Application of Hops (Humulus lupulus L.) and β-Acid Extract to Improve Aerobic Stability and In Vitro Ruminal Fermentation of Maralfalfa Grass Silage

Abstract

The potential of hops (Humulus lupulus L.) and β-acid extract were evaluated for improving the quality of maralfalfa grass (Cenchrus purpureus) silage (with added sorghum grain, sorghum straw, and urea) during aerobic exposure and their residual effects on in vitro ruminal fermentation characteristics. Silage samples and ground hops pellets (Galena and Chinook varieties) as well as β-acid mixtures were incubated at 37 °C for 24 h and then maintained under aerobic exposure for 12 h. The sample pH, counts of filamentous fungi, yeasts, and total coliforms, and volatile fatty acid (VFA) concentrations were determined. Subsequently, in vitro ruminal fermentation was conducted to determine total gas production and concentrations of hydrogen, methane, carbon dioxide, and VFAs. The β-acid treatment controlled yeast populations, but an increase (p < 0.05) in pH values was observed for the Galena and Chinook treatments compared to the Control. However, pH did not differ significantly (p > 0.05) between the Control and the β-acid treatment. Butyric acid concentrations in the silage were lower (p < 0.05) compared to the Control, except in the silage treatment with Galena. In the in vitro ruminal fermentation, the β-acid treatment showed higher butyric acid levels than the Chinook and Galena, but these differences were not significant (p > 0.05). There were no differences (p > 0.05) in methane between the treatments. An increase (p < 0.05) in propionic acid concentration was observed in the in vitro ruminal fermentation with β-acids. It was concluded that β-acids could help reduce silage deterioration during the aerobic phase, reducing the butyric acid and yeast populations, and their residual effect could improve ruminal fermentation, increasing propionate and acetate concentrations.

1. Introduction

Ensiling is the most commonly used method for preserving forages for ruminant feed [1,2] as it requires few additives and minimal infrastructure [3,4]. Broadly speaking, this method is a fermentation process facilitated by the growth of lactic acid bacteria (LAB), in which lactic acid accumulation leads to a pH decrease, creating an inhospitable environment for unwanted microorganisms during the silage process. The presence of undesirable microorganisms and toxins in this type of feed can reduce the quality of the final livestock products (e.g., meat and milk).

The fermentation phase has been extensively studied to improve silage conditions and to obtain a product that effectively preserves the plant material. An excellent grass for ensiling is the tropical-climate grass Maralfalfa, which can produce between 40 and 60 tons of dry matter per hectare per year, providing an abundant source of forage for ruminant animals. It also offers a good level of crude protein at 14%, along with high digestibility and sugar content, making it more palatable than conventional grasses [5]. Frequently, the material to be ensiled has very low nutritional quality, so additives are often included to enhance feed quality. This is the case with urea, which is added to the diet to increase nitrogen availability, allowing the ruminal microbiota to synthesize microbial proteins, thus providing better nutrition for the ruminant [6,7,8]. However, silages with these additives can generate high nutrient availability, making them more susceptible to degradation by opportunistic microorganisms during the aerobic phase [9]. These microorganisms, such as acetic acid bacteria, yeasts, and enterobacteria [10], are present in the plant material before ensiling and remain dormant until the process is complete and favorable conditions return. Other undesirable microorganisms include bacterial spore formers and filamentous fungi, which not only negatively impact nutrient availability but may also contaminate livestock industry products such as meat, milk, and their derivatives, causing serious illnesses in consumers [11].

Commercial chemical additives that improve aerobic stability reduce yeast concentrations after silage is exposed to air [12]. The use of natural products with antimicrobial activity may offer a viable option. The antimicrobial benefit of hops in extending beer shelf life is well-known [13], making them a promising candidate for preventing aerobic silage deterioration. The bitter oils of hops have a known mechanism of action in the growth of Gram-positive bacteria, as they cause permeability in the membrane of these microorganisms, altering nutrient transport and preventing their proliferation. However, some studies indicate that they also have an effect on strains of Escherichia coli. According to Ref. [14], studies have been conducted to exploit the potential of hops as a growth-modulating additive for ruminants, with the goal of replacing the use of ionophores. Results indicated that hops do not alter ruminal microbiota, and reductions in methane production were observed [15]. β-acids extracted from hops are already commercialized and have several applications in the cosmetic industry, medicine, and beer brewing [16,17].

