Effects of a mixture of Lentilactobacillus hilgardii, Lentilactobacillus buchneri, Pediococcus pentosaceus and fibrolytic enzymes on silage fermentation, aerobic stability, and performance of growing beef cattle

Abstract

This study evaluated the effects of novel silage inoculants containing lactic acid bacteria (LAB) and fibrolytic enzymes on ensiling, aerobic stability (AS), and the performance of growing beef cattle. Whole-plant corn forage was either uninoculated (CON) or inoculated with a mixture of LAB containing (cfu g⁻¹ fresh forage) 1.5 × 10⁵L. hilgardii (CNCM I-4785), 1.5 × 10⁵L. buchneri (NCIMB 40788) and 1.0 × 10⁵P. pentosaceus (NCIMB 12455) for a total of 4.0 × 10⁵ cfu g⁻¹ fresh forage LAB (IB), or a combination of IB plus fibrolytic enzymes (xylanase + β-glucanase) (IC). All treatments were ensiled in mini-silos, whereas CON and IC were also ensiled in silo bags for the growth performance study. Total bacteria (TB) counts were lower (P = 0.02) for IC than CON after 14 d of ensiling, whereas TB counts of IC and IB were greater (P ≤ 0.01) than CON after 60 d of ensiling in mini-silos. The LAB in IC and IB ensiled in mini-silos were greater than CON on d 60 (P ≤ 0.01) and 90 (P ≤ 0.001) of ensiling and after 3 d (P ≤ 0.01) of aerobic exposure (AE). Silage pH of IC ensiled in silo bags was lower than CON on d 3 (P < 0.01), 7 (P < 0.001), and 14 (P = 0.02) of AE. Yeast counts were lower for IC than CON in terminal silage (P < 0.001), and after 3 (P < 0.001) and 7 d (P < 0.01) of AE. Acetate (AC) concentrations were higher (P ≤ 0.02) for IC than CON throughout AE, whereas lactate (LA) concentrations of IC were greater than CON on d 3 (P < 0.001), 7 (P < 0.01), and 14 (P < 0.001) of AE. Greater AC concentration and lower yeast counts resulted in greater (P < 0.001) stability for IC ensiled in silo bags than CON after 14 d of AE. Growth performance of steers was similar (P > 0.05) as the nutrient composition of silage was similar across diets. Improved AS of IC could potentially have a greater impact on DMI, production efficiency, and growth performance in large-scale commercial feedlot operations where silage at the silo face may be exposed to air for longer periods of time.

Combining strains of heterofermentative LAB, including Lentilactobacillus buchneriand L. hilgardiicould confer aerobic stability to silages ensiled in silo bags, offering producer flexibility in using silages over a wide range of storage periods.

INTRODUCTION

First generation silage inoculants were designed to accelerate the post-ensiling decline in pH as they contained homolactic lactic acid bacteria (LAB), which produce lactic acid (LA) as the primary fermentation product (Pahlow et al., 2003; Queiroz et al., 2012; Addah et al., 2014). However, the resultant silage often exhibited poor aerobic stability (AS) as yeasts, including Pichia and Candida spp. metabolized LA, creating conditions conducive for the growth of molds (Basso et al., 2012). Upon aerobic exposure, LA assimilating yeasts have a competitive advantage over non-LA assimilating yeasts such as Saccharomyces (Pahlow et al., 2003). Second generation silage inoculants contain both homofermentative and heterofermentative LAB, with Lentilactobacillus buchneri (Lactobacillus buchneri) being the most common heterofermentative LAB. L. buchneri produces acetic acid (AC) during ensiling, which exhibits fungicidal activity and improves AS (Reich and Kung, 2010; Addah et al., 2014). Lentilactobacillus hilgardii (Lactobacillus hilgardii) is also a hetero-fermentative LAB that produces AC during ensiling (Muck et al., 2018; Nair et al., 2020) in as little as 2 weeks after ensiling (Carvalho et al., 2014; Nair et al., 2020). In contrast, AC production is more pronounced after ~60 d of ensiling with L. buchneri (Kleinschmit and Kung, 2006; Muck et al., 2018). Combinations of homo- and hetero-fermentative LAB inoculants have been used to ensure the production of sufficient LA to promote a rapid decline in silage pH and AC to ensure AS (Kleinschmit and Kung, 2006). A combination of heterofermentative LAB consisting of L. hilgardii and L. buchneri could ensure that AS was conferred through the inhibition of yeast shortly after ensiling.

Most of the bacterial strains used as silage inoculants do not possess the fibrolytic enzymes required to liberate carbohydrates from plant cell walls (Addah et al., 2016). To increase fiber digestibility, manufacturers have added fibrolytic enzymes to microbial inoculants (Addah et al., 2016). To our knowledge, the impact of inoculating corn silage with xylanases and β-glucanases in combination with a mixture of hetero-fermentative LAB on fermentation, AS, and growth performance of beef cattle has not been investigated. It was hypothesized that combining an inoculant containing L. hilgardii, L. buchneri, P. pentosaceus with fibrolytic enzymes would improve the AS and feed value of corn silage. The objectives of the study were to evaluate the effects of a mixture of LAB and fibrolytic enzymes on the ensiling and AS of corn silage and on the growth performance of steers fed inoculated and uninoculated corn silage.

MATERIALS AND METHODS

Animal Care and Management

Experimental protocols involving animals were reviewed and approved by the Lethbridge Research and Development Centre (LeRDC) animal care committee (ACC protocol # 1926), and animals were cared for as per the guidelines of the Canadian Council on Animal Care (CCAC, 2009).

Forage Production and Harvesting

Corn (Zea mays; LF 730 CBR; Maizex Seeds Inc., Tilbury, ON) was planted on May 13th, 2019, under irrigation near Lethbridge, AB. Corn was planted at a depth of 4–5 cm, with 38 cm row spacing at a density of 86,500 seeds ha⁻¹. The forage was harvested on October 16th, 2019, after the first killing frost at two-thirds milk line maturity (43.1% DM) and chopped to 9.5 mm with a forage harvester (Claas Jaguar 950 Forage Harvester, Harsewinkel, Germany) equipped with a kernel processor with a roller clearance of 1.0 mm.

