Impact of Inoculation with Pediococcus pentosaceus in Combination with Chitinase on Bale Core Temperature, Nutrient Composition, Microbial Ecology, and Ruminal Digestion of High-Moisture Alfalfa Hay ^†

Abstract

This study evaluated the effects of Pediococcus pentosaceus (PP) and chitinase combinations on the conservation and nutritive value of alfalfa high-moisture hay (HMH). P. pentosaceus [10¹² colony forming unit/g fresh forage] combined with (g/tonne of fresh forage) 1.5 (PP + LC), 7.5 (PP + MC), or 15.0 (PP + HC) g of LANiHay01 chitinase (Exp. 1) or with LANiHay02 (PP + Fe; 1.5 g), LANiHay01 (PP + Pe; 1.5 g), or Sigma (PP + Si; 55 mg) chitinase/tonne (Exp. 2) were used in 2016. In 2017, PP was applied alone or in combination with LANiHay01 at 1.5 g (PP + LC) or 7.5 g (PP + MC) chitinase/tonne (Exp. 3 and 4). Deionized water and propionic acid (4.0 L/tonne of fresh forage in Exp. 1 and 2 and at 6.0 L/tonne of fresh forage in Exp. 3 and 4) were applied as neutral (CON) and positive control (CON+), respectively. The maximum temperature (r² = 0.66) and NH₃-N concentration (r² = 0.80) of the HMH were positively related to total microbial populations. PP + MC had lower (p ≤ 0.05) yeast and mold counts than CON in Exp. 3 and 4 while the neutral detergent fiber degradability was greater (p < 0.01) for PP + MC and PP + LC than CON in Exp. 1 and 3, respectively. P. pentosaceus in combination with chitinase has the potential in conserving the nutrient quality of alfalfa HMH.

1. Introduction

The haying process is intrinsically dependent on local weather conditions at the time of harvest. Wilting forages after harvest to a moisture level of 16–18% [1,2] is essential to minimizing storage losses due to the growth of spoilage microorganisms and associated heat. However, in the last few decades, extreme weather events have made haying on the North American prairies more challenging [3]. Excessive precipitation in the spring and unpredictable rain events throughout the forage-growing season have promoted farmers to adopt alternative practices to preserve high-moisture hay (HMH). As the haying process is significantly dependent on weather conditions, baling HMH grants more flexibility to the farmers when the target DM becomes difficult or impossible to reach without losing the value of the forage stock due to unexpected weather events. Further, haying at higher moisture levels will keep more leaves at baling, thereby reducing the nutritional losses on the ground during tedding and raking of conventional normal-moisture hay production. One of the major factors impacting the quality of alfalfa hay is mold growth, especially when the moisture content of forages at haying is greater than the recommended levels of 12–18%. Organic acids such as formic and propionic acid (PA) have been used to preserve the quality of HMH with varying degrees of success [4]. However, their corrosive nature, high costs, and inconsistent efficacy have made producers reluctant to use organic acids as HMH preservatives [5]. Bacterial inoculants are widely used to enhance the preservation of silages [6,7], but their efficacy in improving the preservation of HMH has not been widely investigated.

Chitin is a common constituent of fungal cell walls [8]. Chitinolytic enzymes such as chitinase have potential applications in the biocontrol of fungi in forage preservation [8]. A recent evaluation of inoculation of alfalfa HMH with a combination of Pediococcus pentosaceus NCIMB 12674 and chitinase (LANiHay01, >6 units of chitinase activity/g) reduced the number of days where the core temperature of hay bales exceeded 30 °C [9]. The combination of P. pentosaceus and chitinase also reduced spoilage and improved neutral detergent fiber (NDF) degradation in vitro. It was hypothesized that refinement of the source and concentration of chitinase in combination with P. pentosaceus would further improve the preservation and NDF digestibility of alfalfa HMH. The objective of this study was to evaluate the efficacy of P. pentosaceus with different sources and concentrations of chitinase on the nutritional and microbiological characteristics and the NDF digestibility of alfalfa HMH preserved in large round bales.

2. Materials and Methods

2.1. Treatments and Experimental Design

A total of four experiments were conducted between 2016 and 2017, where Exp. 1 and Exp. 2 were carried out in 2016, and Exp. 3 and Exp. 4 in 2017. The first experiment (Exp. 1) conducted in 2016 evaluated inoculants containing P. pentosaceus NCIMB 12674 [PP; 10¹² colony-forming unit (cfu)/g)] in combination with three concentrations [(LANiHay01, Lyven, France) at low 1.5 (PP + LC), medium 7.5 (PP + MC), or high 15 (PP + HC) g] of chitinase (>6 units of chitinase activity/g)/tonne fresh first-cut alfalfa, while the second experiment (Exp. 2) assessed P. pentosaceus (10¹² cfu/tonne) in combination with three different chitinases sources at 1.5 g LANiHay02 (Lyven, France; PP + Fe), 1.5 g LANiHay01 (Bio-Cat, Troy, VA, USA, PP + Pe), or 55 mg Sigma pure chitinase (>200 units of chitinase activity/g) from Streptomyces griseus (Sigma Aldrich, St. Louis, MO, USA, PP + Si)/tonne of second-cut alfalfa. Experiments conducted in 2017 assessed P. pentosaceus (10¹² cfu/tonne) alone (PP) or in combination with chitinase (LANiHay01, Bio-Cat, Troy, VA, USA) at 1.5 (PP + LC) or 7.5 (PP + MC) g/tonne first-cut (Exp. 3) or second-cut (Exp. 4) fresh alfalfa forage. Deionized water and PA (68% solution; w/w, Wausau Chemical Corporation, Wausau, WI, USA) were also applied to HMH in all experiments as neutral (CON) and positive controls (CON+), respectively. The PA solution was applied at 4.0 L/tonne of fresh forage in Exp. 1 and 2 and at 6.0 L/tonne of fresh forage in Exp. 3 and 4. Thus, there were five treatments in each experiment. The additives were applied according to the manufacturer’s recommendations. The first cut of alfalfa used in Exp. 1 and 3 was harvested at the early- to mid-bloom stage of the spring growth in mid-June, whereas the second cut of the regrowth used for Exp. 2 and 4 was harvested at early- to mid-bloom in late July from the same fields.

2.2. Forage Production and Harvesting

Alfalfa forage for all experiments was grown near the Agriculture and Agri-Food Canada Lethbridge Research and Development Centre (LeRDC), located at 49.69° N, −112.76° W. Forage production and processing methods for all experiments were similar to that reported previously [9]. Briefly, alfalfa was grown in a non-irrigated silt loam (fine-loamy, mixed, frigid, Typic Ochraqualfs) soil type and harvested using a New Holland Discbine^® 209 (CNH Industrial NV, Amsterdam, The Netherlands) fitted with a rubber chevron roller conditioner. The harvested forage was left in the field in windrows for 2–3 days until the moisture content reached between 23% and 27%. During this period, the harvested forage in each windrow was raked once to achieve uniform moisture distribution. For Exp. 1, 3, and 4, alfalfa hay was sun-dried in the field for 3 days prior to baling without any rainfall, but in Exp. 2, alfalfa remained in the field for 7 days and was subjected to five rain showers (~36 mm) prior to baling. The moisture content of hay was monitored frequently using a portable Farmex hay moisture tester (Aurora, OH, USA) throughout the curing stage. High-moisture hay was baled when the moisture content of harvested forage in the windrows ranged from 23% to 27%. Efforts were made to minimize the variability in forage characteristics due to field location by randomly baling the windrows for each treatment.

2.3. Experimental Procedure

Prior to baling, ingredients for each treatment were mixed with deionized water for 5 min in separate 20-L tanks. A sprayer tank and spray boom with a single T-Jet nozzle were mounted onto a round baler (New Holland BR7900, CNH Industrial NV, Amsterdam, The Netherlands) with net wrapping, as described previously [10]. The spray boom was positioned to cover 90% of the windrow with additives sprayed onto the forage just before it entered the baler chamber. Calculations were made to determine the application rate based on weight and the time for bale formation to achieve an application rate of 1.0 L of deionized water or additives/tonne of forage for CON or treatments and 4.0 L/tonne of PA for Exp. 1 and 2 or 6.0 L/tonne of PA for Exp. 3 and 4. Five bales of approximately 550–600 kg (1.3 × 1.3 m diameter) were made for each treatment in all experiments, except for PP + Si in Exp. 2, where only three bales were made. The tank and sprayer were thoroughly washed with deionized water, and an extra bale was made between each treatment to avoid carryover effects between treatments.

Moisture levels were monitored for both unbaled hay during baling and each bale after baling. Each bale was labeled immediately following production and cored (core size 4.5 × 54 cm; two samples per bale) to obtain a sample from the convex side of the bale. Two Dallas Thermochron iButtons temperature sensors (Embedded Data Systems, Lawrenceburg, KY, USA) were placed into the center of the bale through the core holes (one for each), and the holes were sealed with alfalfa hay of similar moisture and covered with tape. The iButtons recorded temperature at 4-h intervals for the initial 60 d of storage. Bales were removed from the field after baling and stored at least 1.0 m apart in an open shed for 120 d. Each bale was core-sampled from both sides at 30, 60, 90, and 120 d.