By utilizing β-acid extracts, we can more effectively assess their impact in enhancing the aerobic stability of silage. Additionally, these extracts have a higher concentration of antimicrobial compounds. The antimicrobial properties of hops and the minimal impact on the ruminal environment are factors to consider when using hops as an additive to improve silage quality and to extend usability. Once the silage has been removed from the silo and exposed to air, aerobic deterioration can occur. This exposure allows aerobic microorganisms, such as yeasts and molds, to proliferate, leading to nutrient losses and reduced feed quality. The hypothesis was that the addition of hops or hop extract may influence the growth of microorganisms responsible for silage spoilage during air exposure, without negatively affecting the ruminal fermentation process. Therefore, the objective of this study was to evaluate hops as an additive to prevent aerobic silage deterioration of maralfalfa grass silage preparations.

2. Materials and Methods

2.1. Experimental Procedure

The silages used in this study were prepared at the animal science station of the Faculty of Veterinary Medicine and Animal Science at the Autonomous University of Tamaulipas. The maralfalfa grass was produced in the dry tropical climate of Ciudad Victoria, Tamaulipas, in northeastern Mexico. The geographical coordinates are 23°44′06″ N and 99°07′51″ W, at an altitude of 340 m above sea level. Harvest was carried out manually at 100 days of growth. The plants were then chopped with a hammer mill (Swissmex^®, model 610350, Swissmex-Rapid SA de CV, Lagos de Moreno, Guanajuato, Mexico) into pieces approximately 2 cm in size. Four different preliminary silages, Silages 1–4, were considered for further experimentation (

Table 1. Composition of the preliminary maralfalfa grass silages.

	Silage
	1	2	3	4
Maralfalfa grass, %	90.0	85.0	90.0	85.0
Ground sorghum grain, %	8.00	12.0	7.63	11.4
Sorghum straw, %	2.00	3.00	1.63	2.44
Urea, %	-	-	0.75	1.13

). In each treatment, the main ingredients, maralfalfa grass, ground sorghum grain, sorghum straw (harvested by local producers in the region of Ciudad Victoria, Tampaulipas, Mexico), and urea containing 46% nitrogen (Agromex Agrochemical Products, Ciudad Victoria, Tampaulipas, Mexico), were mixed manually and placed in 5 L plastic containers. The contents were manually compacted with a press, and the jars were then hermetically sealed with lids. Four replicates were considered for each treatment. The silages were stored indoors at room temperature (oscillating between 24 and 35 °C) for 90 days. Upon opening the jars, a 4 g sample of silage was taken from each of the four replicate containers per treatment, 10 mL of sterile distilled water was added, and the mixture was shaken in a vortex for 10 min. The pH was then determined using a pH meter (Hannah Instruments, model HI 9017, Arvore-Vila do Conde, Portugal). The silage samples were analyzed for crude protein (CP), ether extract (EE), ash (AOAC, 2023), neutral detergent fiber (NDF), and acid detergent fiber (ADF) with amylase and sulfite according to the methodology of Van Soest et al. (1991) [18]. For dry matter (DM), a forced-air oven (Precision Scientific, GCA Corporation, Chennai, Tamil Nadu, India) was used at a temperature of 55–60 °C for 24 h. For ash determination, a muffle furnace (Thermolyne, Thermo Scientific, Waltham, MA, USA) was used at a temperature of 550 °C for 24 h. All laboratory analyses, as well as the statistical analyses and the writing of this work, were conducted at the Animal Nutrition Laboratory of the Faculty of Animal Science and Ecology at the Autonomous University of Chihuahua.