Mini-Silo Experiment

For mini-silos, harvested corn forage was divided into a total of 15 lots of 25 kg each, with five lots each serving as the replicates. Each lot was placed on separate clean plastic sheets for processing. Lots were either sprayed with distilled water (uninoculated control corn; CON) or with the LAB silage inoculant (Lallemand Specialties Inc., Milwaukee, WI) containing (cfu g⁻¹ fresh forage) 1.5 × 10⁵L. hilgardii (CNCM I-4785), 1.5 × 10⁵L. buchneri (NCIMB 40788) and 1.0 × 10⁵P. pentosaceus (NCIMB 12455) (IB), or a combination of IB plus fibrolytic enzymes (xylanase + β-glucanase) (IC). All inoculants were applied at a rate of 25 mL per lot, providing 4.0 × 10⁵ cfu LAB g⁻¹ fresh forage for IB and 4.0 × 10⁵ cfu LAB g⁻¹ fresh plus fibrolytic enzymes for IC. Separate containers and sprayers were used for each treatment, and gloves were changed for each lot to prevent cross-contamination. Corn sprayed with deionized water at a rate of 25 mL lot⁻¹ served as CON silage. Each lot was thoroughly hand-mixed to ensure uniform inoculation. Mixed forage was weighed, and 2.0 kg was packed into five mini-silos (10.4 cm in diameter × 35.6 cm in height) per lot using a hydraulic press to achieve a density of ~ 240 kg DM m⁻³. One extra mini-silo was also filled from the final mix of each treatment to serve as sample for d 90 so that one each of the five mini-silos per lot served as samples (n = 5) for d 7, 14, 21, and 60 while a total of six mini-silos served as samples for day 90 of ensiling. There was a total of 26 mini-silos for each treatment and a grand total of 78 mini-silos for the study. Each labeled mini-silo was weighed before and immediately after filling and sealing. Silos were stored at ambient temperature (20 °C) and opened after 7, 14, 21, 60, and 90 d of ensiling. Before ensiling (d 0), triplicate samples of fresh forage were collected per treatment for chemical and microbial analyses. Silos were weighed before opening on each sampling day, and recorded weights were used to calculate DM loss. At sampling, the contents of each mini-silo were thoroughly mixed and sub-sampled for chemical and microbial analyses.

Preparation of Whole-Plant Corn Silage in Silo Bags

Chopped corn forage was ensiled in Silobolsa Plastar premium silo bags (2.7 m × 60.0 m; Plaster, San Luis, SA, Buenos Aires, Argentina) using an Ag-Bag bagger (Ag-Bag, St. Nazianz, WI). Inoculant was applied at the bagger using ATV sprayers (AG Spray Equipment, Hopkinsville, KY) at a rate of 40 mL tonne⁻¹, resulting in 4.0 × 10⁵ cfu LAB plus fibrolytic enzymes (xylanase + β-glucanase) g⁻¹ fresh forage of IC. Corn sprayed with deionized water at a rate of 40 mL t⁻¹ served as CON silage. To reduce cross-contamination between bags, spray systems were flushed with 20 L of deionized water each time the treatments were changed. Both CON and IC forages were ensiled in two silo bags, with each bag containing both treatments. A 3-m section of untreated corn forage was marked with spray paint on the outside of the bags to delineate clear separation between CON and IC corn silage. To minimize differences in forage composition due to field location, forage was chopped at random locations within the field. Upon delivery to the bagger, forage was sampled from each truckload with ~ 200 tonnes of each treatment compressed into two silo bags. Forage was proposed to be ensiled for 120 d prior to opening for evaluation of silage quality and AS as described below. However, bags remained ensiled due to COVID-19 restrictions and were not opened for use in the growth performance study until after 418 d of ensiling. Approximately 1 m of silage was removed from the face of each silo and discarded when the bags were first opened.

Determination of the Effect of Inoculants on AS of Whole-Plant Corn Silage

Silage samples from mini-silos opened on d 90 (n = 6), and silo bags opened after 418 d of ensiling were used for the assessment of AS. Subsamples (~ 1.2 kg each) of silage from two mini-silos each (three sets of two mini-silos each per treatment) opened on d 90 of ensiling were used to generate three 4-L insulated containers (13.5 cm in diameter × 30.9 cm in height) per treatment (n = 3). Silage samples collected from multiple regions (5–6 sites) of the silo face from both ends of silo bags on each run (n = 2/silo bag) were combined on a treatment basis and placed into quadruplicate 4-L insulated containers per treatment (n = 4). Insulated containers were covered with two layers of cheesecloth and stored at ambient temperature (20 °C) for 14 d. Silage samples from the silo bags were collected at 3-wk interval for a total of 4 runs (run 1 and 2 from silo bag one and run 3 and 4 from silo bag two) for AS evaluation with four 4-L insulated containers per treatment. Two Dallas Thermochron iButton sensors (Embedded Data Systems, Lawrenceburg, KY) were embedded in the silage at ~ 9.0 cm and ~ 18.0 cm within each container to measure the temperature every 15 min for 14 d. Two sensors were also placed in the room to measure ambient temperature. The contents of each container were sub-sampled the same way after 3, 7, and 14 d of AE for determinations of pH, chemical, and microbial composition.

Determination of Effects of Inoculants on Growth Performance of Feedlot Steers

Animals, treatments, and experimental design

Forty Angus × Hereford steers (291.5 ± 19.8 kg; mean ± SD) were housed in individual feeding pens at the LeRDC individual feeding barn. Upon arrival, steers were ear-tagged, dewormed, and vaccinated against Infectious Bovine Rhinotracheitis, Parainfluenza-3, Heamophilus somnus, and Clostridium spp. as described by Wang et al. (2017). Steers were stratified by weight and assigned to two groups of 20 steers each. Steers in each group were randomly assigned to the CON or IC diets in a completely randomized design.

Steers were fed total mixed rations (TMR) consisting of 65.0% of either CON or IC corn silage, plus 20.0% barley grain, 10.0% canola meal, and 5.0% of a vitamin-mineral supplement (DM basis; Table 4). Both diets (CON and IC) were formulated to meet or exceed the NASEM (2016) nutrient requirements for CP, energy, minerals, and vitamins for beef steers for a targeted gain of 1.3 kg d⁻¹. Diets were also formulated for calcium to phosphorus ratio of 2:1 across treatments. Monensin sodium was provided at 33 mg kg⁻¹ (DM basis) in the vitamin-mineral supplement.

Table 4.

Composition and nutrient analysis of diets containing CON, and IC corn silages used for the growth performance study using feedlot steers

	Treatments1		SEM2	P-value
	CON	IC
Diet ingredient composition (% DM basis)
Corn silage	65.0	65.0	–	–
Barley grain	20.0	20.0	–	–
Canola meal	10.0	10.0	–	–
Supplement3	5.0	5.0	–	–
Diet nutrient composition (% DM basis)
OM	96.1	96.1	0.29	0.98
CP	14.5	14.0	0.43	0.48
ADF	22.0	23.4	0.60	0.08
NDF	42.9	43.7	0.75	0.08
Starch	21.0	20.2	0.56	0.35

CP, crude protein; ADF, acid detergent fiber; NDF, neutral detergent fiber.

¹Treatments included CON, control diet with uninoculated silage; and corn silage inoculated with LAB inoculant containing (cfu g⁻¹ fresh forage) 1.5 × 10⁵Lentilactobacillus hilgardii, 1.5 × 10⁵L. buchneri and 1.0 × 10⁵P. pentosaceus for a total of 4.0 × 10⁵ LAB plus fibrolytic enzymes (xylanase + β-glucanase) (IC).

²SEM, pooled standard error of mean.

³Supplement (as fed basis) contained 30.0% ground barley, 29.1% canola meal,26.0% limestone, 6.5% urea, 5.0% salt, 1.5% canola oil, 1.5% vitamin-mineral premix, 0.3% Rumensin 200 and 0.1% Vitamin E 500.