2.4. Laboratory Analyses

Collected samples were divided into two upon return to the laboratory. One portion was freeze-dried and ground to pass through a 4.0 mm screen using a Wiley Mill (Model 4, Arthur H. Thomas Co., Chadds Ford, PA, USA) for in situ ruminal digestion. The sample was further ground through a 1.0 mm screen for chemical analysis. The second portion was processed immediately for microbial characterization as well as for analysis of fermentation products.

2.4.1. Chemical Analyses

Forage samples were analyzed for DM, organic matter (OM), NDF, acid detergent fiber (ADF), and acid detergent insoluble nitrogen (ADIN), as described previously [9] and following the procedures of the Association of Official Analytical Chemists [11]. Further, forage pH, concentrations of ammonia-N (NH₃-N), and water-soluble carbohydrates (WSC, i.e., glucose equivalents) were also analyzed [9]. Nitrogen was determined by a flash combustion analyzer (Carlo Erba NA 1500, Milan, Italy) equipped with gas chromatography and thermal conductivity detection.

2.4.2. Microbial Characterization

Microbial analyses were carried out [9] using semi-selective nutrient agar (NA; Difco, Detroit, MI, USA) amended with 200 µg/mL cycloheximide for the enumeration of culturable total bacteria (TB) and Sabouraud’s dextrose agar (SDA, Difco, Detroit, MI, USA) containing 100 µg/mL each of tetracycline and chloramphenicol for the enumeration of yeasts and mold, respectively. Plates with colonies in the range of 30–300 were counted.

2.4.3. Determination of Ruminal Digestion of Preserved Forages

Forage samples collected after 120 d of storage were used to evaluate their DM and NDF degradation in the rumen using the in situ procedure. Three ruminally cannulated, non-lactating Angus cows were fed a mixed ration comprising (DM basis) 50% barley silage, 35% alfalfa hay, 12% dry-rolled barley, and 3% standard beef supplement (vitamin premix and mineral). The cows were adapted to the diet for 2 weeks before in situ incubations. All cows used for the in situ study were cared for in accordance with standards established by the Canadian Council on Animal Care [12], and the study was reviewed and approved by the LeRDC Animal Care Committee (Protocol # 1703).

Ground samples (4.0 mm) were pre-loaded (4.0 g) into nylon bags (5 cm × 20 cm, 53-µm pore size, Ankom, Fairport, NY, USA) and heat sealed. Triplicate nylon bags per sample were incubated in the rumen of each cow for 0, 1, 2, 4, 8, 12, 24, 36, 48, and 72 h. Ruminal incubation and the subsequent processing for DM and NDF determination were carried out as previously described [13]. After the in situ incubation, the bags were removed and rinsed in cold water until the rinse water was clear. All bags, including the 0 h bags (bags not incubated), were placed in an automatic washer and washed in cold water for three, 10-min washing cycles. The DM content of the residues was determined after drying at 55 °C for 48 h to calculate the DM degradation. The residues in triplicate bags were combined for NDF analysis using the Ankom200 fiber analyzer to determine NDF degradability.

2.5. Data Collection and Statistical Analysis

The daily temperature recorded by the two iButtons in each bale over the first 60 d of storage was averaged, and high-degree days (HDD) were defined as those days where the average temperature was > 30 °C (HDD30). In addition, average internal temperature (AT) was also calculated as the mean daily internal temperature over the entire 60-d period for each bale. The maximum temperature (Tmax) was defined as the highest internal temperature a bale reached during the first 60-d storage. Microbial populations were expressed on cfu/g of DM basis and were transformed to log₁₀ cfu/g DM prior to statistical analysis. Ruminal DM and NDF disappearances at each sampling point were determined gravimetrically between DM and NDF content pre- and post-ruminal incubation. The degradation parameters of DM were calculated using the equation [14]:

PD = a + b (1 − e ^−c(t−L))

where PD = DM disappeared (proportion) at time t, a = the rapidly degradable fraction (proportion), b = the slowly degradable fraction (proportion), c = the rate at which b is degraded (/h), t = incubation time points (h), and L = lag time (h). The effective degradability (ED) of DM and NDF was calculated following the equation:

ED = a + [bc/(c + k)] e^−(c+k)L

with a, b, c, and L as described above, and k = the ruminal outflow rate (5%/h). The parameters a, b, c, L, and ED were estimated using an iterative least squares procedure using NLIN [15]. The NDF disappearances at 2, 12, 24, 48, and 72 h of ruminal incubation were also compared.

Data in each experiment were statistically analyzed separately by the analysis of variance as a completely randomized design using the PROC MIXED procedure of SAS [15] with treatment as a fixed effect and individual bales as a random effect. Chemical and microbial composition data that were collected at different time points from the same bale were initially analyzed using repeated measures by including storage time and the time × treatment interaction in the model. Because sampling and/or time × treatment interaction effects were found for most of these measurements, all the data were compared at each time point using d 0 data as a covariate.

Multiple-factor regression was performed using the PROC REG procedure of SAS to establish the relationship between bale moisture levels and ambient temperature adjusted bale temperature, microbial population, and maximum bale temperature during 60-d storage, d 0 microbial population and maximum temperature, ammonia-N concentrations and total microbial population during the 120-d storage, microbial population and WSC concentrations during 60 and 120 d of storage, Tmax and NDF, ADFIN, and Tmax and NDF content and effective NDF degradability.

Data from in situ determination were also analyzed using the PROC MIXED procedure of SAS with treatment as a fixed effect and cow and individual bales as random effects. Simple regressions between the relevant variables were conducted, and relationships were plotted using SigmaPlot V15 (Systat Software, Inc., Richmond, CA, USA). Differences among treatments were tested using LSMEANS with Tukey’s method in SAS, with significance declared at p < 0.05 and tendencies at 0.05 < p < 0.10.

3. Results

3.1. Alfalfa HMH

The average DM contents of the alfalfa HMH at baling were 70.8 ± 1.53, 71.7 ± 2.43, 81.7 ± 2.89, and 76.5 ± 2.02% for Exp. 1, 2, 3, and 4, respectively (

Table 1. Dry matter (DM; %), pH, ammonia N content (NH₃-N; mg/g N), and bale temperature of first-cut (Exp. 1) and second-cut (Exp. 2) alfalfa baled above the optimum moisture level without or with the application of various inoculants and stored for 120 d in 2016.

		Exp. 1							Exp. 2
		Treatments							Treatments
Item	Storage Days	CON	CON+	PP + LC	PP + MC	PP + HC	SEM	p-Value	CON	CON+	PP + Fe	PP + Pe	PP + Si	SEM	p-Value
DM	0	72.8	69.1	70.3	72.0	69.9	1.19	0.41	69.7	72.3	70.1	70.9	75.7	3.54	0.71
	30	81.3 a	77.6 b	81.2 b	81.8 b	81.4 b	0.78	0.01	73.5	72.9	76.7	77.3	77.9	2.09	0.16
	60	88.2	86.9	88.4	88.6	89.0	0.72	0.34	90.4	88.4	90.3	88.5	88.6	1.17	0.30
	90	89.7	89.0	89.7	88.8	89.9	0.55	0.51	88.7	89.0	90.1	90.5	90.3	1.45	0.72
	120	89.4	89.3	89.6	90.0	89.6	0.90	0.98	89.5	90.0	89.9	90.2	90.1	0.573	0.85
pH	0	6.20 b	6.08 c	6.26 ab	6.24 ab	6.29 a	0.023	<0.001	7.28 ab	6.66 c	7.44 a	7.34 ab	7.08 b	0.133	<0.001
	30	5.68	5.49	5.80	5.72	5.79	0.077	0.32	7.63	7.45	7.19	7.27	7.27	0.322	0.64
	60	5.96	5.93	5.93	5.85	5.85	0.114	0.93	7.35 b	8.32 a	7.06 b	7.13 b	7.24 b	0.245	0.02
	90	6.00	6.25	6.11	5.84	5.93	0.195	0.39	7.39 ab	7.92 a	6.93 b	7.03 b	7.24 ab	0.286	0.17
	120	6.04	6.20	6.14	5.99	6.12	0.179	0.74	7.36	7.38	7.09	7.13	7.20	0.224	0.70
NH3-N	0	8.9 b	14.5 a	8.8 b	9.1 b	9.3 b	0.77	<0.001	19.8 b	37.0 a	22.9 b	18.3 b	19.0 b	2.54	<0.001
	30	23.3	28.9	32.5	24.6	28.6	3.23	0.31	49.5	52.0	52.4	46.6	57.9	5.16	0.53
	60	29.1	30.5	34.0	28.7	29.1	5.20	0.95	39.6 ab	48.6 a	42.1 ab	44.4 ab	34.8 b	3.12	0.02
	90	26.1	36.1	32.7	24.5	28.1	3.59	0.17	38.7	47.3	48.5	38.5	42.6	4.17	0.14
	120	26.5	34.2	30.3	32.1	27.1	4.11	0.65	40.2	51.6	32.9	35.4	32.8	6.50	0.10
WSC	0	8.6	10.4	8.2	9.3	13.6	1.59	0.12	5.2 a	5.4 a	3.2 b	5.0 ab	4.8 ab	0.70	0.02
	30	13.0 ab	15.2 a	11.2 ab	9.1 b	11.0 ab	1.35	0.05	2.6 b	4.3 a	3.7 ab	4.0 a	5.2 a	0.44	0.00
	60	5.6	8.6	6.1	11.5	11.3	2.59	0.13	4.2	4.8	2.6	4.1	5.0	1.05	0.33
	90	8.5	6.2	5.9	9.7	11.1	2.58	0.39	5.5	6.1	4.8	5.8	4.8	0.89	0.70
	120	6.2	7.9	5.4	6.3	5.3	1.3	0.58	5.6	7.2	4.7	5.8	6.2	0.95	0.24
Temperature
Tmax	-	57.0	61.8	57.9	61.0	58.9	2.17	0.49	55.3 ab	53.8 b	60.5 a	59.7 a	52.2 b	2.45	0.04
AT	-	40.2	40.2	40.2	39.8	39.1	3.01	1.00	21.8	23.4	24.1	25.5	22.7	1.39	0.22
HDD30	-	53.3	50.2	58.7	50.0	49.6	6.18	0.79	18.3	19.1	18.8	21.7	18.9	2.72	0.79

