergent fiber (ADF), whereas cellulose content was calculated as ADF minus lignin; the proportion of hemicellulose, cellulose, and lignin were also calculated on a NDF basis; total acid production was calculated as the sum of lactic, acetic, and propionic acids; and the ratio of lactic:acetic acid and total acid:ethanol was also calculated. Butyric acid was not considered in the calculation of total acids because this acid has no beneficial effect on ensiling process [6]. Otherwise, lactic acid (acid more desired to reduce DM loss) and acetic and propionic acids (antifungal properties) have beneficial role during ensiling

Survey About the Use of Bacterial Inoculants in Brazil: Effects on Silage Quality and Animal Performance

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11

As described earlier, we performed only a descriptive analysis of data found in the studies investigated. For that, we did not consider a minimum or maximum time of fermentation to include the data from each study in the final dataset, because our objective was not to show the fermentation pattern regarding the length of ensiling. From the summarized data, the mean, median, standard deviation, and minimum and maximum values were calculated for all variables. Moreover, the frequency of positive responses from inoculation was also calculated, considering only the means declared statistically different in the studies that comprised the database. The difference between the means of untreated and inoculated silages, when there were positive responses, was also calculated. The criteria considered as positive

Enterobacteria count was not considered in this survey by lack of data, but it is important to state that enterobacteria are the principal competitors against LAB for sugars after the crop is ensiled, and acetic acid is the principal product of enterobacterial fermentation [8]. Conversely, enterobacteria population often declines after ensiling by influence of anaerobiosis and pH

Data were summarized from a total of 29 studies, of which 19, 7, and 7 investigated the effect of hoLAB, heLAB, and a combination between both (mixed), respectively. *Bacillus subtilis* was also investigated in two studies. Considering all treatments, the application rate of bacterial

The ranges of fermentation patterns, *in vitro* digestibility, and aerobic stability are given in **Table 3**. Considering the overall mean, lactic acid and silage pH were unaffected by hoLAB. The concentration of lactic acid was greater by 51.2% when both hoLAB and heLAB were applied than observed in untreated silage. The hoLAB increased by 12.8% the concentration of acid detergent insoluble N (ADIN), suggesting that the temperature of fermentation also increased following inoculation. In addition, hoLAB slightly reduced (−2.8%) the *in vitro* DM digestibility

colony forming units (cfu)/g of fresh forage.

[6].

for each variable are given in **Table 2**.

*2.1.1. Corn silage*

inoculants ranged from 5×104

(IVDMD) of corn silages.

reduction due to the acids produced during fermentation [8].

to 1×109


TN, total nitrogen; WSC, water-soluble carbohydrates; DM, dry matter; LAB, lactic acid bacteria; heLAB, heterofermentative LAB; hoLAB, homofermentative LAB; EE, ether extract; CP, crude protein; NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N; NDF, neutral detergent fiber; ADF, acid detergent fiber; IVDMD, *in vitro* DM digestibility; IVOMD, *in vitro* organic matter digestibility; DMI, DM intake; BW, body weight; OMI, organic matter intake; NDFI, NDF intake; CPI, CP intake; ADG, average daily gain. 1 Adapted from [5].

2 Total acid content was calculated as the sum of lactic, acetic, and propionic acids. 3 Feed efficiency was determined by dividing DMI by ADG.

**Table 2.** Criteria considered as positive (CCP) effect of inoculation for each variable (data are % of DM, unless otherwise stated)1 .

In order to understand the extent to which each type of bacterial inoculant affects silage quality, we summarized data from corn, grass, sugarcane, alfalfa, sorghum, and HMC silages produced in Brazil. All comparisons in this survey were made from studies (at least two studies for each variable) that used a negative treatment (untreated forage—control) against one or more treatments containing bacterial inoculants. Some calculations were made when data were lacking from these publications as follows: hemicellulose content was calculated as neutral detergent fiber (NDF) minus acid detergent fiber (ADF), whereas cellulose content was calculated as ADF minus lignin; the proportion of hemicellulose, cellulose, and lignin were also calculated on a NDF basis; total acid production was calculated as the sum of lactic, acetic, and propionic acids; and the ratio of lactic:acetic acid and total acid:ethanol was also calculated. Butyric acid was not considered in the calculation of total acids because this acid has no beneficial effect on ensiling process [6]. Otherwise, lactic acid (acid more desired to reduce DM loss) and acetic and propionic acids (antifungal properties) have beneficial role during ensiling [6].