Feeding management and measurements

Feed was delivered using a Calan Data Ranger (American Calan, Northwood, NH) to each pen once daily at 0900 h. Steers were fed for ad libitum intake with a target of 5% feed refusal. Individual feeders were observed each morning, and the daily feed allotted was based on the residual feed in the feeder, and the amount fed the previous day. Orts were collected weekly, weighed, and sub-sampled for DM to adjust for DMI. Steers were weighed prior to morning feeding on 2 consecutive days at the beginning and end of an 84-d growth study and every 21 d throughout the study period. Samples of corn silage were collected weekly to measure DM, and the diet was adjusted for DM content if DM of the silage varied by more than 10%. Diets were sampled weekly, and dry rolled barley grain, canola meal, and supplement samples were collected every 2 weeks. All samples of feed and TMR were composited on a monthly basis, and representative samples saved for chemical analysis.

Laboratory Analyses

Microbial analyses of corn silage

Microbial analyses were conducted as described by Nair et al. (2020). Briefly, samples (10 g) of fresh corn forage collected on the day of ensiling, silage from mini-silos, and aerobically exposed silage samples collected on each sampling day from mini-silos and silo bags were added to 90 mL of sterile 70 mM potassium phosphate buffer (pH = 7.0) and agitated for 30 s at 260 × rpm in a Stomacher 400 Laboratory Blender (Seward Medical Limited, London, UK). The suspension was serially diluted (10⁻¹ to 10⁻⁴), and 100 µL aliquots of each dilution were spread in triplicate onto de Man, Rogosa and Sharpe agar (MRS; Dalynn Biologicals, Calgary, AB) for the enumeration of LAB, on to nutrient agar (NA; Dalynn Biologicals, Calgary, AB) for the enumeration of total bacteria (TB) and on to Sabouraud’s dextrose agar (SDA; Dalynn Biologicals, Calgary, AB) for the enumeration of yeast and mold. Both MRS and NA plates contained 200 µg mL⁻¹ of cycloheximide (Dalynn Biologicals, Calgary, AB), while SDA plates contained 100 µg mL⁻¹ each of tetracycline and chloramphenicol. Both MRS and NA plates were incubated at 37 °C for 24–48 h, while SDA plates were incubated at ambient temperature for 72 h. Colonies were counted from plates containing a minimum of 30 and maximum of 300 colonies.

Chemical Analyses

Chemical analyses of samples were conducted as described by Nair et al. (2020). Corn silage (15 g) samples from mini-silos, silo bags, and aerobically exposed silage were mixed with 135 mL of deionized water and blended at full speed for 30 s in a Waring blender (Waring Commercial, Torrington, CT). The suspension was filtered through two layers of cheesecloth, and the pH of the collected fluid was measured twice using a Symphony pH meter (SB70P, VWR, Mississauga, ON). One portion (7.5 mL) of the filtrate was immediately boiled for 10 min to stop fermentation and stored at −20 °C for subsequent analysis of water-soluble carbohydrates (WSC) by the Nelson-Somogyi method (Nelson, 1944) using a Dynatech MRX micro-plate reader (Dynatech Laboratories Inc., Chantilli, VA). The second portion was stored on ice until centrifuged at 10,000 × g for 15 min at 4 °C in a Sorvall Legend Mach 1.6R centrifuge (Thermo electron Corporation, Gormley, ON). The collected supernatant was used for the analysis of VFA, LA, and ammonia, as described by Addah et al. (2016).

Samples of TMR, barley grain, vitamin-mineral supplements, and orts from the growth performance study were first dried at 55 °C for 48 h in a forced air oven, ground to pass through a 1.0-mm screen using a Wiley Mill (Model 4, Arthur H. Thomas Co., Philadelphia, PA) and analyzed in duplicate according to the Association of Official Analytical Chemists [AOAC (2000)] for DM [Method 930.15; AOAC (2000)], ash [(Method 942.05; AOAC (2000)], with ash content used for the determination of OM by subtracting ash from 100%, total nitrogen (N) [method 990.03; AOAC (2000)] using a LECO FP-528 Nitrogen Combustion Analyzer (LECO, St. Joseph, MI), ADF [Method 973.18; AOAC (2000)], NDF using the method of Van Soest et al. (1991) with the addition of α-amylase and sodium sulfite, and starch after the hydrolysis to α-glucose polymers by amyloglucosidase (Megazyme Int. Ltd., Wicklow, Ireland) in combination with a 1,4-α-d-glucan glucanohydrolase (Brennfag Canada Inc., Toronto, ON, Canada) with the samples read on a Thermo Scientific Appliskan (SkanIt Software 2.3) micro-plate reader (Thermo Scientific, Hudson, NH) at a wavelength of 490 nm as described by Addah et al. (2016) and Wang et al. (2017).

Data Calculations and Statistical Analyses

Aerobic stability was defined as the number of hours before the temperature of aerobically exposed silage exceeded ambient temperature by 2 °C (Teller et al., 2012). Microbial populations were estimated as cfu g⁻¹ silage DM and were log-transformed before statistical analysis. The DM loss from mini-silos during ensiling was calculated as DM loss (%) = 100 − [(WO × DMO)/(WC × DMC)] × 100 where WO = weight of forage at opening, DMO = DM content at opening, WC = weight of forage at closure, and DMC = DM content at closure (Assis et al., 2014). Maximum temperature (T_max) was the highest temperature recorded during the 14 days of AE. The duration (h) was measured by adding the length of time (h) the silage temperature was above ambient temperature + 2 °C during the AE while the area above ambient temperature + 2 °C was calculated by multiplying the duration (h) above the ambient temperature + 2 °C (threshold for spoilage) with the temperature above the ambient temperature + 2 °C during the AE and was calculated for d 1–3, 1–7, 8–14, and 1–14 of AE. Dietary NE_m content was calculated based on animal performance using the retained energy formula for medium frame yearling steers [RE = (0.0493 × BW^0.75) × ADG^1.097; NRC, 1996] as per Zinn et al. (2002). Net energy of gain was calculated from NE_m assuming NE_g = NE_m × 0.877–0.41 as per Zinn and Shen (1998). To account for gut fill, BW of steers was reported on a shrunk BW basis (BW × 0.96) (Nair et al., 2019b, 2020).

All data were statistically analyzed following an analysis of variance using the MIXED procedure of SAS (version 9.3.1; SAS Inst. Inc., 2012). For the mini-silo and silo bag AE evaluation, the effect of treatment on nutrient composition, fermentation parameters, and microbial data were assessed using a repeated measures analysis with the effect of treatment (T), days of ensiling or AE (D), and T × D included in the model, with treatment as fixed effects and silo bags (n = 2) and runs (n = 4 for silo bag AE evaluation) as random factors. Mini-silos [n = 5 for d 7, 14, 21, 60 while n = 6 for d 90 of ensiling] and 4-L insulated containers (n = 3 for mini-silo and n = 4 for silo bag study) containing aerobically exposed silage were used as the experimental units for fermentation and AS parameters, respectively. For the performance study, nutrient composition and performance data were analyzed as a completely randomized design with treatment as a fixed effect and individual pen as the experimental unit. Denominator degrees of freedom were determined using the Kenward-Roger option. Means were separated using the Tukey’s test. Significant differences and trends were declared at P < 0.05 and 0.10 > P > 0.05, respectively.