Treatments included CON; uninoculated alfalfa high-moisture hay (HMH) control, CON+; positive control alfalfa HMH treated with propionic acid (4.0 L/tonne fresh forage), PP + LC; alfalfa HMH inoculated with 10¹² colony-forming unit (cfu) Pediococcus pentosaceus (NCIMB 12674) + 1.5 g LANiHay02/tonne fresh forage, PP + MC; 10¹² cfu P. pentosaceus + 7.5 g LANiHay02/tonne fresh forage, PP + HC; 10¹² cfu P. pentosaceus + 15 g LANiHay02/tonne fresh forage (Exp. 1), PP + Fe; 10¹² cfu P. pentosaceus + 1.5 g LANiHay02/tonne fresh forage, PP + Pe; 10¹² cfu P. pentosaceus + 1.5 g LANiHay01/tonne fresh forage, and PP + Si; 10¹² cfu P. pentosaceus + 1.5 g Sigma pure chitinase/tonne fresh forage (Exp. 2). DM; dry matter, NH₃-N; ammonia-nitrogen, WSC, water-soluble carbohydrates, Tmax; maximum temperature, AT, average internal temperature, HDD30; days of average temperature > 30 °C. SEM; standard error of mean. ^a–c Means in rows with different superscripts differ significantly at p < 0.05.

and

Table 2. Dry matter (DM; %), pH, ammonia N content (NH₃-N; mg/g N), and bale temperature of first-cut (Exp. 3) and second-cut (Exp. 4) alfalfa baled above the optimum moisture level without or with the application of various inoculants and stored for 120 d in 2017.

		Exp. 3							Exp. 4
		Treatments							Treatments
Item	Storage Days	CON	CON+	PP	PP + LC	PP + MC	SEM	p-Value	CON	CON+	PP	PP + LC	PP + MC	SEM	p-Value
DM	0	80.4	86.1	78.5	81.5	82.2	1.95	0.13	78.0	76.7	76.6	76.0	75.0	1.02	0.38
	30	89.9	90.5	89.4	90.9	91.0	0.93	0.72	86.7 a	81.6 b	85.1 a	85.0 a	86.5 a	0.70	<0.001
	60	93.3	92.4	92.9	94.4	93.5	0.61	0.27	90.8	89.6	90.7	90.2	91.6	0.78	0.47
	90	93.6	94.7	92.7	93.9	94.0	0.57	0.23	92.4	90.7	91.0	90.4	91.8	0.62	0.21
	120	92.9	93.1	92.3	93.0	92.8	0.36	0.57	91.6	91.0	91.1	92.0	92.0	0.47	0.42
pH	0	6.17 a	5.94 b	6.22 a	6.24 a	6.24 a	0.022	<0.001	6.22	6.10	6.18	6.21	6.20	0.037	0.18
	30	5.86	5.75	5.87	5.78	5.86	0.045	0.22	5.65	5.51	5.50	5.49	5.64	0.048	0.05
	60	5.82 ab	5.69 b	5.96 a	5.77 ab	5.86 ab	0.053	0.03	5.69	5.71	5.74	5.63	5.76	0.049	0.42
	90	5.81	5.72	5.84	5.74	5.80	0.045	0.31	5.75	5.71	5.80	5.69	5.85	0.07	0.51
	120	5.82	5.74	5.84	5.78	5.90	0.041	0.11	5.70	5.74	5.78	5.74	5.84	0.058	0.54
NH3-N	0	7.8 bc	17.4 a	9.0 b	4.8 c	5.5 c	0.83	<0.001	9.4 b	18.3 a	10.7 b	13.6 ab	10.9 ab	1.78	0.02
	30	17.0	18.6	13.3	9.7	10.4	3.07	0.13	16.0 b	24.5 a	20.7 ab	15.1 b	17.7 b	1.70	0.00
	60	12.6	18.7	16.5	8.4	11.3	3.22	0.14	16.6	22.5	23.3	18.3	23.5	2.29	0.09
	90	15.6	17.2	19.3	11.0	12.2	3.36	0.40	15.3 b	23.0 a	20.2 ab	17.5 ab	18.8 ab	1.74	0.03
	120	12.3	23.5	15.3	12.4	11.2	3.26	0.10	17.4	20.8	18.9	17.8	16.5	3.08	0.86
WSC	0	9.4	9.8	8.3	10.8	7.7	0.94	0.19	16.1	18.7	20.0	19.9	18.0	1.32	0.24
	30	20.9	17.4	13.7	13.3	14.1	2.15	0.10	24.4 b	36.7 a	21.8 b	21.5 b	25.1 b	2.45	0.00
	60	20.4	17.7	16.2	16.9	18.8	1.82	0.51	24.6 ab	34.1 a	19.1 b	17.2 b	21.9 ab	3.08	0.01
	90	16.9	19.5	19.7	18.5	18.4	1.67	0.78	26.1 ab	35.2 a	19.7 b	17.1 b	21.5 ab	3.52	0.01
	120	18.1	20.4	16.3	16.2	18.9	1.41	0.22	18.6	18.2	17.0	15.9	14.7	2.73	0.85
Temperature
Tmax	-	46.1	45.5	43.8	44.7	44.2	1.20	0.59	53.1	47.9	53.4	52.2	53.6	2.23	0.37
AT	-	32.6	26.4	29.8	29.0	28.0	1.75	0.14	38.5	35.8	39.5	37.6	38.6	2.04	0.75
HDD30	-	24.6	12.0	22.4	17.4	19.3	4.41	0.28	51.6	50.6	53.6	49.6	50.6	3.11	0.91

Treatments included CON; uninoculated alfalfa high-moisture hay (HMH) control, CON+; positive control alfalfa HMH treated with propionic acid (6.0 L/tonne fresh forage), PP + LC; alfalfa HMH inoculated with 10¹² colony-forming unit (cfu) Pediococcus pentosaceus (NCIMB 12674) + 1.5 g LANiHay02/tonne fresh forage, PP + MC; 10¹² cfu P. pentosaceus + 7.5 g LANiHay02/tonne fresh forage, PP + HC; 10¹² cfu P. pentosaceus + 15 g LANiHay02/tonne fresh forage using first-cut (Exp. 3) and second-cut alfalfa HMH (Exp. 4). DM; dry matter, NH₃-N; ammonia-nitrogen, WSC, water-soluble carbohydrates, Tmax; maximum temperature, AT, average internal temperature, HDD30; days of average temperature > 30 °C; SEM; standard error of mean. ^a–c Means in rows with different superscripts differ significantly at p < 0.05.

). The DM content of all bales increased over the 120-d storage and reached about 90% after 60-d storage.

3.2. Hay Bale Temperatures

Temperatures of the hay bales over the initial 60 d varied between years and among experiments within the year (Table 1 and Table 2 and Figure 1 and Figure 2). Cycles of heating and cooling occurred several times after baling in all experiments, resulting in several heat peaks during the initial 60 d (Figure 1 and Figure 2). The first heat peak appeared after 2–3 d, except for Exp. 1, which occurred 7 d after baling. Generally, the highest heat peak occurred between 10 and 14 d after baling, with the temperature declining thereafter. Overall, the temperature patterns in bales across treatments in each experiment were similar, with the exception of CON+ in Exp. 3 and 4, where the temperature in the first 40 d post-baling was noticeably lower than other treatments (Figure 2).

Differences in Tmax among treatments were observed in Exp. 2, where Tmax of CON+ and PP + Si were lower (p < 0.05) than that of PP + Fe and PP + Pe, whereas in Exp. 4, CON+ had numerically lower Tmax than other treatments. The Tmax across treatments in Exp. 3 was generally lower than other experiments. Exp. 2 and 3 had numerically less HDD30 and numerically lower AT than Exp. 1 and 4. All treatments had similar AT and HDD30 over 60 d in all four experiments, except for Exp. 3, where CON+ and PP + MC had numerically lower AT than CON, while CON+ had numerically lower HDD30 than CON.