As described earlier, we performed only a descriptive analysis of data found in the studies investigated. For that, we did not consider a minimum or maximum time of fermentation to include the data from each study in the final dataset, because our objective was not to show the fermentation pattern regarding the length of ensiling. From the summarized data, the mean, median, standard deviation, and minimum and maximum values were calculated for all variables. Moreover, the frequency of positive responses from inoculation was also calculated, considering only the means declared statistically different in the studies that comprised the database. The difference between the means of untreated and inoculated silages, when there were positive responses, was also calculated. The criteria considered as positive for each variable are given in **Table 2**.

Enterobacteria count was not considered in this survey by lack of data, but it is important to state that enterobacteria are the principal competitors against LAB for sugars after the crop is ensiled, and acetic acid is the principal product of enterobacterial fermentation [8]. Conversely, enterobacteria population often declines after ensiling by influence of anaerobiosis and pH reduction due to the acids produced during fermentation [8].

### *2.1.1. Corn silage*

microorganisms used will determine the success of the application of bacterial inoculants in silages [22]. When that compatibility is better understood, positive responses from inoculation

**CCP Animal performance CCP**

NDIN, % N Decreasing CPI, kg/day Increasing

day

day

day

day

Hemicellulose Decreasing Digestible CPI, kg/

Increasing

Increasing

Increasing

Increasing

**composition**

Propionic acid Increasing ADIN, % N Decreasing Digestible DMI, kg/

Butyric acid Decreasing NDF Decreasing Digestible OMI, kg/

Total acids2 Increasing ADF Decreasing Digestible NDFI, kg/

TN, total nitrogen; WSC, water-soluble carbohydrates; DM, dry matter; LAB, lactic acid bacteria; heLAB,

**Table 2.** Criteria considered as positive (CCP) effect of inoculation for each variable (data are % of DM, unless

matter intake; NDFI, NDF intake; CPI, CP intake; ADG, average daily gain.

Feed efficiency was determined by dividing DMI by ADG.

Total acid content was calculated as the sum of lactic, acetic, and propionic acids.

heterofermentative LAB; hoLAB, homofermentative LAB; EE, ether extract; CP, crude protein; NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N; NDF, neutral detergent fiber; ADF, acid detergent fiber; IVDMD, *in vitro* DM digestibility; IVOMD, *in vitro* organic matter digestibility; DMI, DM intake; BW, body weight; OMI, organic

Ethanol Decreasing Cellulose Decreasing DM digestibility Increasing Total acids:ethanol Increasing Lignin Decreasing OM digestibility Increasing Effluent, kg/t of fresh matter Decreasing IVDMD Increasing NDF digestibility Increasing Gas losses Decreasing IVOMD Increasing CP digestibility Increasing DM losses Decreasing Feed efficiency3 Decreasing LAB, log cfu/g of fresh silage Increasing ADG, kg/day Increasing

pH Decreasing DM, % as fed Increasing DMI, kg/day Increasing Ammonia-N, % TN Decreasing Ash Decreasing DMI, % BW Increasing WSC Increasing EE Increasing OMI, kg/day Increasing Lactic acid Increasing CP Increasing NDFI, kg/day Increasing

**CCP Chemical**

(heLAB) and decreasing

( hoLAB)

( hoLAB) and decreasing

( heLAB)

occur more often.

Acetic acid Increasing

10 Advances in Silage Production and Utilization

Lactic:acetic acid Increasing

Yeasts, log cfu/g of fresh silage Decreasing Molds, log cfu/g of fresh silage Decreasing Aerobic stability, h Increasing Maximum temperature, °C Decreasing

1

2

3

Adapted from [5].

otherwise stated)1

.

**Fermentation and microbiological profile**

> Data were summarized from a total of 29 studies, of which 19, 7, and 7 investigated the effect of hoLAB, heLAB, and a combination between both (mixed), respectively. *Bacillus subtilis* was also investigated in two studies. Considering all treatments, the application rate of bacterial inoculants ranged from 5×104 to 1×109 colony forming units (cfu)/g of fresh forage.