RESULTS

Ensiling Fermentation Characteristics of Whole-Plant Corn Silage

Mini-silo study

The IC (95.4 % DM) and IB (95.5% DM) silages had a greater (P < 0.01) OM content than CON silage (94.8 % DM) after 90 d of ensiling (Table 1). The DM loss (%) was greater (P = 0.02) for IC than CON or IB during ensiling. There was a T × D interaction for ethanol concentration with greater ethanol concentration for IB (2.13 vs. 1.29 mg g⁻¹ DM; P < 0.01) than CON in terminal silage (data not shown). Moreover, there was a T × D interaction (P ≤ 0.02) for TB, and LAB counts during ensiling (Figure 1A, B). Total bacterial counts (log₁₀ cfu g⁻¹ DM) were lower for IC than CON (9.21 vs. 9.46; P = 0.02) on d 14 of ensiling, while IC (8.79) and IB (8.66) had greater (P ≤ 0.01) TB counts than CON (8.02) on d 60 of ensiling. Similarly, LAB counts were greater for IC and IB than CON on d 60 (8.69 and 8.59 vs. 7.93; P ≤ 0.01) and 90 (8.37 and 8.13 vs 6.49; P ≤ 0.001) of ensiling (Figure 1B). Mold was undetected after d 14 of ensiling.

Table 1.

Chemical composition, fermentation products and microbial populations of fresh corn forage, and CON, IC, and IB corn silages ensiled in mini-silos and upon aerobic exposure (AE)

Item5	Corn forage1	Silages after 90 d of ensiling2			SEM3	P-value	Silages after 14 d of AE2			SEM4	P-value
		CON	IC	IB			CON	IC	IB
pH	5.45 ± 0.117	3.84	3.86	3.84	0.021	0.48	6.47	6.23	5.28	0.886	0.58
DM	43.1 ± 1.65	42.5	40.9	41.2	0.7	0.21	42.1	42.4	42.9	0.99	0.72
OM, % DM	95.0 ± 0.67	94.8b	95.4a	95.5a	0.15	< 0.01	NA	NA	NA	NA	NA
CP, % DM	7.56 ± 0.464	7.77	7.67	8.08	0.241	0.61	NA	NA	NA	NA	NA
ADF, % DM	27.0 ± 1.72	27.7	27.8	27.7	0.66	1.00	NA	NA	NA	NA	NA
NDF, % DM	51.4 ± 2.29	50.9	51.1	51.0	0.86	0.99	NA	NA	NA	NA	NA
Starch, % DM	21.0 ± 1.24	17.7	18.3	17.5	1.00	0.84	NA	NA	NA	NA	NA
DM loss, %	NA	3.34b	7.55a	3.8b	1.166	0.02	NA	NA	NA	NA	NA
WSC, mg g−1 DM	133.8 ± 20.5	62.2	47.0	36.5	6.06	0.27	20.8	17.6	18.1	1.87	0.49
Fermentation products mg−1 g DM
Acetate	0.57 ± 0.153	9.78	10.70	9.91	0.689	0.17	0.54b	0.55b	4.48a	0.925	0.02
Propionate	ND	ND	ND	ND	NA	NA	ND	ND	ND	NA	NA
Butyrate	ND	ND	ND	ND	NA	NA	ND	ND	ND	NA	NA
Total VFA	0.57 ± 0.153	9.78	10.70	9.91	0.733	0.18	0.54b	0.55b	5.53a	0.534	< 0.001
Lactate	0.05 ± 0.007	47.0	48.0	47.8	1.47	0.12	4.69b	1.00b	18.1a	2.280	< 0.01
LA:AC ratio	0.10 ± 0.023	4.93	4.51	4.89	0.290	0.06	6.35a	3.56b	4.00b	0.485	0.02
Ethanol	0.05 ± 0.028	1.29b	1.67ab	2.13a	0.111	< 0.01	0.04	0.03	0.03	0.017	0.17
NH3–N	0.27 ± 0.049	0.71	0.73	0.74	0.032	0.39	0.23	0.28	0.30	0.108	0.77
Microbiology, log10 cfu g−1 DM
Total bacteria	7.78 ± 0.569	6.12b	6.42b	7.33a	0.261	0.05	7.82	7.72	7.86	0.719	0.10
LAB	5.45 ± 0.519	6.49b	8.37a	8.13a	0.202	< 0.001	9.16	9.24	9.38	0.341	0.08
Yeasts	7.58 ± 0.147	5.59	5.10	5.19	0.198	0.94	9.20	9.18	9.20	0.161	0.72
Mold	5.42 ± 0.216	ND	ND	ND	NA	NA	ND	ND	ND	NA	NA
Maximum temperature (Tmax), °C	NA	NA	NA	NA	NA	NA	31.9	30.5	30.3	4.45	–
Time to reach Tmax, h	NA	NA	NA	NA	NA	NA	222	283.0	274.3	46.7	–
Aerobic stability6, h	NA	NA	NA	NA	NA	NA	164	202	183.7	46.2	0.85

¹Values for fresh forage were not included in statistical analysis (n = 3).

²Treatments included CON, uninoculated control silage, and corn silage inoculated with lactic acid bacterial (LAB) inoculant containing 1.5 × 10⁵ cfu g⁻¹ fresh forage Lentilactobacillus hilgardii, 1.5 × 10⁵ cfu g⁻¹ fresh forage L. buchneri and 1.0 × 10⁵ cfu g⁻¹ fresh forage P. pentosaceus for a total of 4.0 × 10⁵ cfu g⁻¹ fresh forage LAB with (IC) or without (IB) fibrolytic enzymes (xylanase + β-glucanase).

³SEM, pooled standard error of mean (n = 5).

⁴SEM, pooled standard error of mean (n = 3).

⁵LAB, Lactic acid bacteria; LA:AC ratio, ratio of lactic acid to acetic acid; NA, not applicable; ND, not detected; NH₃-N, ammonia nitrogen; WSC, water-soluble carbohydrates.

⁶Hours after a 2 °C rise in temperature was recorded in the silage mass after exposure to air.

Figure 1.

Impact of uninoculated (Control; CON) or corn silage inoculated with (cfu g⁻¹ fresh forage) lactic acid bacterial (LAB) inoculants consisting of 1.5 × 10⁵L.hilgardii, 1.5 × 10⁵ cfu g⁻¹L. buchneri and 1.0 × 10⁵P. pentosaceus for a total of 4.0 × 10⁵ LAB (IB) and a combination of IB plus fibrolytic enzymes (xylanase + β-glucanase) (IC) on (A) total bacteria (TB) and (B) LAB in terminal silages ensiled in mini-silos and during aerobic exposure (AE; n = 3 for ensiling and 4 for AE). (A) *Denote treatment differences in TB counts in terminal silage and upon AE. The TB counts of IC was lower (P = 0.02) than CON on d 14 of ensiling whereas the TB counts of IB and IC were greater (P ≤ 0.01) than CON on d 60 of ensiling. (B) *Denote treatment differences in LAB counts in terminal silage and upon AE. The LAB counts of IB, and IC were greater than CON on d 60 (P < 0.01), and d 90 (P < 0.001) of ensiling and d 3 (P < 0.01) of AE.

