Influence of microbial inoculation and length of storage on fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage

Journal Pre-proof Influence of microbial inoculation and length of storage on fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage Benjamin A. Saylor (Formal analysis) (Investigation) (Data curation) (Writing - original draft) (Writing - review and editing), Tatiane Fernandes (Investigation) (Writing - review and editing), Halima Sultana (Conceptualization) (Investigation) (Writing - review and editing), Antonio Gallo (Investigation) (Writing - review and editing) (Supervision) (Project administration), Luiz F. Ferraretto (Conceptualization) (Formal analysis) (Data curation) (Writing review and editing) (Supervision) (Funding acquisition)

PII:

S0377-8401(20)30461-2

DOI:

https://doi.org/10.1016/j.anifeedsci.2020.114557

Reference:

ANIFEE 114557

To appear in:

Animal Feed Science and Technology

Received Date:

16 December 2019

Revised Date:

13 May 2020

Accepted Date:

14 May 2020

Please cite this article as: Saylor BA, Fernandes T, Sultana H, Gallo A, Ferraretto LF, Influence of microbial inoculation and length of storage on fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage, Animal Feed Science and Technology (2020), doi: https://doi.org/10.1016/j.anifeedsci.2020.114557

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Influence of microbial inoculation and length of storage on fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage Benjamin A. Saylor,a Tatiane Fernandes,a,b Halima Sultana,a Antonio Gallo,c Luiz F. Ferrarettoa,*

Department of Animal Sciences, University of Florida, Gainesville, FL 32611

b

Department of Animal Sciences, Federal University of Lavras, Lavras, MG, Brazil 32700-000

c

Department of Animal Science, Food and Nutrition (DIANA), Faculty of Agricultural, Food,

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a

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and Environmental Sciences, Università Cattolica del Sacro Cuore, 29122, Piacenza, Italy

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Corresponding author: Luiz Ferraretto

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Department of Animal Sciences Room 202 B Animal Sciences Building

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2250 Shealy Drive Gainesville, FL 32611

Phone: (352) 294 1005

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Email: [email protected]

Highlights     

Microbial inoculants influenced fermentation profile of whole-plant corn silage Microbial inoculants did not improve ruminal starch disappearance after prolonged storage Prolonged storage increased proteolysis and starch disappearance Results suggest greater proteolysis occurred in untreated silage Results suggest that inoculants impact growth of different silage molds

Abstract

Increasing the length of storage of whole-plant corn silage (WPCS) has been shown to increase ruminal in vitro starch digestibility by facilitating hydrolysis of the protein matrix surrounding starch granules. It is possible that microbial inoculants could improve fermentation,

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thereby enhancing proteolytic activity in the silo. Additionally, microbial inoculants have

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potential to reduce or prevent the growth of toxigenic fungi in silage. Therefore, the objective of this study was to determine the effects of storage length and microbial inoculation with

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heterofermentative and homofermentative inoculants containing Enterococcus faecium on the

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fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage, as well as the effect of microbial inoculation on silage mycotoxin concentrations. Whole-

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plant corn (333 ± 10 g of DM/kg as-fed) was ensiled in quintuplicate vacuum pouches untreated (CON) or after the following treatments: E. faecium at 1.5 × 105 cfu/g (EF); Lactobacillus

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plantarum at 1 × 105 and E. faecium at 5 × 104 cfu/g of fresh forage (LPEF); and L. buchneri and Lactococcus lactis at 1.5 × 105 cfu/g (LBLL). Silos were stored for 0, 30, 60, 90 or 120 d. Silage pH was greater with LBLL compared to the other three treatments (P = 0.005). Total acids were greater with LPEF than EF (P = 0.005). Ammonia-N (expressed as g/kg of N) was greatest with

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CON compared to the other treatments (P = 0.001). Concentrations of lactic acid were lower, and concentrations of acetic acid were greater with LBLL compared to the other treatments (P = 0.001). An interaction between microbial inoculation and storage length was observed for soluble CP concentrations as well as ruminal in situ starch disappearance (P = 0.001 and P = 0.012, respectively). Soluble CP (expressed as g/kg of CP) was greater with CON compared to

the other treatments at d 30 and 90, but not different at d 60 and 120. Ruminal in situ starch disappearance was reduced for CON compared to the other three treatments at d 60 and 90. However, at d 120, ruminal in situ starch disappearance was similar across all treatments. Overall, the use of microbial inoculants improved fermentation profile. Microbial inoculation also increased starch disappearance in the earlier stages of fermentation but by 120 d of storage, starch disappearance was similar between inoculated silage and CON. Results from this study

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failed to support the hypothesis that microbial inoculants would reduce mycotoxin

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contamination.

Abbreviations: aNDF, neutral detergent fiber determined using α-amylase and sodium sulfite;

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CP, crude protein; DM, dry matter; in situ SD, ruminal in situ starch disappearance; LAB, lactic

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acid bacteria; N, nitrogen; WPCS, whole-plant corn silage; WSC, water-soluble carbohydrates

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mycotoxins

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Keywords: corn silage, fermentation profile, microbial inoculants, starch disappearance,

1. Introduction

Whole-plant corn silage (WPCS) is an important feed for dairy cattle as it provides energy, primarily in the form of starch, to attend the demands of lactation. However, starch

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digestion in the rumen requires that starch granules be accessible for microbial degradation. Starch accessibility is increased when the intricate starch-protein matrices surrounding starch granules are broken down (Kotarski et al., 1992; McAllister et al., 1993). Increasing the length of storage of WPCS beyond 30 and up to 240 d has been shown to facilitate the breakdown of the protein matrix surrounding starch, thereby increasing ruminal in vitro starch digestibility after 7

h of incubation (Der Bedrosian et al., 2012; Ferraretto et al., 2015a). Because bacterial proteolysis in the silo is the primary mechanism associated with the breakdown of the protein matrix (Junges et al., 2017), the development of microbial inoculants capable of enhancing proteolysis and subsequently starch digestibility is desired, particularly because increasing length of storage in a commercial setting is not always a possibility. Storing silage for a longer period requires that more silage be harvested initially which, in turn, demands additional cropland and

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storage infrastructure (i.e. silos) on the farm.