> The ranges of fermentation patterns, *in vitro* digestibility, and aerobic stability are given in **Table 3**. Considering the overall mean, lactic acid and silage pH were unaffected by hoLAB. The concentration of lactic acid was greater by 51.2% when both hoLAB and heLAB were applied than observed in untreated silage. The hoLAB increased by 12.8% the concentration of acid detergent insoluble N (ADIN), suggesting that the temperature of fermentation also increased following inoculation. In addition, hoLAB slightly reduced (−2.8%) the *in vitro* DM digestibility (IVDMD) of corn silages.


quality often is lower than that produced under temperate conditions [16]. The main problem of corn silage produced under tropical conditions is related with aerobic deterioration [16] when the silos are opened. The elevated temperature occurring in tropical weather is favorable to yeasts' overgrowth [24], which initiates the spoilage of silages by using residual WSC and lactic acid as substrate to growth, with consequent reduction in the nutritive value of silages. In this regard, heLAB should be useful to reduce aerobic deterioration of corn silages, but in general, hoLAB composed 77.2% of all treatments concerning silage inoculants for corn silage. The greater hoLAB utilization likely still reflects the fact that homolactic inoculants were primarily developed as silage additives, and commercial products based on hoLAB are most

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Despite heterolactic inoculation, acetic acid was unaffected, but the aerobic stability of silages was enhanced (+73.4 h), likely because of reductions in the number of yeasts. Nevertheless, heLAB increased ethanol production, gas, and DM losses during fermentation by 103.6, 59.7, and 31.2% compared with untreated silages, respectively. Extensive heterolactic fermentation unavoidably increases DM loss during the time the silo is closed, because additional products (i.e., acetic acid, ethanol, and CO2) are formed besides lactic acid [11]. Furthermore, the concentration of 1,2-propanediol increased 116.7% in silages inoculated with heLAB. *L. buchneri* comprised the main heLAB evaluated in corn silage, and this bacterium is able to produce 1,2-propanediol, coupled with acetic acid, during anaerobic degradation of lactic acid [25]. The ammonia-N concentration of silages inoculated with hoLAB and heLAB, either alone or combined, is in agreement with well-fermented corn silages (range

Considering the overall means, heLAB reduced the NDF content of silages by 6.4%. In many cases, the reductions in NDF content have been attributed to the capacity of *L. buchneri* to produce ferulate esterase, an enzyme that acts on cell wall-releasing ferulic acid [27]. However, only some specific strains of *L. buchneri* have the capacity to produce ferulate esterase [26]. Moreover, a net hydrolysis of hemicellulose did not occur when the values were compared on an NDF basis (C.H.S. Rabelo and R.A. Reis). Thus, the reasons for reduced NDF content of corn silages inoculated with heLAB are still unclear; once DM loss increased, it did not provide better preservation of WSC, which could decrease NDF content by the concentration effect. Heterolactic inoculation also improved IVDMD by 14%, most probably due to a reduction in

Although few studies combining hoLAB and heLAB were carried out in Brazil, overall means revealed increased lactic and acetic concentration when both inoculants were applied on corn silage compared to untreated silage. Combining hoLAB and heLAB may ensure a better fermentation process of corn silage with increased lactic and acetic acid concentration [13], as reported earlier. Consequently, a reduction in DM losses with an increased aerobic stability should be expected, but was not observed. Otherwise, silages treated with both hoLAB and heLAB slightly lowered NDF content and increased IVDMD. Even though the data of this survey about combining hoLAB and heLAB are not encouraged, most likely due to the low number of studies, further researches should consider the investigation of both hoLAB and heLAB for corn silage. The international literature has found a better fermentation process of

available to be assessed compared with heLAB.

from 5 to 7%) [23].

NDF content.

1 DM, dry matter; CP, crude protein; NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N; NDF, neutral detergent fiber; ADF, acid detergent fiber; IVDMD, *in vitro* DM digestibility; IVOMD, *in vitro* organic matter digestibility; TN, total nitrogen; LAB, lactic acid bacteria.

2 Number of means.

3 Standard deviation.

4 Silages inoculated with both heterofermentative and homofermentative bacteria.

5 Total acid content was calculated as the sum of lactic, acetic, and propionic acids.

**Table 3.** Range of fermentation patterns, nutritive value, and aerobic stability of untreated and inoculated corn silages (data are given in % of DM, unless otherwise stated).