Silo Bag Study

The DM content was greater (P = 0.03) for terminal IC than CON silage after 418 d of ensiling (Table 2). Irrespective of treatments, whole-plant corn ensiled for 418 d had numerically lower NDF and WSC content but similar OM, ADF, CP, and starch contents as freshly chopped corn forage (Tables 1 and 2). Viable total bacteria (TB), LAB, yeast, and mold were all numerically lower in terminal silages than in fresh forage, regardless of treatment. Contents of DM, OM, ADF, and starch in terminal silages were similar between the two treatments. However, inoculated silages tended (P= 0.08) to have higher NDF than the control at the end of the long storage period. In terminal silages, the CP and WSC concentrations were lower (P ≤ 0.01) but concentrations of AC and total VFA were greater (P ≤ 0.03) for IC than CON. Similarly, the ratio of LA:AC and ethanol concentrations in terminal silage was lower (P ≤ 0.01) for IC than CON. Moreover, IC had lower (P < 0.001) yeast counts than CON silage.

Table 2.

Chemical composition, fermentation products, and microbial populations of CON, and IC corn silages ensiled in silo bags and upon aerobic exposure (AE)

Item3	Silages after 418d of ensiling1		SEM2	P-value	Silages after 14 d of AE1		SEM2	P-value
	CON	IC			CON	IC
pH	3.78	3.74	0.033	0.58	6.62	4.79	0.351	0.02
DM	39.7	41.4	0.36	0.03	36.1	42.3	1.02	< 0.01
OM, % DM	95.7	95.7	0.07	0.87	NA	NA	NA	NA
CP, % DM	8.25	7.82	0.124	< 0.001	NA	NA	NA	NA
ADF, % DM	27.3	28.2	0.53	0.24	NA	NA	NA	NA
NDF, % DM	44.4	46.0	1.08	0.08	NA	NA	NA	NA
Starch, % DM	21.4	20.4	0.759	0.21	NA	NA	NA	NA
WSC, mg g−¹ DM	41.6	30.3	5.80	< 0.01	10.8	14.9	6.43	0.99
Fermentation products mg−¹ g DM
Acetate (AC)	15.0	19.6	1.06	0.02	0.91	7.04	0.960	< 0.001
Propionate	0.15	0.03	0.065	0.90	0.04	0.001	0.019	0.81
Butyrate	0.08	0.03	0.034	1.00	0.005	0.03	0.006	0.03
Total VFA	15.5	19.9	1.06	0.03	1.40	7.67	0.957	< 0.001
Lactate (LA)	44.6	45.1	3.39	1.00	4.00	28.1	3.87	< 0.01
LA:AC ratio	3.08	2.34	0.27	0.01	4.07	4.24	0.955	1.00
Ethanol	0.73	0.27	0.163	< 0.01	0.54	0.09	0.203	0.33
NH3–N	0.69	0.71	0.016	0.97	0.21	0.32	0.056	0.84
Microbiology, log10 cfu g−¹ DM
Total bacteria	4.91	4.19	0.371	0.64	8.64	6.38	0.610	0.12
LAB	4.89	4.37	0.353	0.94	8.90	6.93	0.508	0.12
Yeasts	5.79	0.77	0.354	< 0.001	9.02	8.61	0.267	0.68
Mold	0.55	1.47	0.416	0.31	6.75	3.96	1.082	0.56
Aerobic stability4, h	NA	NA	NA	NA	79.1	251.4	48.1	< 0.001

¹Treatments included CON, uninoculated control silage, and corn silage inoculated with LAB inoculant containing 1.5 × 10⁵ cfu g⁻¹ fresh forage Lentilactobacillus hilgardii, 1.5 × 10⁵ cfu g⁻¹ fresh forage L. buchneri and 1.0 × 10⁵ cfu g⁻¹ fresh forage P. pentosaceus for a total of 4.0 × 10⁵ cfu g⁻¹ fresh forage LAB plus fibrolytic enzymes (xylanase + β-glucanase) (IC).

²SEM, pooled standard error of mean (n = 4).

³LAB, Lactic acid bacteria; LA:AC ratio, ratio of lactic acid to acetic acid; NA, not applicable; ND, not detected; NH₃-N, ammonia nitrogen; WSC, water-soluble carbohydrates.

⁴Hours after a 2 °C rise in temperature was recorded in the silage mass after exposure to air.

Aerobic Stability of Whole-Plant Corn Silage Ensiled in Mini-Silos and Silo Bags

Mini-silo study

Concentrations of AC and total VFA were greater (P ≤ 0.02) for IB than CON and IC after 14 d of AE (Table 1). Lactate concentration was greater for IB than CON and IC (P < 0.01) while the LA:AC ratio was lower for IC and IB than CON (P = 0.02) in aerobically exposed silages. The LAB counts for IC (8.46 log₁₀ cfu g⁻¹ DM) and IB (8.23 log₁₀ cfu g⁻¹ DM) were greater (P ≤ 0.01) than CON (6.41 log₁₀ cfu g⁻¹ DM) on d 3 of AE (Figure 1B). Aerobic stability did not vary among treatments during the 14 d of AE (Figure 2). Time taken to reach maximum temperature was greater and the maximum temperature reached during AE was relatively lower for IB and IC than CON.

Figure 2.

Impact of uninoculated (Control; CON) or corn silage inoculated with (cfu g⁻¹ fresh forage) LAB inoculants consisting of 1.5 × 10⁵L. hilgardii, 1.5 × 10⁵L. buchneri and 1.0 × 10⁵P. pentosaceus for a total of 4.0 × 10⁵ LAB (IB) and a combination of IB plus fibrolytic enzymes (xylanase + β-glucanase) (IC) on silage temperature during aerobic exposure (AE) of corn silages ensiled in mini-silos (n = 3).

Silo bag study

There was a T × D interaction for silage DM, pH, and yeast counts during 14 d of AE (Figure 3A–C). Silage DM remained greater (P ≤ 0.04) for IC than CON throughout AE. The silage pH of IC was lower than CON on d 3 (3.70 vs. 4.10; P < 0.01), 7 (3.97 vs. 5.82; P < 0.001), and 14 (4.79 vs. 6.62; P = 0.02) of AE. Silage pH was relatively stable for IC over the 14-d AE and was lower for IC than CON after 3, 7, and 14 d (Table 2). There was also a T × D interaction for AC and LA concentrations during AE (Figure 3D, E), with IC having higher AC concentrations than CON over the duration of AE and higher LA concentrations on d 3, 7, and 14. Yeast counts were lower for IC than CON in terminal silage (0.77 vs. 5.79 log₁₀ cfu g⁻¹ DM; P < 0.001), d 3 (2.81 vs 7.82 log₁₀ cfu g⁻¹ DM; P < 0.001) and 7 (6.15 vs 8.83 log₁₀ cfu g⁻¹ DM; P < 0.01) of AE. The AS of IC was greater (P < 0.001) than CON, with IC silages remaining near ambient temperature for a duration three times that of the CON (Figure 4).