Further analysis of the relationships between hay bale average temperatures, bale initial moisture levels, and ambient temperatures showed that after adjustment for differences in ambient temperature, bale temperatures increased with increasing moisture levels (Figure 3). Further, Tmax was positively related to the total microbial population (i.e., bacteria + mold + yeast) after 60 d (Figure 4a; r² = 0.66), with mold populations being responsible for the majority of this response (Figure 4b, r² = 0.28). No relationship was found between the relative presence of the three spoilage microbial populations (TB, yeast, and mold) at the time of baling and any of HDD30, Tmax, or AT. The HDD30 of CON ranged from 18.3 d in Exp. 2 to 53.3 d in Exp. 1. Similarly, the HDD30 of CON+ ranged from 12 d in Exp. 3 to 50.6 d in Exp. 4.

3.3. Fermentation Products

Bales had similar pH across treatments for each experiment, except that the pH of CON+ was lower (p < 0.05) than others at d 0 in Exp. 1, 2, and 3 (Table 1 and Table 2). The pH of the bales in Exp. 2 was numerically higher than that of the bales in other experiments. Further, pH slightly declined over 120-d storage for most of the treatments in Exp. 1, 3, and 4, but not 2. In Exp. 2, there was a numerical increase in TB, yeast, and mold, as well as NH₃-N concentration, while WSC decreased as compared to other experiments. Ammonia-N concentrations increased (p < 0.05) in all bales after 30-d storage as compared to d 0. Additives had no effect (p > 0.05) on NH₃-N concentration in any of the experiments. However, CON+ bales had higher (p < 0.05) NH₃-N concentration than others at d 0 of baling as well over 120 d storage in all experiments.

Regression analysis showed that NH₃-N concentration increased with the total microbial population (TB + yeast + mold) over 120 d (Figure 5a; r² = 0.80). Further analysis showed that among the three microbes measured, NH₃-N was affected by mean bacteria numbers from d 30 to 120 but not yeast or molds (Figure 5b; r² = 0.84). More NH₃-N was also produced as the Tmax of hay bales increased (Figure 5c; r² = 0.60).

The WSC concentration of HM alfalfa hay at baling ranged from 3.2 to 20.9 g/kg DM across experiments. A minimal to moderate increase in WSC concentrations from d 0 to 30 was observed across most treatments in all four experiments. Overall, the WSC concentrations of harvested forages for Exp. 1 and 2 in 2016 were lower than those for Exp. 3 and 4 in 2017.

As expected, the concentrations of WSC in the HMH were negatively affected by the size of the total microbial population (Figure 6a,b; r² = 0.864 and 0.595). As with NH₃-N, the population of TB was the dominant factor associated with a decline in WSC concentration (Figure 6c; r² = 0.750). Combining the results of all four experiments showed that the NDF content increased as the Tmax of the bales increased (Figure 7a: r² = 0.452). The same was also found between maximum temperature and the ADIN content with higher maximum temperatures (Figure 7b; r² = 0.522).

3.4. Microbial Populations

Populations of TB, LAB, yeast, and mold were all numerically greater and remained higher throughout the storage period in Exp. 2 as compared to other experiments (

Table 3. Microbial characterization (log10 colony-forming unit (cfu)/g DM) of first-cut (Exp. 1) and second-cut (Exp. 2) alfalfa baled above the optimum moisture level without or with the application of various inoculants and stored for 120 d in 2016.

		Exp. 1							Exp. 2
		Treatments							Treatments
Item	Storage Days	CON	CON+	PP + LC	PP + MC	PP + HC	SEM	p-Value	CON	CON+	PP + Fe	PP + Pe	PP + Si	SEM	p-Value
TB	0	5.06	5.15	4.95	4.87	5.01	0.26	0.96	8.90	8.76	8.96	8.96	9.14	0.267	0.84
	30	7.42	6.55	7.48	7.36	7.47	0.293	0.17	8.72	8.35	8.64	8.66	8.01	0.222	0.11
	60	7.15	7.12	7.71	7.22	6.98	0.354	0.66	8.55	8.08	8.64	7.95	8.06	0.451	0.53
	90	7.5	7.84	6.96	7.08	7.10	0.381	0.47	8.41	8.42	8.49	8.14	7.72	0.316	0.36
	120	7.14	7.72	7.12	7.00	7.43	0.335	0.57	8.30	7.96	8.10	8.23	8.03	0.481	0.96
Yeast	0	5.36	5.21	5.34	5.40	5.6	0.154	0.51	7.87	7.98	8.20	8.12	7.92	0.115	0.10
	30	6.33	5.81	5.93	5.92	5.79	0.346	0.78	5.56	5.57	5.82	5.39	5.52	0.195	0.41
	60	5.82	5.72	5.59	5.69	5.66	0.204	0.95	5.58	5.45	5.57	5.90	6.00	0.224	0.22
	90	5.83	5.87	5.05	5.27	5.39	0.288	0.20	5.86	5.77	5.74	5.55	5.59	0.246	0.80
	120	5.67	5.10	5.26	5.53	5.7	0.251	0.38	5.71	6.15	5.84	5.79	5.77	0.286	0.72
Mold	0	5.20	5.06	5.17	5.27	5.28	0.061	0.24	6.55 ab	6.22 b	6.80 a	6.68 a	6.14 b	0.185	0.03
	30	5.27	4.95	5.21	5.41	5.21	0.326	0.91	6.76	6.51	6.85	6.87	6.94	0.247	0.56
	60	6.79 a	6.79 a	6.81 a	6.77 a	6.19 b	0.153	0.03	6.17	6.39	5.88	6.13	6.40	0.242	0.49
	90	6.51 a	6.25 ab	5.50 c	5.63 bc	5.74 bc	0.229	0.02	6.57	6.32	6.12	6.17	6.34	0.274	0.57
	120	6.33	6.26	6.28	6.07	6.07	0.131	0.44	6.64	6.43	6.70	6.60	6.34	0.201	0.69
LAB	0	3.29 b	3.82 ab	4.47 a	4.57 a	4.35 a	0.273	0.02	6.54	6.52	6.56	6.42	6.65	0.172	0.88
	120	6.42	6.69	5.99	5.65	6.56	0.477	0.34	7.75	7.75	7.62	7.78	7.11	0.433	0.76

Treatments included CON; uninoculated alfalfa high-moisture hay (HMH) control, CON+; positive control alfalfa HMH treated with propionic acid (4.0 L/tonne fresh forage), PP + LC; alfalfa HMH inoculated with 10¹² cfu Pediococcus pentosaceus (NCIMB 12674) + 1.5 g LANiHay02/tonne fresh forage, PP + MC; 10¹² cfu P. pentosaceus + 7.5 g LANiHay02/tonne fresh forage, PP + HC; 10¹² cfu P. pentosaceus + 15 g LANiHay02/tonne fresh forage (Exp. 1), PP + Fe; 10¹² cfu P. pentosaceus + 1.5 g LANiHay02/tonne fresh forage, PP + Pe; 10¹² cfu P. pentosaceus + 1.5 g LANiHay01/tonne fresh forage, and PP + Si; 10¹² cfu P. pentosaceus + 1.5 g Sigma pure chitinase/tonne fresh forage (Exp. 2). TB; total bacteria, LAB; lactic acid bacteria. SEM; standard error of mean. ^a–c Means in rows with different superscripts differ significantly at p < 0.05.

and

Table 4. Microbial characterization (log₁₀ colony-forming unit (cfu)/g DM) of first-cut (Exp. 3) and second-cut (Exp. 4) alfalfa baled above the optimum moisture level without or with the application of various inoculants and stored for 120 d in 2017.