All silages were close to or inside the ideal range of the DM content (30–35% of DM) recommended for the production of corn silage [6]. Under these conditions, corn plants often exhibit a great amount of WSC and have a low buffer capacity since well managed. Thus, the lack of positive results from homolactic inoculation is likely related to the desired characteristics of corn plants used at ensiling, once all silages (including the untreated) produced a suitable quantity of lactic acid, with an ideal range between 4 and 7% of the DM [23].

Overall, although positive responses from hoLAB inoculation were not observed, hoLAB might be useful to increase lactic acid production and improve fermentation when silage is produced with corn plants harvested with moderately to high DM content (i.e., >37%), because a lack of moisture in dry forages restricts the overall fermentation process [6]. Furthermore, the quality of corn silage produced under tropical conditions is not properly a problem, even though its quality often is lower than that produced under temperate conditions [16]. The main problem of corn silage produced under tropical conditions is related with aerobic deterioration [16] when the silos are opened. The elevated temperature occurring in tropical weather is favorable to yeasts' overgrowth [24], which initiates the spoilage of silages by using residual WSC and lactic acid as substrate to growth, with consequent reduction in the nutritive value of silages. In this regard, heLAB should be useful to reduce aerobic deterioration of corn silages, but in general, hoLAB composed 77.2% of all treatments concerning silage inoculants for corn silage. The greater hoLAB utilization likely still reflects the fact that homolactic inoculants were primarily developed as silage additives, and commercial products based on hoLAB are most available to be assessed compared with heLAB.

Despite heterolactic inoculation, acetic acid was unaffected, but the aerobic stability of silages was enhanced (+73.4 h), likely because of reductions in the number of yeasts. Nevertheless, heLAB increased ethanol production, gas, and DM losses during fermentation by 103.6, 59.7, and 31.2% compared with untreated silages, respectively. Extensive heterolactic fermentation unavoidably increases DM loss during the time the silo is closed, because additional products (i.e., acetic acid, ethanol, and CO2) are formed besides lactic acid [11]. Furthermore, the concentration of 1,2-propanediol increased 116.7% in silages inoculated with heLAB. *L. buchneri* comprised the main heLAB evaluated in corn silage, and this bacterium is able to produce 1,2-propanediol, coupled with acetic acid, during anaerobic degradation of lactic acid [25]. The ammonia-N concentration of silages inoculated with hoLAB and heLAB, either alone or combined, is in agreement with well-fermented corn silages (range from 5 to 7%) [23].

Considering the overall means, heLAB reduced the NDF content of silages by 6.4%. In many cases, the reductions in NDF content have been attributed to the capacity of *L. buchneri* to produce ferulate esterase, an enzyme that acts on cell wall-releasing ferulic acid [27]. However, only some specific strains of *L. buchneri* have the capacity to produce ferulate esterase [26]. Moreover, a net hydrolysis of hemicellulose did not occur when the values were compared on an NDF basis (C.H.S. Rabelo and R.A. Reis). Thus, the reasons for reduced NDF content of corn silages inoculated with heLAB are still unclear; once DM loss increased, it did not provide better preservation of WSC, which could decrease NDF content by the concentration effect. Heterolactic inoculation also improved IVDMD by 14%, most probably due to a reduction in NDF content.

1

2

3

4

5

Number of means.

Standard deviation.

digestibility; TN, total nitrogen; LAB, lactic acid bacteria.

12 Advances in Silage Production and Utilization

(data are given in % of DM, unless otherwise stated).

Silages inoculated with both heterofermentative and homofermentative bacteria.

Total acid content was calculated as the sum of lactic, acetic, and propionic acids.

DM, dry matter; CP, crude protein; NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N; NDF, neutral detergent fiber; ADF, acid detergent fiber; IVDMD, *in vitro* DM digestibility; IVOMD, *in vitro* organic matter

**Table 3.** Range of fermentation patterns, nutritive value, and aerobic stability of untreated and inoculated corn silages

All silages were close to or inside the ideal range of the DM content (30–35% of DM) recommended for the production of corn silage [6]. Under these conditions, corn plants often exhibit a great amount of WSC and have a low buffer capacity since well managed. Thus, the lack of positive results from homolactic inoculation is likely related to the desired characteristics of corn plants used at ensiling, once all silages (including the untreated) produced a suitable

Overall, although positive responses from hoLAB inoculation were not observed, hoLAB might be useful to increase lactic acid production and improve fermentation when silage is produced with corn plants harvested with moderately to high DM content (i.e., >37%), because a lack of moisture in dry forages restricts the overall fermentation process [6]. Furthermore, the quality of corn silage produced under tropical conditions is not properly a problem, even though its

quantity of lactic acid, with an ideal range between 4 and 7% of the DM [23].