Figure 3.

Impact of uninoculated (Control; CON) or corn silage inoculated with (cfu g⁻¹ fresh forage) LAB inoculant containing 1.5 × 10⁵L. hilgardii, 1.5 × 10⁵L. buchneri and 1.0 × 10⁵P. pentosaceus for a total of 4.0 × 10⁵ LAB plus fibrolytic enzymes (xylanase + β-glucanase) (IC) on (A) silage DM, (B) silage pH, (C) yeast counts, (D) acetate (AC) concentration, and (E) lactate (LA) concentration in terminal silages ensiled in silo bags and during aerobic exposure (AE; n = 4). (A) *Denote treatment differences in silage DM during AE. The silage DM of IC was lower than CON in terminal silage (P = 0.03), and on d 3 (P = 0.04), d 7 (P < 0.01) and d 14 (P < 0.01) of AE. (B) *Denote treatment differences in silage pH during AE. The silage pH of IC was lower than CON on d 3 (P < 0.01), d 7 (P < 0.001) and d 14 (P = 0.02) of AE. (C) *Denote treatment differences in yeast counts in terminal silage and upon AE. The yeast counts of IC were lower than CON on d 418 (P < 0.001) of ensiling and d 3 (P < 0.01) and d 7 (P < 0.01) of AE. (D) *Denote treatment differences in AC concentration during ensiling and AE. the AC concentrations were greater for IC than CON on d 418 of ensiling (P = 0.02), d 3 (P < 0.001), d 7 (P < 0.001) and d 14 (P < 0.001) of AE. (E) *Denote treatment differences in LA concentration during aerobic exposure. the LA concentrations were greater for IC than CON on day 3 (P < 0.001), d 7 (P < 0.01) and day 14 (P < 0.001) of AE.

Figure 4.

Impact of uninoculated (Control; CON) or corn silage inoculated with (cfu g⁻¹ fresh forage) LAB inoculant containing 1.5 × 10⁵L. hilgardii, 1.5 × 10⁵L. buchneri and 1.0 × 10⁵P. pentosaceus for a total of 4.0 × 10⁵ LAB plus fibrolytic enzymes (xylanase + β-glucanase) (IC) on silage temperature during aerobic exposure (AE) of corn silages ensiled in silo bags (n = 4).

Maximum temperature (T_max, °C) was lower (P < 0.001), and the time taken to reach T_max (h) was greater (P < 0.001) for IC than CON during AE (Table 3). The duration (h) for which the silage temperatures were above the temperature threshold of ambient temperature + 2 °C was lower (P ≤ 0.01) for IC than for CON when measured for d 1–3, 1–7, 8–14 and 1–14 of AE. Moreover, the area [duration (h) × temperature] under the curve when the silage temperatures were above ambient temperature + 2 °C was lower (P ≤ 0.01) for IC than CON when measured for d 1–3, 1–7, 8–14 and 1–14 of AE.

Table 3.

Temperature data of CON and IC corn silages ensiled in silo bags during 14 d of aerobic exposure (AE)

Item3	Treatments1		SEM2	P-value
	CON	IC
T max, °C	37.0	33.6	1.04	< 0.001
Time to reach Tmax, h	63.9	221.9	39.4	< 0.001
Duration, h
D 1–3	28.3	1.88	9.0	< 0.001
D 1–7	99.4	25.9	27.7	< 0.001
D 8–14	128.2	87.1	24.2	< 0.001
D 1–14	227.7	113.0	49.5	< 0.01
Area, duration (h) × temperature
D 1–3	283.7	1.66	86.8	< 0.001
D 1–7	1095.9	246.5	308.4	< 0.001
D 8–14	1329.1	787.4	381.7	0.01
D 1–14	2425.0	1034.0	684.3	< 0.001

¹Treatments included CON, uninoculated control silage, and corn silage inoculated (cfu g⁻¹ fresh forage) with LAB inoculant containing 1.5 × 10⁵Lentilactobacillus hilgardii and 1.5 × 10⁵L. buchneri and 1.0 × 10⁵P. pentosaceus for a total of 4.0 × 10⁵ LAB plus fibrolytic enzymes (xylanase + β-glucanase) (IC).

²SEM, pooled standard error of mean (n = 4).

³ T _max, maximum temperature attained during AE; time to reach T_max, time (h) taken to reach maximum temperature during AE; Duration, time (h) the treatment silages were above ambient temperature +2 °C during d 1–3, 1–7, 8–14, and 1–14 of AE; Area, area [duration (h) × temperature] under the curve when the silage temperatures were above ambient temperature + 2 °C during d 1–3, 1–7, 8–14, and 1–14 of AE.

Growth Performance of Feedlot Cattle

The nutrient composition was similar (P > 0.05) and averaged 14.2 ± 0.72 % CP, 22.7 ± 1.18 % ADF and 43.3 ± 1.24 % NDF among diets (Table 4). All steers used in the growth performance study were healthy, and no mortality or morbidity occurred during the study. The ADG, DMI, and G:F were unaffected by the silage type and averaged 1.37 ± 0.22 kg d⁻¹, 8.11 ± 0.89 kg, and 0.169 ± 0.02 respectively, across the treatments (Table 5).

Table 5.

Performance of growing beef steers fed CON and IC corn silage ensiled in silo bags

Item2	Treatments1		SEM	P-value
	CON	IC
n, animals	20	20	–	–
Initial shrunk BW3,kg	280.7	278.9	4.31	0.77
Final shrunk BW3, kg	392.9	397.6	6.65	0.62
ADG, kg	1.34	1.41	0.049	0.27
DMI, kg d−1	8.09	8.13	0.201	0.89
DMI as % BW	2.40	2.40	0.041	0.93
G:F4	0.165	0.173	0.0042	0.18
NEm, Mcal kg−¹ DM5	1.78	1.82	0.027	0.23
NEg, Mcal kg−¹ DM5	1.15	1.19	0.024	0.25

¹Treatments included control diet with uninoculated corn silage (CON), inoculated (cfu g⁻¹ fresh forage) with LAB inoculant containing 1.5 × 10⁵Lentilactobacillus hilgardii, 1.5 × 10⁵ cfu g⁻¹L. buchneri and 1.0 × 10⁵P. pentosaceus for a total of 4.0 × 10⁵ LAB plus fibrolytic enzymes (xylanase + β-glucanase) (IC).

²ADG, average daily gain; DMI, dry matter intake; G:F, gain:fed ratio, NE_m and NE_g, net energy of maintenance and gain respectively, SEM, standard error of mean.

³Shrunken BW calculated as 96% of live weight (NRC, 2000).

⁴G:F is calculated as ADG/DMI.

⁵Calculated based on performance (Zinn and Shen, 1998; Zinn et al., 2002).