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Silage inoculants generally fall into one of two categories. Homofermentative bacterial inoculants, which contain bacteria that almost exclusively produce lactic acid, have been shown

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to effectively reduce silage pH and improve DM recovery (Muck and Kung, 1997). Examples of

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homofermentative LAB commonly used in silage inoculants include Lactobacillus plantarum, Pediococcus pentosaceus, and Lactococcus lactis (Kung et al., 2003). Heterofermentative

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bacterial inoculants, which contain bacteria capable of producing both lactic and acetic acid, have been shown to improve silage aerobic stability. Lactobacillus buchneri, the

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heterofermentative strain most commonly used in silage inoculants, improves aerobic stability by degrading moderate amounts of lactic into acetic acid which has antifungal properties (Oude Elferink et al., 2001). Typically, L. buchneri is paired with one or more homofermentative bacterial strains in combination inoculants, one of the common bacterial species included in

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these pairings is Lactococcus lactis (Ellis et al., 2016). Lactococcus lactis inhibits the growth of clostridia and lowers the risk of butyric acid fermentation. The LAB commonly used in homoand heterofermentative silage inoculants generally have reduced proteolytic activity, however (McDonald et al., 1991). Recently, there have been suggestions that direct-fed microbials containing strains of E. faecium could improve ruminal starch digestibility; this phenomenon

may be due to its proteolytic activity, but literature confirming this hypothesis is unavailable. E. faecium is a homolactic organism traditionally included with heterolactic bacteria and other homolactic bacteria in combination silage inoculants (Kung et al., 2018). As Enterococcus spp. grow faster than Lactobacillus spp., they may dominate the initial stages of silage fermentation to prevent the growth of undesirable microorganisms (Muck et al., 2018). If E. faecium has proteolytic activity in addition to its ability to control the early active fermentation period, there

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is potential for E. faecium to increase silage quality and starch digestibility. Jalc et al. (2009a)

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reported greater concentrations of ammonia-N in orchardgrass silage inoculated with E. faecium compared to silage inoculated with L. fermentum and L. plantarum which is suggestive of

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increased proteolysis. However, a similar effect was not observed with WPCS (Jalc et al.,

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2009b).

In addition to fermentation profile and starch digestibility, the presence of mycotoxins in

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corn silage is an additional indicator of overall silage quality (Kung et al., 2018). Mycotoxins in silage can originate from the pre- or post-harvest contamination of plant material with toxigenic

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molds and can adversely affect the performance and health of livestock (Ogunade et al., 2018). Although the use of microbial inoculants cannot reduce concentrations of mycotoxins present on plants at harvest, they have potential to reduce or prevent the growth of toxigenic fungi in silage (Ogunade et al., 2018).

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The objective of this study was to determine the effects of storage length and microbial

inoculation with heterofermentative and homofermentative inoculants containing E. faecium on the fermentation profile, nitrogen fractions, and ruminal in situ starch disappearance of WPCS. The mycotoxin contamination of silages after 120 days of ensiling was also analyzed. Our hypothesis was that the use of microbial inoculants would improve the fermentation profile and

starch disappearance of WPCS, while reducing mycotoxin contamination. In addition, we hypothesized that the starch disappearance effect would be increased with inoculants containing E. faecium. 2. Materials and Methods 2.1. Silage preparation and treatments Whole-plant corn was harvested at the University of Florida Dairy Unit (Gainesville, FL)

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on June 23, 2017 when the kernel maturity stage was between one-half and two-thirds of the

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milk line. Five WPCS samples were collected, one at a time, at harvest from five random

locations within the same field to correspond with five replicates per treatment. Each of the five

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samples (one from each location) was manually homogenized and allocated into 21 samples of

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approximately 600 g each using a quartering technique: homogenous samples were divided into four equal subsamples. Two subsamples were saved for later treatment, whereas the other two

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subsamples were re-homogenized and re-divided. The process was repeated until 21 subsamples of approximately 600 g were prepared. One subsample was frozen for nutrient characterization.

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Throughout the manuscript these subsamples for nutrient characterization will be referred to as unfermented samples to avoid confusion with 0 d samples. Each of the remaining 20 samples was randomly assigned to one of 20 individual treatments. Treatments were a combination of four microbial inoculation treatments and five storage lengths. Microbial inoculation treatments

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were as follows: (1) a control with no added inoculant (CON); (2) 1.5 x 105 cfu of Enterococcus faecium CH212 per g of WPCS (EF; Chr. Hansen Animal Health and Nutrition, Milwaukee, WI); (3) 1 x 105 cfu of Lactobacillus plantarum and 5 x 104 cfu of Enterococcus faecium CH212 per g of WPCS (LPEF; Biomax® SB; Chr. Hansen Animal Health and Nutrition, Milwaukee, WI); and (4) 1.5 x 105 cfu of Lactobacillus buchneri LB1819 and Lactococcus lactis O224 per g

of WPCS (LBLL; SiloSolve® FC; Chr. Hansen Animal Health and Nutrition, Milwaukee, WI). Inoculation rates were based on cfu/g of wet material as recommended by the manufacturer. Before the experiment, bacterial populations were counted by pour plating on de Man, Rogosa, Sharpe agar (Oxoid, Basingstoke, UK). Bacterial counts were used to ensure that targeted inoculant application rates were met. Samples of WPCS were individually treated with 50 mL of distilled water (CON) or inoculant solution (EF, LPEF, or LBLL) before ensiling. All samples

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were vacuum heat sealed in nylon-polyethylene standard barrier vacuum pouches (0.09-mm

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thickness, 25.4 x 35.6 cm; Doug Care Equipment Inc., Springville, CA) using an external clamp vacuum machine (Bestvac; distributed by Doug Care Equipment Inc., Springville, CA) and

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stored for 0, 30, 60, 90 or 120 d. Thus, the experiment consisted of 20 treatments (4 microbial

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inoculant treatments × 5 storage length treatments) and 100 mini-silos (five replications per inoculant and storage length combination). All mini-silos were filled and sealed within 30 min

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after harvest, with a total of 180 min for the entire experiment. Mini-silos with a designated storage length of 0 d were sealed after inoculant treatment and immediately frozen. All other

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mini-silos were stored at room temperature (approximately 20°C) in the dark until the targeted storage length was reached.