Although few studies combining hoLAB and heLAB were carried out in Brazil, overall means revealed increased lactic and acetic concentration when both inoculants were applied on corn silage compared to untreated silage. Combining hoLAB and heLAB may ensure a better fermentation process of corn silage with increased lactic and acetic acid concentration [13], as reported earlier. Consequently, a reduction in DM losses with an increased aerobic stability should be expected, but was not observed. Otherwise, silages treated with both hoLAB and heLAB slightly lowered NDF content and increased IVDMD. Even though the data of this survey about combining hoLAB and heLAB are not encouraged, most likely due to the low number of studies, further researches should consider the investigation of both hoLAB and heLAB for corn silage. The international literature has found a better fermentation process of corn silage accompanied of a greater aerobic stability when hoLAB and heLAB were simultaneously used [13, 14, 15]. These responses may ensure most suitable nutritive value of silage and lead some beneficial on animal response.

Despite heterolactic inoculation, greater frequencies of positive responses were observed for IVOMD, aerobic stability, number of LAB and yeasts, IVDMD, and acetic acid. In addition, the greatest magnitudes of response were observed for the concentration of 1,2-propanediol,

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The low number of means for some variables contributed to large values for the frequency of positive responses, as well as the difference between untreated and inoculated silages. However, the data clearly showed that heLAB in corn silage, composed mainly of *L. buchneri*, were biologically effective. *L. buchneri* has been shown to enhance the aerobic stability of silages by increasing the production of acetic acid, which decreases the growth of spoilage microorganisms [29]. Acetic acid has antifungal characteristics [30], and heterolactic inoculation may be particularly important in silages produced under tropical conditions, as elevated temper-

Data were summarized from a total of 45 studies, of which 40, 4, and 6 investigated the effect of hoLAB, heLAB, and a combination of both (mixed), respectively. In these studies, several tropical grasses were investigated: 18 studies with *Pennisetum purpureum* (Elephant grass cv. Napier and Cameroon), 12 studies with *Panicum maximum* (Guinea grass cv. Mombasa and Tanzania), 11 studies with *Brachiaria brizantha* (Palisadegrass cv. Marandu, Xaraes, and Piata), 3 studies with *Cynodon dactylon* (Bermudagrass), 2 studies with *Cynodon nlemfuensis* (Stargrass), and 1 study with *Brachiaria decumbens*. Considering all treatments, the application rate of silage

to 8×1010 cfu/g of fresh forage.

The range of fermentation patterns, *in vitro* digestibility, and aerobic stability are given in **Table 5**. Considering the overall mean, homolactic inoculation increased the concentration of lactic acid by 29.4%, leading to a pH drop from 4.75 (untreated silage) to 4.47. The main purpose to use hoLAB is ensuring a rapid pH decline in earlier times of fermentation (often the first 2 days of ensiling) because the greater production of lactic acid [6]. Indeed, pediococci, streptococci, and lactobacilli comprised the majority commercial homolactic inoculants investigated in Brazilian studies, and they lead to the rapid production of lactic acid and great sugar-tolactic acid conversion efficiency [6]. Otherwise, after the stable phase of fermentation is reached, similar pH can be reported between untreated and inoculated silage with hoLAB [6]. The DM losses and ammonia-N concentration decreased 11.4 and 11.7%, respectively, due to the use of hoLAB. The reduction observed for ammonia-N is likely due to a rapid drop in pH, avoiding proteolysis by the plant, and the action of undesirable microorganisms, such as clostridia. Furthermore, the ADIN content decreased 15.1% due to homolactic inoculation. Results from the present survey agree with the international literature, wherein inoculation with hoLAB generally results in a faster rate of fermentation, less proteolysis, more lactic acid, less acetic and butyric acids, less ethanol, and a greater recovery of energy and organic matter(OM) [9]. Moreover, the data from this survey suggest that homolactic inoculation is most effective in tropical grass silages, compared to other crops. Homolactic inoculation was also most effective in improving the fermentation process of grass silages, compared with corn and sorghum silages in temperate climates [31]. The reasons for that are because the reduced

aerobic stability, and molds.