2.2. Treatments, Microbial Growth

The study was conducted in Chihuahua, Chihuahua, Mexico, located at latitude 28°35′10.9″ N, longitude 106°6′26.6″ W, and an altitude of 1440 m above sea level. From the preliminary silage study, the second replicate of Silage 2 exhibited the lowest pH (4.38) among the 16 experimental replicates, a desirable characteristic of high-quality silage, and it was therefore selected for further experimental studies. After opening the plastic container, the silage was exposed to air for 12 h, simulating aerobic degradation during the exposure time to air that can occur in livestock feed production. The effects of the following treatments were tested: (1) Control (4 g of silage); (2) 4 g of silage plus 0.03 g of Chinook hops; (3) 4 g of silage plus 0.03 g of Galena hops; and (4) 4 g of silage plus 0.03 g of β-acid extract (hops antimicrobial components). The Chinook and Galena hops were acquired as pellets (38.58 and 36.02% dry matter, respectively) from Lupulin Exchange (Charlottesville, VA, USA). The composition of the hops, with α-acid contents of about 12 and 13%, and β-acid contents of about 3 and 9%, respectively, has been described previously [19]. The α-acids and β-acids from hops are reported to exhibit antimicrobial properties [20,21,22], with β-acids more specifically affecting amino acid-degrading, hyper-ammonia-producing bacteria [22]. Treatment mixtures were placed in 50 mL conical centrifuge tubes (three replicates per treatment per incubation time), to which 10 mL of sterile distilled water was added then mixed and incubated aerobically at 37 °C, and were examined at 0, 6, and 24 h for effects on the growth of microbial populations. According to the sampling schedule, three tubes per treatment were removed, from which 1 mL of sample fluid was used for serial dilutions in a sterile 0.4 M sodium phosphate solution, with a pH of 6.4, to determine effects on the growth of microbial populations within the air-exposed silage. The remaining sample from the tubes was removed at 24 h and dried in an oven at 65 °C for 24 h then ground to be used as a substrate in in vitro ruminal fermentation studies. The hops varieties Chinook and Galena were donated by the United States Department of Agriculture, Agricultural Research Service, Forage Animal Production Research Unit, Lexington, Kentucky.

Yeasts were plated on Petri dishes with Malt Extract Agar (BD Difco™, Sparks, MD, USA) and incubated for 24 h at 28 °C. Colony-forming units per milliliter (CFU/mL) were visually counted. Coliforms were plated on Petrifilm 3M (Minnesota Mining and Manufacturing Company, St. Paul, MN, USA) and incubated at 37 °C. pH was measured at all sampling times using a pH meter (Hannah Instruments), and ammonia concentration was determined [23] using UV-Vis spectrophotometry (Varioskan Flash v4.00.53, Thermo Scientific, Waltham, MA, USA). Butyric acid, acetic acid, and propionic acid concentrations were determined by gas chromatography with flame ionization detection. For this purpose, a Claurus 400^® gas chromatograph (Perkin Elmer, Waltham, MA, USA) equipped with a Varian capillary column CP-wax58 (FFAP) CB (15 m × 0.53 mm, 0.5 µm) was used. The initial oven temperature was raised to 115 °C, maintained for one minute, then increased by 10 °C per minute until reaching 190 °C. In this method, three isothermal minutes were given, and the carrier gas was helium at a constant pressure of 3 psi [24].