2.2. Sample preparation and analysis

After the designated length of storage was reached, vacuum pouches were weighed (to

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determine fermentative DM losses) and opened. Fermentative DM losses were calculated as the difference between the weight of the plant material placed in each mini silo at ensiling and the weight of the silage after the designated length of storage, corrected for the DM content of the forage and its respective silage. The material was homogenized, and one half of each sample was immediately frozen at -20°C to stop fermentation and stored until processed for analysis. All

samples were frozen for at least 14 d to ensure protocol similarity among all treatments. The other half was dried at 60°C for 48 h in a forced-air oven to determine DM content then ground to pass through a 6-mm screen in a Wiley mill (A. H. Thomas Scientific, Philadelphia, PA). Unfermented samples were dried at 60°C for 48 h in a forced-air oven to determine DM content then ground to pass through a 1-mm screen in a Wiley mill (A. H. Thomas Scientific, Philadelphia, PA) before being sent to Rock River Laboratory, Inc. (Watertown, WI) for analysis

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of CP (method 990.03; AOAC, 2012), ether extract (method 920.39; AOAC, 2012), starch (Hall,

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2015), water-soluble carbohydrates (Dubois et al., 1956), ash (method 942.05, AOAC, 2012) and NDF determined using α-amylase and sodium sulfite (aNDF; method 2002.04, AOAC, 2012)

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and inclusive of residual ash.

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All experimental dried and frozen samples were sent to Rock River Laboratory, Inc. (Watertown, WI) for nutrient, fermentation profile, and starch disappearance analysis. Frozen

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samples were analyzed for organic acids. A 20 g subsample of undried and unground silage was diluted 10-fold (mass basis) in double distilled water, blended for 30 s in a high-speed blender,

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and filtered through a filter funnel with a 2-mm filter screen. The extract was collected and analyzed for pH using a pH meter (Thermo-Orion Dual Star; Thermo Fisher Scientific Inc., Waltham, MA) fitted with a glass pH electrode (Thermo-Orion 9172BNWP; Thermo Fisher Scientific Inc.). After pH was measured, the extract was centrifuged (750 × g) for 20 min at

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25°C, and the supernatant was combined with 1.0 mL of calcium hydroxide solution and 0.5 mL of copper sulfate solution and re-centrifuged as described previously. Supernatant was analyzed for organic acids and alcohols using high-performance liquid chromatography with isocratic pump, auto-sampler, column heater, and refractive index detector (Waters Corporation 1515, 2707, Heater, and 2414 respectively; Waters Corporation, Milford, MA) and a reverse-phase ion

exclusion column (Bio-Rad Aminex HPX-876H; Bio-Rad Laboratories, Hercules, CA). Flow rate and temperature were set at 0.6 mL/min and 35°C, respectively. The mobile phase used was 0.015 N H2SO4 / 0.25 mM EDTA. Measurements of ammonia-N were performed using a pH/ion selective electrode meter (Thermo-Orion 9172BNWP; Thermo Fisher Scientific Inc.) fitted with an ammonia-specific electrode (OrionTM High-Performance Ammonia Electrode; Thermo Fisher Scientific Inc.) that

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was equipped with a hydrophobic gas-permeable membrane. The membrane allows dissolved

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ammonia to diffuse across, separating ammonia-N from the rest of the solution. Undried and

unground sample (5 g) was diluted in 100 mL of distilled water and mixed for 30 min using a

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magnetic stir plate. The probe was submerged into the solution and 1 mL of 10 N NaOH was

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added and ammonia-N was recorded. Mold and yeast enumeration were performed on Petrifilm Rapid Yeast and Mold Count (RYM) Plates (3M, Saint Paul, MN) using the technique described

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by Adesogan et al. (2004). Mold species identification was performed visually by trained technicians who conducted a morphological examination of microscopic fungal structures as

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described in St. Germain and Summerbell (2011).

Dried silage samples were analyzed for CP and starch as described previously, boratephosphate soluble CP (Krishnamoorthy et al., 1982), absolute DM by oven-drying at 105°C for 3 h (method 2.2.2.5; NFTA, 1993), and ruminal in situ starch disappearance at 7 h (in situ SD).

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For in situ SD, samples were dried and ground to pass through a 6-mm screen in a Wiley mill (A. H. Thomas Scientific, Philadelphia, PA), weighed into filter bags with a pore size of 50 µm, and incubated for 7 h in three cannulated lactating Holstein cows (1 bag per cow, yielding three analytical replicates) fed a diet consisting of (DM basis) 70% forage (mixture of corn silage and alfalfa haylage) and 30% concentrate. The 7 h incubation time-point is the industry standard for

measuring starch disappearance in corn silage and high-moisture corn samples in the United States and was chosen so data could be compared with the existent research. Additional dried 120 d samples were sent to Università Cattolica del Sacro Cuore (Piacenza, Italy) for analysis of mycotoxins. Mycotoxins were extracted from 3 g of a dried silage sample using a 30 mL extraction solution (i.e., 20 mL of 1% acetic acid in acetonitrile and 10mL deionized water containing 3.34 g sodium acetate trihydrate), as described by Gallo et al.

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(2016). The mixture was shaken for 45 min, and 4 g of anhydrous magnesium sulfate was then

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added to obtain phase separation. After shaking for 2 min and centrifugation at 4500 × g for 10 min, the upper acetonitrile phase was recovered. The extract was then diluted 5-fold (0.1 mL

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brought to 0.5 mL) by addition of acetonitrile:water 30:70 (v/v), and passed through a filter

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(Millipore Corporation, Bedford, MA, HV 0.45 µm). A 5 µL sample was injected into the HPLC-MS/MS system, using the system and procedures described by Gallo et al. (2015).

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2.3. Statistical analysis

Microbial data and mycotoxin concentrations were log-transformed prior to statistical

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analysis. Microbial data are presented as log10 values; the mycotoxin concentrations are reported as their natural values, but all terms in the ANOVA refer to log-transformed values. Nutrient, soluble CP, and ammonia-N concentrations, ruminal in situ starch disappearance, and yeast and mold counts data were analyzed as a completely randomized design

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with a 4 × 5 factorial arrangement of treatments using PROC GLIMMIX of SAS (SAS Institute Inc., Cary, NC). The model included microbial inoculation, storage length, and their interactions as fixed effects. Means were determined using the LSMEANS statement, and treatment means were compared using the Bonferroni t-test option after a significant overall treatment F-test. The Bonferroni t-test is a sequentially rejective test based on the Holm-Bonferroni method (Holm,

1979). Orthogonal contrasts were used to evaluate ensiling effects (0 d vs. 30 d). If significant, interaction effects were partitioned using the SLICE option to study the effect of microbial inoculation within each day of storage length. Organic acids, alcohols, pH, and DM loss data were analyzed without d 0 as a completely randomized design with a 4 × 4 factorial arrangement of treatments using the same model, with the exception that orthogonal contrasts to evaluate ensiling effects (0 d vs. 30 d) were not

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performed. Mycotoxin data (collected from d 120 samples only) were analyzed as a completely

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randomized design. The model included microbial inoculation as a fixed effect. Statistical significance and trends were declared at P < 0.05 and P > 0.05 to P < 0.10, respectively.