		Exp. 3							Exp. 4
		Treatments							Treatments
Item	Storage Days	CON	CON+	PP	PP + LC	PP + MC	SEM	p-Value	CON	CON+	PP	PP + LC	PP + MC	SEM	p-Value
Total bacteria	0	6.38	6.34	6.42	6.84	6.76	0.176	0.21	5.19	6.13	6.21	5.91	6.06	0.319	0.16
	30	6.70	6.72	5.94	6.91	6.10	0.269	0.08	5.93	7.04	5.85	6.28	6.23	0.448	0.38
	60	6.42	6.29	5.75	5.95	6.54	0.365	0.53	6.06	6.62	6.87	5.76	6.09	0.398	0.31
	90	6.14	6.34	5.39	6.39	4.83	0.607	0.32	5.79	5.77	6.58	5.84	6.20	0.352	0.42
	120	5.78	6.34	4.61	6.47	3.92	0.754	0.11	5.92	6.45	6.41	6.64	6.55	0.374	0.70
Yeast	0	6.33 b	6.58 ab	7.23 a	7.31 a	7.35 a	0.208	0.01	4.14	4.84	4.75	4.79	4.84	0.180	0.06
	30	4.33	4.60	5.39	2.98	4.27	1.021	0.59	0.00	1.83	0.00	1.70	0.00	0.691	0.14
	60	5.55	5.42	5.72	5.15	4.97	0.204	0.21	3.79 a	4.57 a	2.62 ab	1.64 ab	0.00 b	0.807	0.01
	90	5.55	5.24	5.72	5.17	4.97	0.205	0.11	5.27	5.22	4.79	5.54	5.54	0.604	0.90
	120	6.18 a	5.92 a	6.21 a	5.87 a	1.05 b	0.500	<0.001	3.36	5.88	5.49	5.93	4.64	0.819	0.18
Mold	0	5.84	6.21	5.94	6.29	5.92	0.144	0.16	5.61	6.06	5.71	5.89	5.83	0.124	0.15
	30	6.38	5.97	6.26	6.18	6.40	0.169	0.32	6.72 a	5.47 b	6.41 ab	6.66 a	6.54 ab	0.281	0.03
	60	6.27	5.98	6.35	6.39	6.33	0.121	0.16	6.79	6.77	6.46	6.8	6.67	0.107	0.17
	90	6.28 ab	5.76 b	6.47 a	6.39 a	6.42 a	0.137	0.01	6.11	6.43	4.77	6.06	5.81	0.573	0.33
	120	6.45 a	5.84 a	6.49 a	6.65 a	1.28 b	0.584	<0.001	6.99	6.99	6.70	6.94	6.85	0.119	0.40
LAB	0	3.38 b	3.51 b	4.19 a	4.32 a	4.64 a	0.213	<0.01	3.93 b	5.02 ab	5.80 a	5.09 ab	5.62 a	0.291	<0.01
	120	4.16	2.84	4.17	5.00	2.62	0.907	0.34	4.58	5.32	5.63	5.10	5.47	0.574	0.74

Treatments included CON; uninoculated alfalfa high-moisture hay (HMH) control, CON+; positive control alfalfa HMH treated with propionic acid (6.0 L/tonne fresh forage), PP + LC; alfalfa HMH inoculated with 10¹² cfu Pediococcus pentosaceus (NCIMB 12674) + 1.5 g LANiHay02/tonne fresh forage, PP + MC; 10¹² cfu P. pentosaceus + 7.5 g LANiHay02/tonne fresh forage, PP + HC; 10¹² cfu P. pentosaceus + 15 g LANiHay02/tonne fresh forage using first cut (Exp. 3) and second cut alfalfa HMH (Exp. 4). TB; total bacteria, LAB; lactic acid bacteria. SEM; standard error of mean. ^a,b Means in rows with different superscripts differ significantly at p < 0.05.

). Treatments had no effect on TB in all experiments except for a trend (p = 0.08) for greater TB counts on d 30 for PP + LC than PP + MC in Exp. 3 (Table 4). Yeast populations were unaffected by treatments in Exp. 1 and 2, but there was a trend (p = 0.10) for greater yeast counts for PP + Fe than CON on d 0 in Exp. 2 (Table 3). Conversely, a higher (p < 0.05) yeast population was observed for PP, PP + LC, and PP + MC than for CON at d 0 in Exp. 3. However, bales treated with PP + MC had lower (p < 0.05) yeast counts after 120 d of storage than other treatments. Similarly, there was a trend (p = 0.06) for greater yeast counts for CON+ and PP + MC than CON on d 0 in Exp. 4. Additionally, yeast counts were lower (p < 0.001) for PP + MC than CON and CON+ on d 60.

The HMH bales had similar mold populations at d 0 in all experiments except for Exp. 2, where CON+ and PP + Si had lower (p < 0.05) mold counts than PP + Fe and PP + Pe (Table 3). Mold populations for bales treated with PP + HC at d 60 and PP + LC, PP + MC, and PP + HC at d 90 in Exp. 1, PP + MC treatment at d 120 in Exp. 3, and CON+ at d 30 in Exp. 4 were all lower (p < 0.05) than CON. Combining data from all four experiments, the average yeast concentration between d 30 and 120 after baling was lower (p < 0.01) than at d 0. However, compared to CON, the extent of this decrease tended to be greater for PP + HC (p = 0.096) in Exp. 1 and PP + MC (p = 0.091) in Exp. 4, and was greater for PP + LC (p < 0.05) and PP + MC (p < 0.001) in Exp. 3. This trend was also observed in the mold population for PP + MC (p = 0.096) and PP + HC (p = 0.054) in Exp. 1 and PP + MC in Exp. 3.

The population of LAB was higher (p < 0.05) for PP and PP + chitinase treatments than for CON at d 0 in Exp. 1, 3, and 4. In addition, the concentration of LAB was greater (p < 0.01) at d 120 than at d 0 for all treatments in Exp. 1 and 2 but not in Exp. 3 and 4.

3.5. Chemical Composition

The OM concentrations were similar across treatments, with only minor variation over 120-d storage in all experiments (Supplementary Tables S1 and S2). Regardless of treatment and storage days, alfalfa bales in Exp. 2 had numerically higher NDF and ADF but lower WSC than in the other experiments. Further, the NDF and ADF content increased over 120 d of storage across treatments in all experiments. However, the concentrations of total CP and ADIN varied among treatments and experiments but remained relatively stable over 120 d. For all experiments, treatments had minimal effects on the chemical composition of HMH, with most of those occurring in the first 60 d of storage.

3.6. Ruminal DM Degradation

Treatments had mixed effects on DM degradability (

Table 5. Dry matter (DM) and neutral detergent fiber (NDF) ruminal degradation kinetics parameters of the first-cut (Exp. 1) and second-cut (Exp. 2) alfalfa baled above the optimum moisture level without or with the application of various inoculants stored for 120 days in 2016.

	Exp. 1							Exp. 2
	Treatments							Treatments
Item	CON	CON+	PP + LC	PP + MC	PP + HC	SEM	p-Value	CON	CON+	PP + Fe	PP + Pe	PP + Si	SEM	p-Value
DM
PD	75.6	74.9	74.2	76.1	75.7	0.86	0.40	66.0 b	67.2 a	65.6 b	65.9 b	68.0 a	0.69	<0.001
a	31.6 b	33.5 a	31.9 ab	29.8 c	32.4 ab	0.684	<0.01	22.3 c	29.2 a	26.1 b	26.9 ab	26.8 ab	1.18	<0.001
b	43.9 ab	41.4 b	42.4 b	46.5 a	43.4 ab	1.27	0.02	43.7 a	37.9 b	39.6 b	38.9 b	40.9 ab	1.37	<0.01
c	0.056	0.052	0.053	0.049	0.054	0.0068	0.82	0.058 b	0.069 a	0.068 a	0.069 a	0.066 ab	0.0056	0.13
L	0.13 ab	2.09 a	2.20 a	0.00 b	1.90 a	0.628	0.03	1.1 b	3.3 a	2.4 ab	3.2 a	1.3 ab	0.94	0.10
ED (k = 0.05)	47.7 a	47.0 ab	45.4 b	48.6 a	46.9 ab	0.81	0.03	40.1 b	41.8 ab	41.2 b	40.0 b	44.4 a	1.17	0.03
NDFD
2 h	12.2 b	14.9 b	19.5 a	19.1 a	19.3 a	1.05	<0.001	19.2 b	21.3 b	25.5 a	20.7 b	20.6 b	1.97	<0.01
12 h	27.2 b	26.8 b	28 ab	31.2 a	31.1 a	1.86	0.02	24.4 c	28.6 ab	30.9 a	26.9 b	30.5 a	1.14	<0.001
24 h	38.9 b	37.8 b	38.1 b	40.6 ab	42.5 a	2.74	0.06	37.2 b	40.5 a	42.5 a	41.3 a	40.5 a	2.20	<0.001
48 h	49.3 b	48.3 b	49.6 b	53.1 a	53.0 a	2.17	0.01	50.4 b	50.8 b	52.4 a	51.0 b	51.4 ab	0.65	0.02
72 h	54.8 a	51.9 b	55.0 a	55.4 a	56.9 a	0.99	<0.01	51.6	52.0	53.1	51.5	51.7	0.70	0.29
ED (k = 0.05)	40.8 bc	37.9 c	42.7 ab	44.7 a	45.6 a	1.32	<0.001	40.1 c	42.1 ab	43.7 a	41.8 b	42.6 ab	0.74	0.001

Treatments included CON; uninoculated alfalfa high-moisture hay (HMH) control, CON+; positive control alfalfa HMH treated with propionic acid (4.0 L/tonne fresh forage), PP + LC; alfalfa HMH inoculated with 10¹² colony-forming unit (cfu) Pediococcus pentosaceus (NCIMB 12674) + 1.5 g LANiHay02/tonne fresh forage, PP + MC; 10¹² cfu P. pentosaceus + 7.5 g LANiHay02/tonne fresh forage, PP + HC; 10¹² cfu P. pentosaceus + 15 g LANiHay02/tonne fresh forage (Exp. 1), PP + Fe; 10¹² cfu P. pentosaceus + 1.5 g LANiHay02/tonne fresh forage, PP + Pe; 10¹² cfu P. pentosaceus + 1.5 g LANiHay01/tonne fresh forage, and PP + Si; 10¹² cfu P. pentosaceus + 1.5 g Sigma pure chitinase/tonne fresh forage (Exp. 2). PD; potential degradability, a; rapidly degradable fraction, b; slowly degradable fraction, c; rate at which b is degraded (/h), L; lag time, ED; effective degradability, NDFD; neutral detergent fiber disappearance. SEM; standard error of mean. ^a–d Means in rows with different superscripts differ significantly at p < 0.05.

and

Table 6. Dry matter (DM) and neutral detergent fiber ruminal degradation kinetics parameters of the first-cut (Exp. 3) and second-cut (Exp. 4) alfalfa baled above the optimum moisture level without or with the application of various inoculants and stored for 120 d in 2017.