*2.1.2. Tropical grass silage*

inoculant ranged from 5×104

atures are favorable for yeast growth [24].

The frequency and difference of positive responses found in corn silages from homolactic and heterolactic inoculations are given in **Table 4**. Considering only homolactic inoculation, the greatest frequency of positive responses occurred for aerobic stability, lactic acid, DM content, IVDMD, number of yeasts, and IVOMD. Furthermore, the greatest differences in response were observed for lactic acid, effluent production, and aerobic stability. The greater frequency of positive responses observed for aerobic stability is likely to be related to the low number of trials used to generate the data. According to **Table 3**, the average aerobic stability was greatest for heLAB among all treatments. Conversely, increases in the concentration of lactic acid, DM content, and IVDMD suggest better preservation of soluble sugars during ensiling. The hoLAB have been used to reduce variation in the ensiling process, usually by accelerating the post-ensiling decline in pH, while improving DM and nutrient retention [28].


1 DM, dry matter; CP, crude protein; NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N; NDF, neutral detergent fiber; ADF, acid detergent fiber; IVDMD, *in vitro* DM digestibility; IVOMD, *in vitro* organic matter digestibility; TN, total nitrogen; LAB, lactic-acid bacteria.

**Table 4.** Summary of positive responses of silage inoculants on the fermentation patterns, nutritive value, and aerobic stability of corn silages (data are given in % of DM, unless otherwise stated).

Despite heterolactic inoculation, greater frequencies of positive responses were observed for IVOMD, aerobic stability, number of LAB and yeasts, IVDMD, and acetic acid. In addition, the greatest magnitudes of response were observed for the concentration of 1,2-propanediol, aerobic stability, and molds.

The low number of means for some variables contributed to large values for the frequency of positive responses, as well as the difference between untreated and inoculated silages. However, the data clearly showed that heLAB in corn silage, composed mainly of *L. buchneri*, were biologically effective. *L. buchneri* has been shown to enhance the aerobic stability of silages by increasing the production of acetic acid, which decreases the growth of spoilage microorganisms [29]. Acetic acid has antifungal characteristics [30], and heterolactic inoculation may be particularly important in silages produced under tropical conditions, as elevated temperatures are favorable for yeast growth [24].

#### *2.1.2. Tropical grass silage*

corn silage accompanied of a greater aerobic stability when hoLAB and heLAB were simultaneously used [13, 14, 15]. These responses may ensure most suitable nutritive value of silage

The frequency and difference of positive responses found in corn silages from homolactic and heterolactic inoculations are given in **Table 4**. Considering only homolactic inoculation, the greatest frequency of positive responses occurred for aerobic stability, lactic acid, DM content, IVDMD, number of yeasts, and IVOMD. Furthermore, the greatest differences in response were observed for lactic acid, effluent production, and aerobic stability. The greater frequency of positive responses observed for aerobic stability is likely to be related to the low number of trials used to generate the data. According to **Table 3**, the average aerobic stability was greatest for heLAB among all treatments. Conversely, increases in the concentration of lactic acid, DM content, and IVDMD suggest better preservation of soluble sugars during ensiling. The hoLAB have been used to reduce variation in the ensiling process, usually by accelerating

the post-ensiling decline in pH, while improving DM and nutrient retention [28].

DM, dry matter; CP, crude protein; NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N; NDF, neutral detergent fiber; ADF, acid detergent fiber; IVDMD, *in vitro* DM digestibility; IVOMD, *in vitro* organic matter

**Table 4.** Summary of positive responses of silage inoculants on the fermentation patterns, nutritive value, and aerobic

and lead some beneficial on animal response.

14 Advances in Silage Production and Utilization

1

digestibility; TN, total nitrogen; LAB, lactic-acid bacteria.

stability of corn silages (data are given in % of DM, unless otherwise stated).

Data were summarized from a total of 45 studies, of which 40, 4, and 6 investigated the effect of hoLAB, heLAB, and a combination of both (mixed), respectively. In these studies, several tropical grasses were investigated: 18 studies with *Pennisetum purpureum* (Elephant grass cv. Napier and Cameroon), 12 studies with *Panicum maximum* (Guinea grass cv. Mombasa and Tanzania), 11 studies with *Brachiaria brizantha* (Palisadegrass cv. Marandu, Xaraes, and Piata), 3 studies with *Cynodon dactylon* (Bermudagrass), 2 studies with *Cynodon nlemfuensis* (Stargrass), and 1 study with *Brachiaria decumbens*. Considering all treatments, the application rate of silage inoculant ranged from 5×104 to 8×1010 cfu/g of fresh forage.