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3. Results

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P-values for all microbial inoculation × storage length interactions are in Table 1. Nutrient composition of unfermented WPCS is in Table 2. Main effects of microbial inoculation

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and storage length, found in Tables 3 and 4, respectively, are presented only if the interaction effect was not significant (P > 0.05). Silage pH was greater (P = 0.01) with LBLL than with the

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other three treatments. A significant effect of microbial inoculation on concentrations of lactic acid, acetic acid, and total acids was observed. Lactic acid concentrations were lower (P = 0.001) and acetic acid concentrations were greater (P = 0.001) in silage treated with LBLL compared to the other treatments. Concentrations of total acids were greatest in LPEF and lowest in EF.

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Ethanol concentrations were greater (P = 0.009) in LPEF than in LBLL. A microbial inoculation × storage length interaction was observed (P < 0.05) for 1,2-propanediol concentrations (Figure 1). Concentrations of 1,2-propanediol were not detected at d 30 for all treatments. From d 60 to 120, 1,2-propanediol concentrations were greater (P < 0.05) in LBLL than in the other treatments.

There was a significant effect of microbial inoculation on ammonia-N concentrations. Concentrations of ammonia-N were greatest in CON compared to the other inoculant treatments. Dry matter loss, concentrations of WSC, and counts of yeast and mold were unaffected (P > 0.10) by microbial inoculation. The identity of the detected molds at d 0 is in Supplementary Table 1. Regardless of microbial inoculant treatment, Monascus spp. was above 60% of total molds. Penicillium spp. and Fusarium spp. were also identified across all treatments, whereas

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Mucor spp. was identified solely on LBLL samples.

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An inoculation × storage length interaction was observed for concentrations of DM (P = 0.03) and is shown in Figure 2. Concentrations of DM were similar among all treatments from d

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0 to d 60. However, at d 90, DM concentrations were greater in CON than in the other

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treatments. At d 120, DM concentrations were greater in EF than in the other treatments. An interaction between microbial inoculation and storage length was also observed for

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concentrations of CP (P < 0.01; data not presented in tables or figures). At d 0, CP concentrations were greatest in LPEF and EF (averaging 83.4 g/kg of DM) and lowest in CON

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(77.5 g/kg of DM). There was no difference in CP concentrations between treatments at d 30. At d 60, CP concentrations were greater in EF than in the other treatments (88.1 vs. 83.3 g/kg of DM, on average). At d 90, concentrations of CP were greater in LBLL than in the other treatments (93.9 vs. 86.2 g/kg of DM, on average). At d 120, CP concentrations were lower in

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CON compared to the other treatments (80.6 vs. 86.6 g/kg of DM, on average). Microbial inoculation × storage length interactions were also observed (P < 0.05) for

soluble CP concentrations and in situ SD. The interaction between microbial inoculation and storage length for concentrations of soluble CP is shown in Figure 3. Overall, soluble CP concentrations in CON were greater than the other three inoculant treatments on d 30 (584 vs.

445 ± 16 g/kg of CP, on average, respectively) and d 90 (595 vs. 491 ± 16 g/kg of CP, on average, respectively), and greater than EF and LBLL on d 60 (552 vs. 462 ± 16 g/kg of CP, on average, respectively) and d 120 (575 vs. 508 ± 16 g/kg of CP, on average, respectively). The interaction between microbial inoculation and storage length for in situ SD is shown in Figure 4. In situ SD was similar in all treatments from d 30 to d 120 with the exception of CON. In situ SD was significantly lower in CON compared to the other three treatments at d 90. By d 120,

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however, in situ SD was similar across all treatments.

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The effect of microbial inoculation on concentrations of mycotoxins in corn silage stored for 120 d is in Table 5. Although low levels of roquefortine C and alternol monomethyl ether

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were detected in all samples, values were numerically similar among treatments. Concentrations

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of fusaric acid, and altenuene were unaffected by microbial inoculation. Tentoxin concentrations were greater in LBLL compared to the other treatments (P = 0.001). Mycotoxins analyzed but

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not detected in this study included: citrinin, deoxynivalenol, fumonisin B1, fumonisin B2, gliotoxin, monacolins, mycophenolic acid, Penicillium roqueforti (PR) toxin, and tenuazonic

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acid. 4. Discussion

Concentrations of nutrients in unfermented WPCS were within the range of those commonly reported in WPCS trials (Ferraretto and Shaver, 2015). Corn silage contains

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considerable levels of WSC (Ferrero et al., 2018) which favor the growth and development of lactic acid-producing bacteria (Kung et al., 2018). This, in combination with a low buffering capacity (Ferrero et al., 2018), allows for a sharp reduction in pH during initial storage. The inoculant LBLL contained a combination of L. buchneri and Lactococcus lactis. L. buchneri is heterofermentative and well-known to degrade moderate amounts of lactic into acetic acid and

1,2-propanediol (Oude Elferink et al., 2001). Consequently, an increase in pH is a frequently observed in silage treated with L. buchneri (Kleinschmit and Kung, 2006a). Our results corroborate with these premises as inoculation with LBLL resulted in greater acetic acid and 1,2propanediol concentrations, increased pH, and lower concentrations of lactic acid. In agreement with our results, Gallo et al. (2018) reported an increase in pH, and a similar increase in acetic acid and 1,2-propanediol concentrations when corn silage was inoculated with L. buchneri and

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Lactococcus lactis. Similarly, Kleinschmit and Kung (2006b) reported an increase in 1,2-

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propanediol concentrations with prolonged storage of WPCS treated with L. buchneri. The lactate-converting ability of L. buchneri has been shown to be pH dependent, and acidic

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conditions are often needed for this conversion to occur (Oude Elferink et al., 2001).

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Ethanol concentrations observed in this study were within the normal range for WPCS (Kung et al., 2018). Yeasts and some bacteria metabolize pyruvate into either ethanol or acetate

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depending on the redox balance of the cell (Rooke and Hatfield, 2003). Moreover, high ethanol concentrations in silage are often associated with high numbers of yeasts since ethanol is one of

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their main fermentation products (Pahlow et al., 2003; Kung et al., 2018). High concentrations of acetic acid (a good antifungal agent) may explain the reduced ethanol concentrations in LBLL. Other microbes, such as enterobacteria, may be responsible for the differences in ethanol concentrations, but this is only speculation as we did not analyze for enterobacteria in this study.