	Exp. 3							Exp. 4
	Treatments							Treatments
Item	CON	CON+	PP	PP + LC	PP + MC	SEM	p-Value	CON	CON+	PP	PP + LC	PP + MC	SEM	p-Value
DM
PD	71.1 a	71.0 a	70.6 ab	71.1 a	69.2 b	0.48	<0.01	76.0	74.6	72.8	73.7	73.9	1.35	0.21
a	28.6	29.6	29.0	28.3	29.3	0.56	0.41	34.1	33.8	33.2	32.4	33.1	0.85	0.45
b	42.5 a	41.5 ab	41.7 ab	42.8 a	39.8 b	0.78	0.001	41.1	40.8	39.7	41.3	40.8	1.73	0.73
c	8.01	8.01	7.55	7.39	7.47	0.595	0.40	6.65	6.49	8.27	7.23	6.72	1.16	0.09
L	0.0 b	0.28 ab	0.28 ab	0.11 ab	0.56 a	0.18	0.03	1.08	1.03	2.11	1.79	1.46	0.521	0.43
ED (k = 0.05)	51.1	50.7	48.7	47.6	49.3	1.78	0.11	51.0	50.7	49.5	49.7	48.2	1.55	0.13
NDFD
12 h	19.7 b	26.5 a	18.5 b	26.1 a	17.7 b	1.46	<0.001	31.3	30.2	28.8	30.1	29.2	2.67	0.58
24 h	30.8 b	38.8 a	32.8 b	37.2 a	32.0 b	2.37	<0.001	39.2 b	39.3 b	46.3 a	41.9 ab	41.2 b	1.17	<0.001
36 h	43.7 b	49.3 a	43.8 b	47.0 a	41.3 b	1.35	<0.001	48.7 b	49.1 b	51.1 ab	51.2 ab	52.9 a	1.52	<0.01
48 h	45.4 b	51.0 a	45.5 b	50.2 a	43.3 b	1.01	<0.001	55.0 a	53.7 ab	51.7 b	54.9 a	55.6 a	0.7	0.001
72 h	45.2 d	50.9 ab	48.7 bc	53.4 a	46.6 cd	1.08	<0.001	56.7	56.1	55.2	56.6	54.6	1.19	0.17
ED (k = 0.05)	32.9 b	39.3 a	33.4 b	38.6 a	32.2 b	1.21	<0.001	40.7	37.7	35.7	38.3	37.4	3.13	0.19

Treatments included CON; uninoculated alfalfa high-moisture hay (HMH) control, CON+; positive control alfalfa HMH treated with propionic acid (6.0 L/tonne fresh forage), PP + LC; alfalfa HMH inoculated with 10¹² colony-forming unit (cfu) Pediococcus pentosaceus (NCIMB 12674) + 1.5 g LANiHay02/tonne fresh forage, PP + MC; 10¹² cfu P. pentosaceus + 7.5 g LANiHay02/tonne fresh forage, PP + HC; 10¹² cfu P. pentosaceus + 15 g LANiHay02/tonne fresh forage using first cut (Exp. 3) and second cut alfalfa HMH (Exp. 4). PD; potential degradability, a; rapidly degradable fraction, b; slowly degradable fraction, c; rate at which b is degraded (/h), L; lag time, ED; effective degradability, NDFD; neutral detergent fiber disappearance. SEM; standard error of mean. ^a–c Means in rows with different superscripts differ significantly at p < 0.05.

). Overall, the PD, which is the sum of fractions a (rapidly degradable) and b (slowly degradable) and ED of DM in Exp. 2, were numerically lower than that of other experiments. PP + MC had a lower (p < 0.05) proportion of fraction a, greater (p < 0.05) proportion of fraction b, and greater (p < 0.05) ED and PD than PP + LC. Compared to CON, CON+ had no effect on PD or ED of DM, except that CON+ had increased PD of DM in Exp. 2. Further, PP + LC decreased (p < 0.05) ED of DM and fraction b compared to CON in Exp. 1. However, PP + Pe increased (p < 0.05) the fraction a and the degradation rate c of fraction b in Exp. 2. Further, the PD of DM was increased by PP + Si. The ED of DM was also increased (p < 0.05) by PP + Si. The PD was reduced for PP + MC in Exp. 3 due to a lower proportion of fraction b compared to CON, while treatments did not affect the PD or ED in Exp. 4.

3.7. Ruminal NDF Degradation

Across experiments, the ED of NDF observed in Exp. 1 and 2 was numerically greater than that obtained in Exp. 3 and 4 (Table 5 and Table 6). Overall, the effects of treatments on NDF ruminal degradation were greater than their effects on DM ruminal degradation. Application of PA (CON+) at baling increased (p < 0.05) ruminal NDF digestion at 12 and 24 h, and its ED compared to CON in Exp. 2. Similar results were also observed in Exp. 3 but not in Exp. 1 and 4, although greater NDFD for PP was observed after 24 h of incubation in Exp. 4. Among the four experiments that assessed PP + low dosage (1.5 g/tonne) of LANiHay01 chitinase, increased NDF ruminal digestion as compared to CON was observed in Exp. 2 and 3, but not in Exp. 1 and 4. In contrast, applying PP + MC increased the NDF digestion in Exp. 1 but not in Exp. 3 or 4. Treatment PP + HC was assessed in Exp. 1 only and was observed to increase (p < 0.01) the NDF digestion, similar to that of PP + MC. In addition, the combinations of PP and three sources of chitinase (LANiHay01, LANiHay02, and Sigma pure chitinase) in Exp. 2 indicated that combinations of PP and low dosage (1.5 g/tonne) of these three chitinases improved NDF digestibility after 12 and 24 h of incubation, but not after 48 h of incubation. Regression analysis found that the NDF content of alfalfa hay had a quadratic (p < 0.05) effect on the ED of NDF (Figure 8a; r² = 0.731). When NDF content was lower than 50%, the ED of NDF increased as NDF content increased. However, when the NDF content was higher than 50%, its ruminal ED decreased as the NDF content increased. Further analysis showed that Tmax in the range of 40–64 °C had a positive effect on the ED of NDF (Figure 8b; r² = 0.505). Similarly, there was a positive correlation between NDFD and the average TB counts between d 30 and 120 (Figure 8c; r² = 0.605).

4. Discussion

4.1. Harvesting Alfalfa as High-Moisture Hay

Baling alfalfa at higher than the conventional 12–18% moisture levels reduces the wilting period and expands the time window that forages can be baled. This could reduce the impact of precipitation on the harvest window and forage quality [9]. The range in moisture levels (18.3–29.2%) of high-moisture alfalfa hay in the current studies was within the range reported by others [1,9,16]. However, it should be noted that moisture levels of HMH bales generally varied by around 5–10 units between experiments and years in the present study. Weather conditions (wind, temperature, relative humidity, etc.), field topography, operation and handling of haying equipment, and swath width will significantly influence the degree distribution of moisture within forages [17,18,19]. These authors also reported that a wide windrow (72% of cut width) immediately after cutting will improve the quality of alfalfa haylage compared to narrow windrows (25% of cut width). These climatic and management variabilities will contribute to large variations within the same experiment and among experiments. An increase in the DM content of hay bales during storage is also consistent with the findings of previous studies using alfalfa HMH [9,20]. Moist hay undergoes several heating peaks, and this heating process leads to additional moisture loss during storage [21].

4.2. Hay Bale Temperatures

The temperature increase in the first 40 d of post-baling was numerically lower for CON+ in Exp. 3 and 4, while the temperatures of hay bales for all the treatments remained similar for Exp. 1 and 2. It should be noted that PA was applied at 4.0 L/tonne in Exp. 1 and 2 and 6.0 L/tonne in Exp. 3 and 4. Applying PA at a concentration of 6.0 L/tonne decreased heating within bales. A relatively lower increase in temperature for CON+ across experiments in the present study is somewhat contrasting to that reported previously [9], which reported higher internal temperatures in a 3-year study where HMH treated with PA at 4.0 L/tonne was compared to that inoculated with Pediococcus or Pichia and stored for 60–180 d. However, it should be noted that the pH of hay bales treated with PA ranged from 6.5 to 7.3 after 60–180 d of storage in that study [9], while in the present study, CON+ had an average pH of 5.7. These results indicate that the application of PA at a concentration of 4.0 L/tonne does not sufficiently reduce forage pH to a level that inhibits spoilage microorganisms. The pH of CON+ hay bales in Exp. 1, 2, and 3 was lower than other treatments and numerically lower in Exp. 4 at baling (d 0). It is logical to assume that PA applied at 6.0 L/tonne lowered the pH of HM alfalfa hay to a level that inhibited plant respiration and spoilage microorganisms during storage.