DISCUSSION

Lentilactobacillus hilgardii (CNCM I-4785) was originally isolated from sugarcane (Avila et al., 2014) and is a relatively new addition to the heterofermentative LAB that are being employed as silage inoculants. The fermentation profile of L. hilgardii is similar to L. buchneri in that it increases AC concentrations and lowers yeast populations in silages and has been shown to improve the AS of sugarcane, sorghum, and corn silages (Avila et al., 2014; Carvalho et al., 2014; Ferrero et al., 2019; Gomes et al., 2021). Unlike L. buchneri which results in a pronounced increase in AC concentrations after ~60 to 90 d of ensiling, L. hilgardii has been reported to increase the AC concentrations within 2–3 wk of ensiling to levels that improve the AS of silages (Carvalho et al., 2014; Muck et al., 2018; Reis et al., 2018). Recent studies utilizing silage inoculants containing a combination of L. hilgardii with L. buchneri have reported potential synergism between these two heterofermentative species (Ferrero et al., 2019; Nair et al., 2020). Ferrero et al. (2019) reported an improvement in AS of corn silages inoculated with a combination of L. hilgardii with L. buchneri than uninoculated silage as early as d 15 of ensiling while having similar AC concentrations across treatments. These authors also reported similar or improved AS for corn silages inoculated with L. hilgardii and L. buchneri and ensiled for up to 250 days relative to that inoculated with or without the individual inoculants. However, the results were not consistent when studies were carried out in multiple crop years (Ferrero et al., 2019). In the present study, even though the treatments did not differ in AC concentrations throughout the fermentation or AE in mini-silos, the inoculant resulted in nearly a three-fold increase in AS as compared to control silage ensiled in silo bags. Moreover, a combination of homo- and hetero-fermentative LAB inoculants has been reported to reduce the number of yeasts in corn silage ensiled for up to 282 d (Kleinschmit and Kung, 2006). Though yeast counts did not differ across treatments during ensiling or AE in mini-silos, the yeast counts were lower for IC than CON ensiled in silo bags. Thus, the novel combination of both homo- and hetero-fermentative LAB and the combination of hetero-fermentative LAB strains used in the present study has the potential for rapid fermentation and improved AS over a wide range of storage periods ranging from a few weeks to over a year of ensiling.

The LAB strains used in the present study lack fibrolytic activity. The inclusion of a cocktail of fibrolytic enzymes in silage inoculants is one approach to increase the availability of WSC for fermentation to LA and to potentially improve ruminal fiber digestibility. However, the impacts of a combination of LAB silage inoculants and fibrolytic enzymes on silage fermentation, AS, and beef cattle performance have been inconclusive. Zahiroddini et al. (2004) reported improvement in feed efficiency of steers fed barley silage inoculated with a combination of exogenous fibrolytic enzymes and strains of Pediococcus, Lactobacillus and Enterococcus spp. In contrast, Schaefer et al. (1989) reported no effect of such a combination on the growth performance of growing steers fed corn silage. Factors including the strains of LAB used, nature of the enzyme cocktail, type of forage ensiled, ensiling process, and the duration of ensiling can have significant impacts on the fermentation profile, AS, and feed value of inoculated silages (Chen et al., 1994; Muck et al., 2018). For example, Chen et al. (1994) reported numerically lower ADF and NDF and greater hemicellulose concentrations for hay-crop silages (containing timothy, alfalfa, ladino clover, and arlington red clover) inoculated with an enzyme-inoculant mixture containing xylanase and LAB strains as compared to uninoculated forage. However, the ADF and NDF concentrations were numerically greater, and hemicellulose concentrations were significantly greater for inoculated corn silage treated with a similar enzyme-inoculant mixture as compared to uninoculated corn silage. These authors also reported that treatment of hay-crop silage with an enzyme-inoculant mixture increased the rapidly degradable NDF and the rate of degradation of slowly degradable NDF fractions while significantly reducing the slowly degradable NDF constituents. In contrast, there was no effect of enzyme-inoculant treatment on NDF degradation parameters of corn silage (Chen et al., 1994). These results indicate that the enzyme-inoculant mixture may have an impact on fiber degradability in the rumen depending on the extent of change in cell wall components. Moreover, extended ensiling periods has been reported to increase starch digestibility while having minimal impact on NDF digestibility (Ferraretto, 2016).

Effects of Inoculants on Ensiling Fermentation and AS of Whole-Plant Corn Silages

The average DM content of corn forage at the time of ensiling in the present study was slightly greater (43.1 ± 1.65%; mean ± SD) than the recommended range (32.0–38.0%) for ensiling (Guyader et al., 2021) as the corn forage was harvested after the first killing frost. Guyader et al. (2021) reported that delaying harvest until after freezing would increase the DM content of whole-plant corn as a plant cell lysis and subsequent desiccation. However, the greater DM content of corn forage at the time of ensiling did not negatively impact the ensiling fermentation as the silage pH in mini-silos decreased from 5.45 ± 0.12 to 4.01 ± 0.05 by d 7 and 3.89 ± 0.06 by d 14 of ensiling in mini-silos and 3.69 ± 0.05 after 418 days of ensiling in silo bags.

The decrease in silage pH is brought about by the metabolic activity of epiphytic microbes and the inoculated LAB, utilizing the WSC as a substrate during ensiling. The average WSC concentration of 133.8 ± 20.5 mg g⁻¹ DM across treatments at the time of ensiling is greater than that reported in similar studies for corn forage in our laboratory (Nair et al., 2019b, 2020). Guyader et al. (2021) reported that severe frost might inhibit the translocation of sugars to the ears and the conversion of sugars to starch in the kernels, preventing kernel filling. Starch content of corn forage in the present study (21.0 ± 1.24; % DM basis) was lower (23.7 ± 2.8; % DM basis) than that reported by Nair et al. (2020) and significantly lower than the starch content (32.7 ± 3.05; % DM basis) of corn silages typically reported in western Canada or the mid-west United States (Chibisa and Beauchemin, 2018; Hilscher et al., 2019). It is logical to assume that the killing frost prevented the conversion of WSC to starch in the kernels in the present study, resulting in relatively greater WSC content and lower starch content in corn forage at the time of ensiling.

The concentration of residual WSC in terminal silages (90 d of ensiling) averaged 48.6 ± 16.8 mg g⁻¹ DM across treatments in mini-silos. It should also be noted that residual WSC concentration of CON silage in mini-silos was nearly double the concentration in silo bags (62.2 vs. 30.3 mg g⁻¹ DM) whereas the concentration of WSC of IC in mini-silos was nearly 1.5-fold greater than in silo bags (47.0 vs. 30.3 mg g⁻¹ DM). As reported previously, silages in mini-silos were ensiled for 90 d whereas silages in silo bags were ensiled for 418 d. Moreover, the ensiling environment in mini-silos is more homogeneous than that achieved in commercial scale bag silos (Duniere et al., 2017). It is logical to assume that prolonged ensiling and less than ideal ensiling conditions in silo bags likely resulted in greater utilization of WSC, resulting in lower residual WSC in bagged silage.