2.3. In Vitro Ruminal Fermentation

For in vitro ruminal fermentation, the selected silage (see above) was ground in an electric mill (Thomas-Wiley Laboratory Mill, Model 4, Thomas Scientific, Chadds Ford, PA, USA) to pass through a 1.0 mm sieve. Test tubes (18 mL) were used, with three replicates per treatment, containing 0.2 g of silage from hour 24 of the previous experiment, which had been previously dried (Control, Chinook, Galena, and β-acid extract). Ruminal microbial inoculum was obtained from three Holstein heifers with an average weight of 350 ± 1.5 kg, previously fed for 7 days with corn silage and water ad libitum. All animals used in this study were cared for according to the Internal Regulations of Bioethics and Animal Welfare of the Faculty of Animal Science and Ecology of UACH (2018) [22]. Ruminal fluid was extracted via an esophageal tube before the first feeding (8:00 a.m.), collected in a thermos pre-warmed to 39 °C and hermetically sealed, and transported to the laboratory where it was filtered through muslin, always under a stream of CO₂. The inoculum was then anaerobically distributed (10 mL/vial) under a stream of O₂-free CO₂ with the addition of a buffer solution 2:1 [25], and the vials were immediately sealed and incubated for 24 h at 110 rpm and 39 °C in an Orbital Shaker Incubator (New Brunswick Model Innova 4000, Nijmegen, The Netherlands). Additionally, three vials containing no substrate were prepared as blank controls. The chemical composition of the silage fed to the donor animals was as follows: 93.81% DM, 7.07% CP, 2.72% EE, 53.63% NDF, 30.53% ADF, and 10.58% ash, pH 4.38.

The molar concentrations of VFAs were determined by gas chromatography with flame ionization detection, following the previously described procedure [24]. After 24 h of incubation, the vials were placed on ice to stop the fermentation process and to proceed with sample analysis. Total gas production was measured (mmol/mL) using a FESTO^® pressure transducer (Siemens, Munich, Germany). Subsequently, 1 mL of gas sample was taken from each tube to determine its composition using a GOW-MAC series 580 gas chromatograph (GOW-MAC Instrument Co., Bethlehem, PA, USA) equipped with a Carbosphere^® packed column, 80/100. 5682PC. Nitrogen was used as the carrier gas at a flow rate of 20 mL/min, and levels (mmol/mL) of hydrogen, methane, and carbon dioxide were determined.

pH was measured with a pH meter (Hannah Instruments), and ammonia concentrations were determined following the technique described by Broderick and Kang [23] using UV-Vis spectrophotometry (Varioskan Flash, Thermo Scientific v4.00.53).

2.4. Statistical Analysis

2.4.1. Selection of Experimental Silage (Statistical Analysis)

A completely randomized design was used for preliminary Silages 1–4. The determinations of pH, DM, ash, EE, NDF, ADF, and CP were compared using a completely randomized analysis of variance performed using the GLM procedure of SAS v9.4 (SAS, 2014) with Duncan’s multiple range tests. The model used is shown in the following equation:

Yi = μ + Ti + εij

(1)

where Yi = the dependent variable; µ = overall mean; Ti = treatment effect (i = 1, 2, 3, 4); and εij = experimental error.

2.4.2. Treatments, Microbial Growth, and Fermentation (Statistical Analysis)

A completely randomized design with a factorial arrangement was used, with the factors being the hops treatment applied to the silage and time, as well as their interaction. An analysis of variance was performed using the GLM procedure of SAS v9.4 (SAS, 2014). The adjusted model included the effects of treatment, fermentation time, and the interaction between these two factors.

The equation for the adjusted model was as follows (Equation (2)):

Yi = μ + Ti + tj + Ti × tj + εij

(2)

where Yi = the dependent variable; µ = overall mean; Ti = treatment effect (i = 1, 2, 3, 4); tj = time (1, 2, 3); Ti xtimes tj = treatment × time interaction; and εij = experimental error.

Differences in log10 CFU/mL were measured at each sampling time for microorganisms, and the accumulations of ammonium and butyric acid were analyzed to determine the effects of the treatment using a completely randomized analysis of variance with least significant difference (LSD) mean separation.

2.4.3. In Vitro Ruminal Fermentation (Statistical Analysis)

A completely randomized design was used to evaluate the hops treatments applied. The analysis of variance was performed using the GLM procedure of SAS v9.4 (SAS, 2014). The determination of pH and the net accumulations of acetic, propionic, and butyric acids, as well as hydrogen and methane, were analyzed to evaluate the effects of the hops treatment using Duncan’s multiple range tests. The model follows the formulation given in Equation (1).