The lower Tmax across treatments in Exp. 3 is likely due to the lower moisture content of the HMH in this experiment. The average moisture content (%) of hay bales across treatments in Exp. 3 was 18.3 relative to 29.2, 28.3, and 23.5 for Exp. 1, 2, and 4, respectively. Greater moisture levels promote plant cellular respiration and microbial activities, resulting in the heating of forages during storage [20]. The temperature profile of treatments had no real correlation between years and studies in the present research. Similar variability in temperature profiles for alfalfa treatments in multi-year studies were also reported previously [9]. These authors reported that the bale temperature profiles were not consistent with the main trend from previous years in terms of the effects of treatments on AT, HDD30, and Tmax [9]. This could likely be due to the natural variation in forage and harvest management, climatic conditions at the time of forage handling, and the difference in experimental conditions among studies.

An increase in average bale temperatures with increasing moisture levels in the present study, when the temperatures of the bales were adjusted for ambient temperature, was likely due to the increased microbial activity at the elevated moisture levels. Natural plant respiration and an increase in bacterial population following baling result in an increase in temperatures within bales in about 2 weeks [22]. High-moisture bales may result in excessive microbial activity, resulting in temperatures in excess of 65.5 °C, leading to heat damage of hay and spontaneous combustion. A greater impact on Tmax within the heated bales as a result of mold populations over the initial 60 d of storage signifies the role played by molds in hay spoilage. It is worth noting that no relationship was found between any of the three microorganisms (TB, yeast, and mold) at d 0 of baling and any of the three temperature parameters by 60 d of storage (HDD30, Tmax, and AT). Interestingly, the forage in Exp. 2 was rained on several times after harvest and was left to dry in the field for over 7 days before baling. The forage was baled at a relatively lower DM content (71.7 ± 2.43% vs. 70.8 ± 1.53%, 81.7 ± 2.81%, and 76.5 ± 1.09% for Exp. 1, 3, and 4, respectively), and the bales remained “wetter” until day 30 of storage (75.7 ± 2.30% vs. 80.7 ± 1.73%, 90.3 ± 0.68%, and 85.0 ± 2.04% for Exp. 1, 3, and 4, respectively). However, the temperature parameters (AT and HDD30) were lower across treatments in Exp. 2, although the yeast and mold counts were relatively greater, and the hay bales contained higher moisture than in other experiments during the first few weeks of storage. It is possible that the increased microbial activity might have resulted in greater heat production in the bales had the forage in Exp. 2. been baled sooner, as heat is trapped and dissipated much slower in large round bales with greater forage density [23]. As the forage was left to dry down for an extended period in the field, the heat generated likely dissipated faster in the windrows. A quadratic effect of the total microbial population (bacteria + yeast + mold) at d 0 on the maximum temperature was observed. It is possible that the changes in the hay conditions during storage could alter the nature and composition of the microbial population over time, with a possible shift in their metabolic activity [24]. It was reported that the bacterial and fungal populations in standing crops vary from that of cut forage during field curing as the moisture content of the forage drops to 20–30%. Fungal growth is inhibited once the moisture content of the bales is below 15% [24], though specific genera and species of fungi can grow and predominate hay bales at any given time. It is logical to assume that the shifts in the predominant microbial population in the bales could impact nutrient utilization and heat production during storage.

Variability in HDD30 ranging from 15.5–55.8 d for uninoculated HMH over a 3-year study has been reported previously [9]. These authors also reported HDD30 ranging from 12.4–55.2 d for alfalfa HMH treated with PA. As discussed above, the degree of heat production in stored forages is determined by the biochemical activities of epiphytic microbes in HMH, coupled with the environmental conditions during storage, such as daily temperatures, precipitation, humidity, and wind speed. However, the mechanism by which these environmental conditions impact the epiphytic microbial activities in stored forages is poorly defined. Further evaluation of the epiphytic microbial population dynamics during storage using next-generation sequencing and the use of metagenomic data could potentially explain some of the observations in these studies.

4.3. Fermentation Products

The pH of hay bales across experiments, except Exp. 2 in the present study, was ≤ 6.29 on d 0 (day of baling). The pH of the hay bales reported in the present study is very similar (6.03–6.30) to the pH range reported [9] for alfalfa HMH treated with microbial inoculants and stored for 60−180 d. These authors also reported hay pH ranging from 6.08–6.32 for conventional moisture hay, 6.06–7.39 for uninoculated alfalfa HMH, and 6.03–7.29 for alfalfa HMH treated with PA. The pH of hay bales in Exp. 2 ranged from 6.66–7.44. This numerical increase in bale pH in Exp. 2 was likely because the harvested forage was rained on prior to baling. Though the impact of short periods of rain on the pH of harvested forages has been minimal, extended rain events in the fields can significantly impact forage pH [25]. These authors reported a pH of 7.43 for alfalfa subjected to extended wetting and wilting periods compared to freshly harvested forages (6.48). Similarly, the numerical increase in the microbial population and NH₃-N concentrations and a similar decrease in WSC concentrations for hay bales in Exp. 2 is also a likely reflection of the impact of rain prior to baling. Forages damaged by rain are at risk of clostridial fermentation, resulting in the production of ammonia and butyric acid as the end products of fermentation [26]. These authors also reported a linear decrease in WSC concentrations with simulated rainfall. The reduction in WSC concentrations was much more severe (2.72–3.04%) for forages subjected to rain and subsequent prolonged field curing (~8 d) relative to fresh forages [25].

Greater NH₃-N concentrations for PA-treated alfalfa HMH observed in our study are consistent with previous studies when alfalfa HMH was treated with PA or buffered PA (BPA) [9,20]. An increase in NH₃-N indicates the deamination of free amino acids during microbial growth [27]. However, the application of PA decreased forage pH to around 5.7, likely reducing microbial activity. This is further supported by the observation that the WSC concentrations of CON+ were higher than any of the other treatments. These results contrast with Jin et al. [9], who reported a decrease in WSC and an increase in NH₃-N concentrations for alfalfa HMH treated with 4.0 L/tonne forage PA. Higher NH₃-N concentrations in alfalfa hay treated with buffered PA are reported to be due to the presence of ammonium hydroxide as a buffering agent rather than a result of increased proteolysis in the forage [20]. The greater impact of mean bacterial numbers from d 30–120 on NH₃-N suggests that it arose as a result of microbial activity, with bacteria being principally responsible.

The range in WSC concentrations (3.2–18.7 g/kg DM) for alfalfa HMH reported in the present study was similar to that reported in a previous multi-year study [9]. Further, an increase in WSC concentrations during the storage of alfalfa hay bales has also been reported [20,28]. An increased WSC concentration from 13.1 g/kg for freshly baled alfalfa hay to 31.5 g/kg after 60 d of storage was reported previously [20]. A similar increase in WSC concentrations for ensiled forages has also been reported for grasses [29], cereal grains [30], and legumes [28]. Enzymatic hydrolysis of structural carbohydrates to soluble sugars likely accounts for the increase in WSC concentrations during storage. However, the WSC concentrations varied between experiments and years in the present study. While the WSC concentrations of hay bales, in general, increased from d 0 to 30 across experiments and years, WSC concentrations were lower on d 0 and remained lower throughout storage in Exp. 2, reflecting the duration the forages remained in the windrows before baling. The difference in WSC concentrations between experiments and years might have been caused by factors including the interaction between environmental and agronomic factors with plant development that would impact alfalfa nutrient composition and quality [31,32]. These authors reported that environmental factors such as temperature, water deficit, solar radiation, and soil nutrient availability impact forage nutrient composition and quality by altering the leaf/stem ratio modifications in plant development and changes in the chemical composition of plant parts even if harvested at the same stage of development [31,32].

4.4. Microbial Populations and Chemical Compositions

A relatively greater microbial count (TB, yeast, and mold) in Exp. 2 likely reflects the rainfall that occurred during this time. The occurrence of rain or high temperatures during drying and the contamination of forages with soil [33] have been reported to increase the number of epiphytic microflora in harvested forages. The microbial numbers in the present study are consistent with those reported previously for alfalfa hay treated with combinations of Pediococcus or Pichia and chitinase and stored for 60 or 120 d [9].