The concentration of AC was similar among treatments and averaged 10.1 ± 1.49 mg g⁻¹ DM in terminal silage ensiled in mini-silos. Although TB and LAB counts were higher for IC and IB than CON in terminal silages, similar fermentation profiles across treatments indicate that the ensiling process proceeded through similar pathways across silage treatments in mini-silos. Moreover, similar residual WSC and AC concentrations in terminal silages resulted in similar AS of treatments ensiled in mini-silos. Conversely, though relatively lower than the AC concentrations in similar studies (Nair et al., 2019b, 2020), IC had greater AC concentration and lower yeast counts than CON in terminal silage ensiled in silo bags. It should be noted that the mini-silos were ensiled for 90 d whereas the silo bags were opened after 418 d of ensiling. It is logical to assume that the conversion of LA to AC during storage by hetero-fermentative LAB strains such as those used in the present study could add to the AC concentrations. It is also worth noticing that the LA concentrations of terminal silages were numerically lower for silages ensiled in silo bags than mini-silos across treatments.

The results showed that yeast and mold count in IC silage were less than the threshold (1 × 10⁵ g⁻¹ of silage) typically associated with silage spoilage (Pahlow and Zimmer, 1985; O’Kiely et al., 1987). It has been commonly recognized that upon AE, growth of yeast in terminal silage is the key factor contributing to silage instability, with AC inhibiting yeast growth. Therefore, the greater AC concentration and lower yeast counts in IC, together with lower residual WSC (which is the essential substrate for yeast growth), likely contributed to the greater AS of IC compared to CON silage. Nair et al. (2019a) also reported that the end products of fermentation, including AC, LA, and residual WSC concentrations in terminal silages had a significant impact on the AS of corn silage. Further studies to determine the effect of inoculants on the microbiome of silage could further elucidate the mechanisms whereby inoculants improve AS.

T _max was the highest temperature recorded during the AE and was relatively lower for IC than for CON. Similarly, the time required to attain T_max was greater for IC than for CON. Corresponding to these observations, the duration (h) for which the silage temperature remained above ambient temperature + 2 °C and the area under the curve for temperatures above ambient temperature + 2 °C were also lower for IC than for CON silage during AE. A decrease in the DM content of CON from 39.6% in terminal silage to 36.1% after 14 d of AE in the present study likely indicates DM losses due to spoilage, as indicated by the greater yeast concentrations throughout AE. As the losses of the nutritive and feeding value of the silage during feed-out may be more significant than that during ensiling (Borreani et al., 2018), even if animal performance were not significantly improved with IC, having more nutritionally uncompromised silage available as feed would still contribute to overall farm profitability.

It is important to note that the silage used in this study was ensiled for more than 400 d because of the restriction imposed by the COVID-19 pandemic. This long ensiling duration would not be a common practice in the industry, and there are few studies that have investigated the implications of long-term ensiling. Arriola et al. (2011) investigated the effect of inoculants with hetero-lactic or homo-lactic and hetero-lactic bacteria on the fermentation and quality of corn silage in a 575-d mini-silo experiment. Unfortunately, only terminal silage was used in that study, and therefore, it was not possible to define the effects of the extended ensiling length on the efficacy of inoculants on ensiling. However, it has often been proposed that silage remains stable almost indefinitely if low pH and anaerobic conditions are maintained. Our results using mini-silos showed that application of the inoculants and ensiling for 90-d resulted in a stable silage pH of about 3.85 between d 20 and 60, a value similar to that measured in bagged silage after 418 d of ensiling. The similar pH value between d 20 and 60 for whole-plant corn silage ensiled in mini-silos was also reported in Nair et al. (2019a, 2019b) and in our previous publications with other small-cereal crop silages. These findings suggest that silages with an extended ensiling period maintain properties that are similar to silages that are ensiled for more typical shorter durations.

Effects of Inoculant on Growth Performance of Feedlot Steers

Steers fed CON or IC diets did not differ in any of the growth performance parameters measured, reflecting the similar nutrient composition of diets across treatments. Both diets contained > 14% CP, thereby meeting the CP requirement for growing steers for the weight and growth rate in the present study. Moreover, even though there was a tendency for greater NDF content for IC than CON silage, and similar tendencies for ADF and NDF content in the diets, the DMI or DMI as a % of BW of steers fed IC or CON diets did not differ. Feeding inoculated silage with enhanced AS did not result in improved animal growth performance compared to the uninoculated silage, likely because consumed silages were not subject to 3-d of AE, a duration for which CON and IC silage remained stable. Aerobic spoilage of silages, in addition to the loss of DM and nutrients, can result in lower palatability and depress feed intake in cattle (Kung, 2010; Borreani et al., 2018). Moreover, the proliferation of pathogenic and/or undesirable microorganisms during spoilage can impact animal health and performance (Borreani et al., 2018). Both CON and IC were aerobically stable at the time of feeding during the growth performance study as evidenced by similar DMI and DMI as a % of BW across treatments. Uninoculated CON silage was aerobically stable for slightly more than 3 d. It should also be noted that silo bags have less exposed silo face than that would be typical for bunker silos or silage piles. Moreover, the rate of removal of silage from the silo face in the present study was about 30 cm d⁻¹ (data not shown) which was more than the rate of removal required for preventing the aerobic spoilage of silages during feed-out (Muck et al., 2003; Heguy et al., 2016). As ensiling conditions in commercial scale silos are less strictly controlled due to larger silo face, slower silage removal rate, and longer duration of AE, it is logical to predict that IC could result in higher amounts of edible silage DM due to the expected better control of spoilage. It may ultimately offer greater flexibility to the producer for his bunker’s face management with good quality forage all along the feed-out period.

CONCLUSIONS

Whole-plant corn ensiled with inoculant containing homo- and hetero-fermentative LAB and fibrolytic enzymes for 418 d had greater concentrations of AC and total VFA, but lower concentrations of WSC, ethanol, and yeast and lower LA:AC ratio than uninoculated silage. Inoculant greatly improved AS of corn silage. Longer periods of ensiling did not negatively impact the nutrient composition, microbial populations in terms of total bacteria, LAB, yeast, and mold or AS of whole-plant corn silage ensiled in silo bags. Steers fed silages did not vary in growth performance as CON and IC silages exhibited similar composition. Further metabolism studies evaluating the ruminal and total tract nutrient digestibilities would provide detailed information on the impact of addition of fibrolytic enzymes in silage inoculants on forage cell wall digestibility and potential improvement in feed value. Potential benefits of feeding IC with greater AS would likely be more prominent in large commercial feeding operations with prolonged AE of silage at the silo face if slower silage removal rates were practiced.

Glossary

Abbreviations

AC

acetic acid

ADICP

acid detergent insoluble crude protein

AE

aerobic exposure

CON

control

G:F

feed efficiency

IB

bacterial inoculant

IC

inoculant complex

LA

lactic acid

LAB

lactic acid bacteria

LeRDC

Lethbridge Research and Development Centre

MRS

de Man, Rogosa, and Sharpe agar

NA

nutrient agar

NASEM

National Academies of Sciences, Engineering, and Medicine

NDICP

neutral detergent insoluble crude protein

SDA

Sabouraud’s dextrose agar

TB

total bacteria

TMR

total mixed ration

WSC

water-soluble carbohydrate

Conflict of Interest

The authors declare that there are no conflicts of interest.