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Gallo et al. (2018) observed a similar reduction in ethanol concentrations when treating corn silage with L. buchneri and Lactococcus lactis. The observed effect of microbial inoculation on ammonia-N concentrations suggest

increased proteolysis and deamination in CON. According to Kung et al. (2003), inoculation with lactic acid-producing bacteria reduces proteolysis by inhibiting clostridia and plant

proteases through rapid acidification. Plant proteases are responsible for hydrolyzing plant proteins into peptides, free amino acids, and amides (Muck et al., 2003). Active plant proteases could explain the greater ammonia-N levels in the untreated silage. Gallo et al. (2018) observed greater ammonia-N concentrations in untreated corn silage compared to corn silage treated with L. buchneri and Lactococcus lactis. Daniel et al. (2018), in a study with farm-scale silos, reported greater ammonia-N concentrations in untreated silage compared to silage treated with a

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combination of homofermentative inoculants. Overall, our results fail to support our initial

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hypothesis that inoculation with E. faecium would enhance proteolytic activity in the silo. Li et al. (2018) inoculated Pennisetum sinese silage with a variety of fibrolytic enzymes, cellulolytic

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fungi, and bacteria and found that silage inoculated with E. faecium (isolated from yak rumen

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fluid) contained ammonia-N concentrations similar to silage inoculated with L. plantarum suggesting similar proteolytic activity. Conversely, Jalc et al. (2009a) reported significantly

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greater concentrations of ammonia-N in orchardgrass silage inoculated with E. faecium compared to the same silage inoculated with L. fermentum and L. plantarum suggesting

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increased proteolysis with E. faecium. However, the same effect on ammonia-N was not observed in corn silage (Jalc et al., 2009b). Contrary to what was observed in this study, Gallo et al. (2018) found that inoculation with L. buchneri and Lactococcus lactis tended to increase losses of DM. The production of

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acetic acid and other volatile organic compounds by heterolactic bacteria during fermentation has potential to increase fermentative DM losses (Kung et al., 2018). Losses of DM reported in this study are likely a result of the production of carbon dioxide and other volatiles during storage and the use of heat during oven drying. Heat during oven drying will volatilize alcohols, acetate, propionate, and a portion of the lactate (Woolford, 1984). Furthermore, vacuum-sealed bags

were utilized as mini-silos in our study. Johnson et al. (2005) revealed that polyethylene vacuum bags used as laboratory-scale silos are more permeable to CO2 than O2 (155 vs. 40 cm3/m2/d). This, in combination with greater CO2 concentrations in the silo relative to those in the atmosphere, might explain the loss in CO2 from the system and the subsequent reduction in silo weight (DM loss) following a period of prolonged storage. Although the production of acetic acid and other volatile organic compounds by heterolactic bacteria during fermentation may

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increase DM losses (Kung et al., 2018), this response did not occur in the present study. Similar

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to what was observed in this study, Gallo et al. (2018) found that WSC concentrations and counts of yeast and mold were unaffected by inoculation with L. buchneri and Lactococcus lactis.

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Interactions between microbial inoculation and storage length for concentrations of DM

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and CP were observed in this study but differences were minimal which agree with previous literature (Der Bedrosian et al., 2012; Windle et al., 2014; Ferraretto et al., 2015b). It is well-

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documented that as the duration of storage of corn silage increases, proteolysis in the silo continues, resulting in increased concentrations of ammonia-N, soluble CP, and increased starch

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digestibility (Der Bedrosian et al., 2012; Young et al., 2012; Ferraretto et al., 2015a; Ferraretto et al., 2015b; Ferraretto et al., 2016). Similarly, the interaction between microbial inoculation and storage length for concentrations of soluble CP and in situ SD in this study suggest that, across all treatments, proteolysis continued for the duration of ensiling. However, proteolytic activity

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was greatest in the untreated silage and lowest in EF and LBLL. The apparent contradiction between soluble CP and ammonia-N concentrations and in situ SD in CON is difficult to explain. Soluble CP and ammonia-N concentrations have been reported to be positively related to ruminal in vitro starch digestibility (Der Bedrosian et al., 2012; Ferraretto et al., 2015b). Both plant and microbial proteases in the silo are capable of degrading plant proteins to peptides and free amino

acids (Muck et al., 2003). However, in a study conducted by Junges et al. (2017), bacteria were found to contribute to 60% of the proteolytic activity occurring in reconstituted corn grain silage. The subsequent deamination of amino acids by silage microbes results in an accumulation of ammonia-N (Grum et al., 1991). Hoffman et al. (2011) observed a decrease in zein protein concentrations, as well as an increase in concentrations of soluble CP and ammonia-N, when high-moisture corn was ensiled for 240 d. Hoffman et al. (2011) suggested that proteases in the

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silo were responsible for degrading the zein protein matrix surrounding starch granules in corn

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kernels. Because the protein matrix is hydrophobic and represents a physicochemical barrier to amylolytic rumen microorganisms, degradation of the matrix with prolonged ensiling can

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improve ruminal starch disappearance (Hoffman et al., 2011). The fact that in situ SD in CON

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was reduced at d 60 and 90 suggests that proteolysis was occurring but just not in the kernel. Perhaps epiphytic and plant proteases in CON were acting on stover proteins during the

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intermediate stages of fermentation, then, when stover protein became limiting, proteolysis in the kernel portion of the silage was activated, thereby increasing the in situ SD of CON at d 120.

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A similar delay in the increase in starch digestibility for untreated corn silage was observed by Windle et al. (2014). In their study, corn plants harvested at two different maturities were stored either untreated or treated with one of two doses of an experimental protease for 0, 45, 90, or 150 d. Windle et al. (2014) found that after 45 d of storage, in vitro starch digestibility

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was greatest in corn silage treated with the highest level of protease. However, by 150 d, in vitro starch digestibility was not different between the untreated and treated silage. Windle et al. (2014) concluded that the protease treatment accelerated proteolysis during ensiling, so that the corn silage treated with protease reached starch digestibility levels after 45 d of storage that were only obtained in untreated silage after 150 d. These results suggest that accelerated proteolytic

activity in the silo, which could result from either the addition of exogenous enzymes or microbial inoculants, has potential to improve starch digestibility at earlier stages of ensiling, but that after prolonged storage, starch digestibility of untreated silage may reach levels similar to those in treated silage. Contrary to what was observed in our study, however, Windle et al. (2014) found ammonia-N and soluble CP concentrations to be consistently lower in untreated corn silage compared to silage treated with protease.