The yeast counts did not vary in Exp. 1 and 2, where P. pentosaceus was combined with chitinase. No effect of additives on yeast counts indicates that the source or rate of application of chitinase did not impact hay spoilage microorganisms during storage under the conditions of these experiments. Reduced fungal population in alfalfa HMH inoculated with P. pentosaceus at a rate of 5.7 × 10⁵ cfu/g forage DM alone and baled at 20–25% moisture has been reported previously [34]. However, a similar response was not observed when alfalfa/hay/clover mixture varying in moisture levels (20–35% moisture) levels was inoculated with P. pentosaceus at a rate of 5 × 10⁵–5 × 10⁶ cfu/g fresh forage [35]. Pediococcus pentosaceus is a homofermentative LAB that is increasingly used for food and feed conservation and as probiotics due to its ability to produce short-chain fatty acids and bacteriocins [36]. Pediococcus pentosaceus has been used as a bacterial inoculant for different silages to enhance the ensiling fermentation and improve silage quality [36,37,38]. However, there are only a few studies assessing the effects of P. pentosaceus on HMH or haylage preservation with varying success [9,34,35,39], likely due to the different experimental conditions and different strains of P. pentosaceus being used. This study, together with others, suggests that the efficacy of P. pentosaceus in conserving alfalfa HMH is likely strain-dependent and possibly impacted significantly by the type of forage and the haying conditions. The average moisture content of HMH at baling was similar to the range reported previously [34]. Conversely, a relatively lower yeast count (4.4 log₁₀ cfu/g DM forage) for alfalfa HMH treated with a combination of P. pentosaceus and chitinase as compared to untreated CON (5.13 log₁₀ cfu/g DM forage) after 180 d of storage was reported previously, while the yeast counts did not vary after 60 d of storage in other experiments. It should be noted that the rate of application of chitinase in the previous study [9] was 1.5 g/tonne DM forage, while the present study had three application rates [1.5 g (LC), 7.5 g (MC) and 15 g (HC)]. It has been suggested that the success of inoculation depends on the type and viability of microbes in the inoculant, the number and type of epiphytic microbes, the method of application, forage characteristics, and processing [40]. This study indicated that interactions between the inoculated bacteria and chitinase may also have played a role. The lower yeast counts for PP + MC in Exp. 3 and 4 indicate that applying chitinase at 7.5 g/tonne forage DM in combination with P. pentosaceus reduces spoilage by yeast when the forage moisture levels range between 20–25%.

While PP + MC had no effect on yeast counts in Exp. 1 and 2, yeast counts were lowest for PP + MC on d 120 in Exp. 3 and d 60 in Exp. 4. Similarly, while PP + HC resulted in the lowest mold counts on d 60, PP + LC resulted in the lowest mold counts on d 90 in Exp. 1. It should also be noted that the TB counts were unaffected by PP + chitinase. Overall, these results indicate that PP + chitinase could reduce the number of yeast and mold in alfalfa HMH without negatively impacting bacterial populations, but the impact on fungal populations varied between the concentrations of chitinase and the duration of hay storage between experiments. This variation could likely be due to differences in experimental conditions, including hay moisture content, dose of chitinase, heating of the bales, and the prevailing climatic conditions during these experiments. In an evaluation of the impact of P. pentosaceus and Pichia anomala in combination with chitinase on the preservation of high-moisture alfalfa hay, a similar numerically lower yeast count for alfalfa HMH inoculated with PP + chitinase after storage of 60 to 180 d was reported in experiments conducted over 3 years [9].

The higher number of LAB in Exp. 2 than in other studies, together with the observation of similar LAB populations across treatments at d 0, was likely due to the increased natural LAB populations due to prolonged field curing as a result of rain events. It was reported that prolonged wilting time increased the number of lactic acid bacteria in forages [41,42]. The LAB concentrations at day 120 were greater than day 0 for CON in all experiments, but this was not consistent for inoculant treatments. The variability in LAB counts likely indicates a natural decline in LAB population during the storage period [20] as the continued detection of microbes and the variation in their numbers during the storage period is a reflection of the changing conditions within the bales. The lower LAB counts at d 120 in Exp. 3 might be related to the lower Tmax of < 45° and average HDD30 of < 20, as bacterial growth is favored by temperature and humidity. This was also consistent with the TB numbers from the same experiment. It should also be noted that the average moisture of fresh forage at baling in Exp. 3 was 18.2%, which was the lowest moisture across all four experiments. The moisture content of forage at baling is reported to have the highest impact on the severity of heating during storage [2]. These authors reported that the threshold moisture levels for acceptable storage for large round bales are 16–18%, while that for small rectangular bales is about 20%. Greater than the recommended moisture levels at haying will result in high temperatures and mold growth. This is also consistent with the findings of this study, as illustrated in Figure 3. It is logical to assume that the relatively lower moisture content of hay in Exp. 3 resulted in lower microbial activity and fewer heating events during storage.

The effects of additives on bacteria, yeast, and mold were inconsistent across experiments in the present study. As indicated previously, the discrepancy among the experiments might be due to the different experimental conditions and variations in sampling. To minimize the influence of variation in initial microbial populations, yeast and mold counts at d 0 of baling were compared with the respective mean of each microorganism between d 30 and d120 of storage. The results showed that compared to d 0, yeast concentration decreased after 30 d post-baling regardless of treatment in Exp. 2, 3, and 4. However, the significant reduction in yeast and mold counts during storage across experiments suggests these additives were effective in minimizing the growth of undesirable microbes in high-moisture alfalfa hay, likely due to a combination of the effect of bale temperature, water activity, chemical composition, and ecological succession and interaction of microbes during storage.

Multiple heating cycles likely reflect a corresponding variation in microbial population during the initial 60 d of storage. However, except for yeast in Exp. 2, 3, and 4, the other microorganisms did not change over the initial 60 d of storage. This may indicate that sampling dates for microbial enumeration did not coincide with the peak periods in the heating cycle. A more dynamic sampling scenario may be needed to examine the relationship between changes in microbial population and heating within the bales.

Regardless of treatments and storage days, alfalfa bales in Exp. 2 had numerically higher NDF and ADF but lower WSC concentrations than in other experiments. This is likely due to the loss of soluble nutrients caused by the rain and the prolonged duration of the swath remaining in the field. An increase in fiber constituents and ash for forages that were damaged by natural rainfall in the field, followed by an extended wilting period, has been previously reported [25]. Leaching of highly soluble, non-structural carbohydrates during rain events has been reported to increase fiber and fiber-bound protein concentrations [43]. The generally increased NDF and ADF contents over the 120-d storage period in this study are consistent with previous studies [9]. These authors reported an increase in NDF concentration from 398 ± 28.4 g/kg to 543.8 ± 2.9 g/kg and ADF concentration from 296 ± 21.7 g/kg to 358.8 ± 18.7 g/kg after 180 d of storage of alfalfa HMH. The increase in the fiber fractions likely reflects the proportionate variation in nutrient composition due to DM losses during storage.

4.5. Ruminal DM and NDF Degradation

The PD of DM observed in the present study was similar to that reported previously [9,20]. However, the PD of DM in Exp. 2 was lower than in other experiments. This is likely caused by the precipitation that the alfalfa in Exp. 2 experienced prior to baling, which is also supported by the data on chemical (e.g., higher NDF and lower WSC) and microbial composition. Leaching of soluble carbohydrates and proteins due to wet weather during the haying process can decrease the digestibility of conserved forage by 6–40% [39].

The observation of an increase in NDF digestion by combining PP and chitinase is consistent with our previous findings [9]. However, the treatments (PP + chitinase) varied in their impact on NDF digestion [44]. This discrepancy indicates that the efficacy of this inoculant in improving fiber digestion is likely affected by experimental conditions. This is likely because, as the Tmax increased, the NDF concentration also increased, which resulted in greater ED of NDF. Further, the growth of molds was found to contribute to the heat generated inside the hay bales, which is somewhat contrasting to the previous observations [24], which indicated that moldy hay is often heat-damaged, but fungi alone cause little heat damage. However, it should be noted that the microbial dynamics, including that of yeast and mold, may vary between normal moisture and HMH, impacting hay temperatures and spoilage. The improvement in fiber digestion of alfalfa hay by the combination of PP + chitinase is likely through multiple mechanisms involving alterations in microbial composition, thereby affecting hay bale temperature and resulting in alteration of NDF composition. An increase in NDF digestibility in vitro for HM alfalfa hay treated with LAB + chitinase has been reported previously [9]. These authors also reported that LAB + chitinase resulted in a greater proportion of soluble fraction of DM in HM alfalfa hay than in uninoculated-control HM alfalfa hay, indicating that the soluble nutrients in uninoculated-control HM alfalfa hay were utilized by microbes while they were preserved by LAB + chitinase in the inoculated HM alfalfa hay.

5. Conclusions

Combining the results from experiments conducted in this study showed that HMH bale temperature was positively correlated to the initial moisture levels and total spoilage microbial populations, and that ammonia-N was positively but WSC was negatively correlated with the total microbial population of the hay bales. Applying a combination of P. pentosaceus and chitinase at the rates of 10¹² cfu P. pentosaceus plus 1.5–7.5 g chitinase/tonne fresh forage during baling limited the heating of HMH bales, as indicated by the lowered HDD30, and minimized the fungal population, as indicated by the reduced yeast populations during storage as well as the improved ruminal NDF digestion, although variations existed among experiments. Combinations of P. pentosaceus and chitinase have the potential as an additive to conserve alfalfa HMH.