The range of fermentation patterns, *in vitro* digestibility, and aerobic stability are given in **Table 5**. Considering the overall mean, homolactic inoculation increased the concentration of lactic acid by 29.4%, leading to a pH drop from 4.75 (untreated silage) to 4.47. The main purpose to use hoLAB is ensuring a rapid pH decline in earlier times of fermentation (often the first 2 days of ensiling) because the greater production of lactic acid [6]. Indeed, pediococci, streptococci, and lactobacilli comprised the majority commercial homolactic inoculants investigated in Brazilian studies, and they lead to the rapid production of lactic acid and great sugar-tolactic acid conversion efficiency [6]. Otherwise, after the stable phase of fermentation is reached, similar pH can be reported between untreated and inoculated silage with hoLAB [6]. The DM losses and ammonia-N concentration decreased 11.4 and 11.7%, respectively, due to the use of hoLAB. The reduction observed for ammonia-N is likely due to a rapid drop in pH, avoiding proteolysis by the plant, and the action of undesirable microorganisms, such as clostridia. Furthermore, the ADIN content decreased 15.1% due to homolactic inoculation. Results from the present survey agree with the international literature, wherein inoculation with hoLAB generally results in a faster rate of fermentation, less proteolysis, more lactic acid, less acetic and butyric acids, less ethanol, and a greater recovery of energy and organic matter(OM) [9]. Moreover, the data from this survey suggest that homolactic inoculation is most effective in tropical grass silages, compared to other crops. Homolactic inoculation was also most effective in improving the fermentation process of grass silages, compared with corn and sorghum silages in temperate climates [31]. The reasons for that are because the reduced

WSC concentration and epiphytic bacteria populations found prior to ensiling in those crop, which commits the ensiling process [31]. In our survey, although homolactic inoculation consistently improved the fermentation parameters of tropical grass silages, a small effect was observed on the nutritive characteristics, and IVDMD was only slightly improved (+1.5%).

Despite heterolactic inoculation, *L. buchneri* was the only heLAB evaluated in the studies that impaired silage quality by increasing pH, ammonia-N, and NDF and reducing crude protein (CP). The responses to inoculation with *L. buchneri* may be crop specific, as evidenced by a meta-analytical study that showed higher effectiveness when applied in corn silages, com-

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Overall, there were not consistent results by combining hoLAB and heLAB for grass silage. Utilization of both hoLAB and heLAB reduced the pH and ammonia-N concentration in silage; however, DM losses increased by 13.2%. The CP content also increased (+20.8%) following inoculation with both hoLAB and heLAB. Although NDF content increased 10.9% due to inoculation, IVDMD also improved by 23.6%. The number of studies assessing both hoLAB and heLAB as silage inoculants for grass is still very low, but the results reported in this survey suggest a suitable strategy to improve fermentation process along with enhanced silage

The ash content of grass silages had an elevated value in all treatments (>9.5%) suggesting contamination, probably by soil, during the ensiling process. Tractors are utilized to transport the harvested forage, fill the silo, and compact the forage mass. Normally, soil in the tractor's tire might be deposited in the forage mass. Moreover, soil contamination is often responsible

There was no comparison regarding positive responses and differences for heLAB and control silages (**Table 6**), because only a few studies used this group of bacteria (**Table 5**). Homolactic inoculation had the greatest frequency of positive responses for IVOMD, gas losses, acetic acid, lactic acid, and lactic:acetic acid. Furthermore, the greatest differences of response were observed for lactic:acetic acid, yeasts, WSC, and lactic and propionic acids. The increased production of lactic acid allowed by homolactic inoculation reduced gas and DM losses, after CO2 production ceases and, consequently, preserved a greater amount of soluble sugars,

Regarding association of both hoLAB and heLAB, the greatest frequency of positive responses was observed for butyric acid and DM losses. In addition, the greatest differences in the response observed for the concentration of butyric acid, effluent production, and DM losses is

The data from this survey suggest that hoLAB should be the only group used for the ensiling of grass, because this group had the greatest frequency of positive responses compared to

Data were summarized from a total of 50 studies, of which 21, 40, and 7 investigated the effect of hoLAB, heLAB, and a combination of both (mixed), respectively. Considering all treatments,

The range of fermentation parameters, *in vitro* digestibility, and aerobic stability are given in

to 2.5×1010 cfu/g of fresh forage.

for the increased number of Clostridia and Bacilli in the ensiling forage [33, 34].

pared with grass and small-grain silages [29].

digestibility.

increasing silage digestibility [6].