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It is important to note that vacuum-sealed bags were utilized in the present study. This

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technology results in the rapid removal of most, if not all, oxygen and aerobic epiphytic

microbes at ensiling which may restrict the development of yeasts during the aerobic phase.

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Furthermore, the use of vacuum-sealed bags promotes a rapid acidification. These effects

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combined may impact the activity of plant proteases and subsequently soluble CP concentrations. Therefore, extrapolation of our results to large-scale, commercial silage systems

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should be done with caution. It is imperative to note, however, that a previous study with approximately 6,000 high-moisture corn samples from various commercial settings (i.e. large-

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scale silos) conducted by our laboratory (Ferraretto et al., 2014) revealed a similar pattern of increasing soluble CP and ammonia-N concentrations, as well as increased ruminal in vitro starch digestibility as storage length progressed. These results corroborate the findings from our current study.

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Low pH and low oxygen conditions during ensiling inhibit the growth of toxigenic fungi,

thereby reducing the production of mycotoxins in well-fermented silage (Pahlow et al., 2003). Although levels of mycotoxin contamination were minimal in this study, our analysis revealed the presence of some mycotoxins associated with Penicillium (roquefortine C), Fusarium (fusaric acid) and Alternaria (altenuene, alternol monomethyl ether, and tentoxin). Roquefortine

C has been observed to cause reproductive disorders, mastitis, and lack of appetite in cattle fed silages containing 0.2 to 1.5 mg of the toxin (Auerbach et al., 1998). The toxicological effects of fusaric acid and the Alternaria-produced toxins in ruminants have yet to be confirmed (Ogunade et al., 2018). Except for tentoxin, microbial inoculation failed to have an effect on mycotoxin concentrations. Gallo et al. (2018) found that inoculation with L. buchneri and Lactococcus lactis reduced contamination of corn silage with Penicillium-related mycotoxins and increased fusaric

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acid contamination after 32 d of ensiling. No such effect of microbial inoculation was observed

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in this study. Concentrations of tentoxin, however, were found to be greater in silage treated with LBLL, even though this mycotoxin was detected at low concentrations. Since Alternaria spp. are

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usually categorized as field fungi, they are believed to be unable to grow under typical silage

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conditions, such as low pH and oxygen concentrations and high carbon dioxide levels (Storm et al., 2014). To the best of our knowledge, the present study is the first to report an increase in this

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Alternaria-produced mycotoxin during ensiling. This response deserves further investigation related to the interactions among bacterial, fungal, and other microorganisms in silage. Results

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from this study fail to support the hypothesis that microbial inoculants would reduce mycotoxin contamination. Further research is needed to determine how silage inoculants affect different fungal populations and the production of mycotoxins. 5. Conclusions

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Microbial inoculation with L. buchneri and Lactococcus lactis increased acetic acid and

1,2-propanediol concentrations. Contrary to our hypothesis, microbial inoculation with E. faecium or a combination of L. plantarum and E. faecium did not improve fermentation profile; but fermentation patterns were adequate for all treatments. Furthermore, although greater ruminal in situ starch disappearance was observed for all microbial inoculants after 90 d of

ensiling compared to the control, no differences were observed after 120 d. Last, microbial inoculants affected the growth of different molds but had little effect on mycotoxin concentrations. Further research is warranted to elucidate the mechanism by which silage inoculants affect different fungal populations. Author Statement

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B. A. Saylor – formal analysis, investigation, data curation, writing – original draft, writing – review & editing

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T. Fernandes – investigation, writing – review & editing

H. Sultana – investigation, writing – review & editing, supervision, project administration

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A. Gallo – conceptualization, investigation, writing – review & editing

Conflict of interest

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L. F. Ferraretto – conceptualization, formal analysis, data curation, writing – review & editing, supervision, funding acquisition

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All authors declare no potential conflicts of interest.

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Acknowledgments

Appreciation is extended to Mr. Todd Pritchard (University of Florida Dairy Research

Unit, Gainesville, FL) for assisting with the harvesting of corn silage. This work was supported by the USDA National Institute of Food and Agriculture (Hatch project 1016004), and Chr Hansen A/S (Denmark). Furthermore, the Brazilian Federal Agency for Post-Graduate Education

(CAPES; Brasilia, DF, Brazil) provided the financial support of the scholarship of Tatiane Fernandes. References Adesogan, A. T., N. Krueger, M. B. Salawu, D. B. Dead, and C. R. Staples. 2004. The influence of treatment with dual purpose bacterial inoculants or soluble carbohydrates on the fermentation and aerobic stability of bermudagrass. J. Dairy Sci. 87:3407-3416.

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Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers, and F. Smith. 1956. Colormetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. https://doi.org/10.1021/ac60111a017

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Ferraretto, L. F., K. Taysom, D. M. Taysom, R. D. Shaver, and P. C. Hoffman. 2014. Relationships between dry matter content, ensiling, ammonia-nitrogen, and ruminal in vitro starch digestibility in high-moisture corn samples. J. Dairy Sci. 97:3221-3227. https://doi.org/10.3168/jds.2013-7680

Ferrero, F., S. Piano, E. Tabacco, and G. Borreani. 2018. Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages. J. Sci. Food Agric. 99:2530–254. https://doi.org/10.1002/jsfa.9463 Gallo, A., G. Giuberti, T. Bertuzzi, M. Moschini, and F. Masoero. 2015. Study of the effects of PR toxin, mycophenolic acid and roquefortine C on in vitro gas production parameters

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Nielsen, K. L. Witt, and F. Masoero. 2018. Effect of inoculation with Lactobacillus buchneri LB1819 and Lactococcus lactis O224 on fermentation and mycotoxin

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production in maize silage compacted at different densities. Anim. Feed Sci. Technol. 246:36-45. https://doi.org/10.1016/j.anifeedsci.2018.09.009 Grum, D. E., W. L. Shockey, and W. P. Weiss. 1991. Electrophoretic examination of alfalfa silage proteins. J. Dairy Sci. 74:146–154. https://doi.org/10.3168/jds.S0022-

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Kleinschmit, D. H. and L. Kung Jr.. 2006b. The effects of Lactobacillus buchneri 40788 and Pediococcus pentosaceus R1094 on the fermentation of corn silage. J. Dairy Sci. 89:3999-4004. https://doi.org/10.3168/jds.S0022-0302(06)72443-2 Kotarski, S.F., R. D. Waniska, and K. K. 1992. Starch hydrolysis by the ruminal microflora. J. Nutr. 122: 178–190. https://doi.org/10.1093/jn/122.1.178 Krishnamoorthy, U., T. V. Muscato, C. J. Sniffen, and P. J. Van Soest. 1982. Nitrogen fractions

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chemical, microbial, and organoleptic components of silages. J. Dairy Sci. 101:4020–

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Science and Technology. D. R. Buxton, R. E. Muck, and R. E. Harrison, ed. Amer. Soc. Agron., Crop Sci. Soc. Amer., Soil Sci. Soc. Amer., Madison, WI.

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McAllister, T.A., R. C. Phillippe, L. M. Rode, and K. J. Cheng. 1993. Effect of the protein matrix on the digestion of cereal grains by ruminal microorganisms. J. Anim. Sci. 71: 205–212. https://doi.org/10.2527/1993.711205x

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Muck, R. E. and L. Kung Jr. 1997. Effects of silage additives ensiling. Pages 187–199 in Proc. Silage: Field to Feedbunk. NRAES-99. Natural Resource, Agriculture, and Engineering Service, Ithaca, NY. Muck, R. E., L. E. Moser, and R. E. Pitt. 2003. Postharvest factors affecting ensiling. Pages 251– 304 in Silage Science and Technology. D. R. Buxton, R. E. Muck, and J. H Harrison, ed. American Society of Agronomy, Madison, WI.

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Muck, R. E., E. M. G. Nadeau, T. A. McAllister, F. E. Contreras-Govea, M. C. Santos, and L.

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Kung Jr. 2018. Silage review: Recent advances and future uses of silage additives. J. Dairy Sci. 101:3980–4000. https://doi.org/10.3168/jds.2017-13839

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Rooke, J. A. and R. D. Hatfield. 2003. Biochemistry of ensiling. Pages 95 -139 in Silage Science and Technology. D. R. Buxton, R. E. Muck, and J. H Harrison, ed. American Society of Agronomy, Madison, WI. St. Germain, G. and R. Summerbell. Identifying Fungi – A Clinical Laboratory Handbook. 2nd ed. Star Publishing Co., Inc., Redwood City, CA; 2011. Storm, I. M. L. D., R. R. Rasmussen, and P. H. Rasmussen. 2014. Occurrence of pre- and post-

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(Basel) 6:2256–2269. https://doi.org/10.3390/toxins6082256

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harvest mycotoxins and other secondary metabolites in Danish maize silage. Toxins

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fermentation and nutritive value of corn silage harvested at different dry matter contents

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and ensiled for various lengths of time. J. Dairy Sci. 97:3053–3060. https://doi.org/10.3168/jds.2013-7586

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Woolford, M. K. 1984. The silage fermentation. New York: Marcel Dekker, 2 ed. 350 p. Young, K. M., J. M. Lim, M. C. Der Bedrosian, and L. Kung Jr. 2012. Effect of exogenous

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protease enzymes on the fermentation and nutritive value of corn silage. J. Dairy Sci. 95:6687– 6694.

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http://dx.doi.org/10.3168/jds.2012-5628

Table 1. P-values for all microbial inoculation × storage length interactions.

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Ruminal in situ starch disappearance at 7 h

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P-value 0.03 0.61 0.01 0.001 0.22 0.01 0.09 0.45 0.35 0.64 0.03 0.19 0.43 0.97 0.73

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Item DM, g/kg as-fed WSC1, g/kg of DM CP, g/kg DM Soluble CP, g/kg of CP Ammonia-N, g/kg of N in situ SD2, g/kg of starch pH Lactic acid, g/kg of DM Acetic acid, g/kg of DM Total acids, g/kg of DM 1,2-propanediol, g/kg of DM Ethanol, g/kg of DM Yeast count, log cfu/g Mold count, log cfu/g DM loss, g/kg of DM 1 Water-soluble carbohydrates

Table 2. Nutrient composition of unfermented whole-plant corn.1 Item DM, g/kg as-fed CP, g/kg of DM aNDF2, g/kg of DM Starch, g/kg of DM WSC3, g/kg of DM Ether extract, g/kg of DM Ash, g/kg of DM 1 n=5

Mean 333.0 80.0 420.0 290.0 102.0 25.0 48.0

SD 10.0 2.0 27.0 46.0 11.0 3.0 2.0

aNDF = NDF determined with heat-stable alpha-amylase and inclusive of residual ash.

3

WSC = water-soluble carbohydrates

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Table 3. Effect of microbial inoculation on fermentation profile and nutrient composition of whole-plant corn silage, when averaged over storage length.1,2 SEM P-value 0.008 0.005 0.11 0.001 0.09 0.001 0.15 0.005 0.12 0.009 0.5 0.38 0.7 0.52 3.0 0.43 0.8 0.001 6.7 0.15

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Treatments were CON – distilled water; EF - Enterococcus faecium CH212 at 1.5 x 105 cfu/g of

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LBLL 3.82a 57.3b 24.9a 85.4ab 3.10b 3.36 3.29 59.0 48.7b 74.9

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Item CON EF LPEF b b pH 3.79 3.79 3.79b a a Lactic acid, g/kg of DM 66.0 62.3 66.4a b b Acetic acid, g/kg of DM 19.6 18.2 19.6b Total acids, g/kg of DM 89.3ab 83.8b 90.8a ab ab Ethanol, g/kg of DM 5.58 6.78 9.01a Yeast count, log cfu/g 3.34 3.41 3.46 Mold count, log cfu/g 3.27 3.31 3.40 3 WSC , g/kg of DM 65.0 64.0 63.0 Ammonia-N, g/kg of N 52.7a 46.2b 48.7b DM loss, g/kg of DM 58.6 63.9 77.8 a,b Means with different superscript letters differed (P ≤ 0.05).

fresh forage; LPEF - 1 x 105 cfu/g of Lactobacillus plantarum and 5 x 104 cfu of Enterococcus

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faecium CH212/g of fresh forage; and LBLL - 1.5 x 105 cfu of Lactobacillus buchneri LB1819

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and Lactococcus lactis O224/g of fresh forage ensiled from 0 to 120 d. Main effects are presented only if the interaction with storage length was not significant (P >

3

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0.05).