*2.1.3. Sugarcane silage*

**Table 7**.

likely to be related to the low number of studies evaluated.

heLAB and to utilization of hoLAB and heLAB combined.

the application rate of silage inoculants ranged from 2.5×104

In some cases, adding homolactic inoculants reduced the aerobic stability of silages, because the lactic acid they produce is used as a growth substrate by yeasts that initiate spoilage [32]. However, unexpectedly the aerobic stability of tropical grass silages increased from 59.5 to 114 h when hoLAB were applied at ensiling, which is likely to be due to the greater production of acids and a lower pH, inhibiting the growth of aerobic microorganisms. But this is only a hypothesis and perhaps factors other than fermentation end products likely contributed to increase the aerobic stability of grass silages treated with hoLAB.


1 DM, dry matter; CP, crude protein; NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N; NDF, neutral detergent fiber; ADF, acid detergent fiber; IVDMD, *in vitro* DM digestibility; IVOMD, *in vitro* organic matter digestibility; WSC, water-soluble carbohydrates; TN, total nitrogen; LAB, lactic-acid bacteria. 2

Number of means. 3 Standard deviation.

4

Silages inoculated with both heterofermentative and homofermentative bacteria.

5 Total acid content was calculated as the sum of lactic, acetic, and propionic acids.

**Table 5.** Range of fermentation patterns, nutritive value, and aerobic stability of untreated and inoculated grass silages (data are given in % of DM, unless otherwise stated).

Despite heterolactic inoculation, *L. buchneri* was the only heLAB evaluated in the studies that impaired silage quality by increasing pH, ammonia-N, and NDF and reducing crude protein (CP). The responses to inoculation with *L. buchneri* may be crop specific, as evidenced by a meta-analytical study that showed higher effectiveness when applied in corn silages, compared with grass and small-grain silages [29].

Overall, there were not consistent results by combining hoLAB and heLAB for grass silage. Utilization of both hoLAB and heLAB reduced the pH and ammonia-N concentration in silage; however, DM losses increased by 13.2%. The CP content also increased (+20.8%) following inoculation with both hoLAB and heLAB. Although NDF content increased 10.9% due to inoculation, IVDMD also improved by 23.6%. The number of studies assessing both hoLAB and heLAB as silage inoculants for grass is still very low, but the results reported in this survey suggest a suitable strategy to improve fermentation process along with enhanced silage digestibility.

The ash content of grass silages had an elevated value in all treatments (>9.5%) suggesting contamination, probably by soil, during the ensiling process. Tractors are utilized to transport the harvested forage, fill the silo, and compact the forage mass. Normally, soil in the tractor's tire might be deposited in the forage mass. Moreover, soil contamination is often responsible for the increased number of Clostridia and Bacilli in the ensiling forage [33, 34].

There was no comparison regarding positive responses and differences for heLAB and control silages (**Table 6**), because only a few studies used this group of bacteria (**Table 5**). Homolactic inoculation had the greatest frequency of positive responses for IVOMD, gas losses, acetic acid, lactic acid, and lactic:acetic acid. Furthermore, the greatest differences of response were observed for lactic:acetic acid, yeasts, WSC, and lactic and propionic acids. The increased production of lactic acid allowed by homolactic inoculation reduced gas and DM losses, after CO2 production ceases and, consequently, preserved a greater amount of soluble sugars, increasing silage digestibility [6].

Regarding association of both hoLAB and heLAB, the greatest frequency of positive responses was observed for butyric acid and DM losses. In addition, the greatest differences in the response observed for the concentration of butyric acid, effluent production, and DM losses is likely to be related to the low number of studies evaluated.

The data from this survey suggest that hoLAB should be the only group used for the ensiling of grass, because this group had the greatest frequency of positive responses compared to heLAB and to utilization of hoLAB and heLAB combined.