WSC - water-soluble carbohydrates

Table 4. Effect of storage length on fermentation profile and nutrient composition of whole-plant

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corn silage, when averaged over inoculant treatments.1

Item

pH Lactic acid, g/kg of DM Acetic acid, g/kg of DM Total acids, g/kg of DM Ethanol, g/kg of DM Yeast count, log cfu/g Mold count, log cfu/g WSC5, g/kg of DM

0d NA3 NA NA NA NA 4.96 4.53 122.0

30 d 3.82a 57.7c 11.3c 73.3c 2.96b <3.0 <3.0 51.0ab

P-value2 60 d 3.82a 62.0b 20.6b 85.1b 9.91a <3.0 <3.0 56.0a

90 d 3.82a 66.6a 25.6a 96.1a 5.66ab <3.0 3.1 43.0bc

120 d 3.74b 65.7ab 24.7a 94.9a 5.92ab <3.0 <3.0 41.0c

SEM 0.01 0.11 0.09 0.15 0.12 -4 3.0

E NA NA NA NA NA 0.001 0.001 0.001

S 0.001 0.001 0.001 0.001 0.002 0.001

Ammonia-N, g/kg of N 12.4 48.3c 60.0b 66.1a DM loss, g/kg of DM NA 69.3ab 87.3a 49.0b a,b Means with different superscript letters differed (P ≤ 0.05). 1

58.7b 69.7ab

0.9 6.7

0.001 NA

Main effects are presented only if the interaction with inoculant treatment was not significant (P

> 0.05). E = ensiling effect (0 vs. 30 d), S = storage length effect (30 vs. 60 vs. 90 vs. 120 d)

3

NA - Not assessed

4

Statistical analysis unable to be performed since exact fungal counts below 3.0 log cfu/g are

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WSC - water-soluble carbohydrates

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unknown.

0.001 0.001

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Table 5. Effect of microbial inoculation on concentrations of mycotoxins in whole-plant corn silage stored for 120 d.1,2

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Item CON EF LPEF LBLL SEM P-value 3 Roquefortine C, µg/kg of DM 0.50 0.50 0.50 0.50 Fusaric acid, µg/kg of DM 22.22 30.88 48.58 7.58 1.75 0.17 Altenuene, µg/kg of DM 9.45 5.00 5.00 5.00 1.37 0.41 Alternol monomethyl ether, µg/kg of DM 5.00 5.00 5.00 5.00 b b b a Tentoxin, µg/kg of DM 1.48 0.50 1.21 8.18 1.45 0.001 1 Mycotoxins detected in at least one sample are indicated. Mycotoxins analyzed but not detected were: citrinin, deoxynivalenol,

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fumonisin B1, fumonisin B2, gliotoxin, monacolins, mycophenolic acid, Penicillium roqueforti (PR) toxin, and tenuazonic acid. For each treatment, the indicated concentration is the average for each mycotoxin, including samples in which the mycotoxin was not

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detected (in which case, the instrumental detection limit was used).

Treatments were CON – distilled water; EF - Enterococcus faecium CH212 at 1.5 x 105 cfu/g of fresh forage; LPEF - 1 x 105 cfu/g of

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Lactobacillus plantarum and 5 x 104 cfu of Enterococcus faecium CH212/g of fresh forage; and LBLL - 1.5 x 105 cfu of Lactobacillus buchneri LB1819 and Lactococcus lactis O224/g of fresh forage. Statistical analysis unable to be performed.

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Supplementary Table 1. Identity of detected molds found in whole-plant corn silage at d 0.

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Mold ID, % of total molds Monascus Penicillium Fusarium Aspergillus Mucor Inoculation treatment1 spp. spp. spp. spp. spp. CON 63 34 3 0 0 LPEF 77 19 4 0 0 EF 71 23 6 0 0 LBLL 67 8 15 0 10 1 Treatments were CON – distilled water; LPEF –1 x 105 cfu/g of Lactobacillus plantarum and 5 x 104 cfu of Enterococcus faecium CH212/g of fresh forage; EF - Enterococcus faecium CH212 at 1.5 x 105 cfu/g of fresh forage; and LBLL - 1.5 x 105 cfu of Lactobacillus buchneri LB1819 and Lactococcus lactis O224/g of fresh forage.

CON

EF

LPEF

LBLL

16.0

a

14.0

a

12.0 10.0 a

8.0 6.0

b

4.0 0.0

60 90 Storage length, d

120

-p

30

of

b

b b

2.0

b b

ro

1,2-propanediol, g/kg of DM

18.0

Figure 1. Effects of microbial inoculation and storage length on 1,2-propanediol concentration

re

of whole-plant corn silage. Means within the same day with different letters (a, b) differ (P <

lP

0.05). Effects of microbial inoculation (P = 0.001), storage length (P = 0.001), and their

Jo

ur na

interaction (P = 0.03); SEM = 0.15.

CON

EF

LPEF

LBLL

350.0

DM, g/kg as-fed

a

340.0 a 330.0 b 320.0

b b b

b

of

310.0

30

60 Storage length, d

90

120

-p

0

ro

300.0

re

Figure 2 . Effects of microbial inoculation and storage length on dry matter concentration of whole-plant corn silage. Means within the same day with different letters (a, b) differ (P < 0.05).

Jo

ur na

0.03); SEM = 0.48.

lP

Effects of microbial inoculation (P = 0.10), storage length (P = 0.001), and their interaction (P =

CON a

600.0 550.0

b b

450.0

b

a

350.0

a

300.0

b bc c

b

a ab bc c

b b

c

of

400.0

LBLL

a a

500.0

250.0

LPEF

200.0 30

60 Storage length, d

90

120

-p

0

ro

Soluble CP, g/kg of CP

EF

re

Figure 3. Effects of microbial inoculation and storage length on soluble CP concentration of whole-plant corn silage. Means within the same day with different letters (a, b, c) differ (P <

lP

0.05). Effects of microbial inoculation (P = 0.001), storage length (P = 0.001), and their

Jo

ur na

interaction (P = 0.001); SEM = 1.57.

CON

EF

LPEF

LBLL

900.0

a

a a

850.0 800.0

a b

750.0 700.0

b b

of

in situ SD, g/kg of starch

950.0

30

60 Storage length, d

90

120

-p

0

ro

650.0

Figure 4. Effects of microbial inoculation and storage length on ruminal in situ starch

re

disappearance at 7 h of whole-plant corn silage. Means within the same day with different letters

lP

(a, b) differ (P < 0.05). Effects of microbial inoculation (P = 0.004), storage length (P = 0.001),

Jo

ur na

and their interaction (P = 0.01); SEM = 1.84.