3. Results

3.1. Preliminary Silages: Bromatological Analysis

Silage 3 treatment showed the highest (p < 0.05) pH compared to the other treatments, followed by the silages without urea (Silages 1 and 2), which did not differ (p > 0.05) from each other (

Table 2. Proximate analysis¹ of preliminary maralfalfa grass silages (DM basis).

	Silages
	1	2	3	4	SEM	p-Value
pH	6.08 b	6.00 b	8.19 a	5.06 c	0.13	<0.01
Ash %	10.4 b	10.2 b	12.4 a	11.8 a	0.68	<0.05
Ether extract %	2.66	2.65	3.65	2.68	0.22	0.07
Crude protein %	8.92 b	5.71 c	6.67 c	11.9 a	0.61	<0.01
NDF %	61.8	56.7	63.9	61.7	14.0	0.09
ADF %	37.5 ab	29.8 b	42.4 a	38.5 ab	11.2	<0.05

Means in rows and with different superscript letters are statistically different (p < 0.05). NDF, neutral detergent fiber. ADF, acid detergent fiber.

). Silage 1 exhibited a higher (p < 0.05) DM content than Silage 3. Silage 3 presented a higher (p < 0.05) ADF content than Silage 2.

Silage 4 showed a higher (p < 0.05) CP content than the other treatments, followed by Silage 1. The lowest (p < 0.05) CP content was observed in Silages 2 and 3, which did not differ (p > 0.05) from each other. No treatment effect (p > 0.05) was found for EE and NDF content.

3.2. In Vitro Fermentation and Microbial Growth: Characteristics in the Aerobic Phase

3.2.1. pH Values

In the β-acid treatment at 6 h, the pH increased (p < 0.05) compared to the Control, while the other treatments (Galena and Chinook) did not show increases (p > 0.05). At 24 h, the Control exhibited a lower (p < 0.05) pH compared to the Chinook and Galena treatments (Figure 1).

3.2.2. Microbial Counts

No growth of E. coli/Coliforms was observed in any of the evaluated treatments. Yeast growth at 24 h in the aerobic phase of the evaluated silages is given in

Table 3. Yeast counts in silages containing 0.03 g of Chinook hops, Galena hops, and β-acid extract.

Treatment	Time			SEM	p-Value
	0	6	24
Control	3.90 a	4.82 ac	5.40 b	0.11	0.03
Chinook	3.90 a	4.48 a	5.05 bc
Galena	3.90 a	4.60 a	4.78 a
β-acids	3.90 a	3.94 a	3.74 a

^abc Means (CFU/g of silage) in columns and with different letters are significantly different (p < 0.05).

.

A significant interaction effect between treatment and time was found for yeast counts on malt agar (p = 0.027). The β-acid treatment showed a reduction in CFU/g; however, this decrease was not statistically significant (p > 0.05), effectively maintaining yeast populations under the Control treatment compared to the other treatments evaluated. In contrast, a significant increase in yeast populations was observed at 24 h for the Control and Chinook treatments (p < 0.05). The Galena treatment showed a similar trend to the Control and Chinook treatments, but the increase observed was not statistically significant compared to the values recorded at earlier sampling times (p > 0.05). As expected, the highest yeast count was observed in the Control treatment at 24 h (p < 0.05). An increase (p < 0.05) in yeast populations over time was evident, with the lowest counts at hour 0 and the highest at 24 h (Table 3).

3.2.3. Ammonia Concentrations

No interaction (p = 0.16) was found between the addition of hops and β-acids and the times evaluated in the study (

Table 4. Ammonia, acetic acid, and propionic acid concentrations in silages with the addition of 0.03 g of Chinook hops, Galena hops, and β-acid extract.

	Ammonia (µmol/g)	Acetic Acid (µmol/g)	Propionic Acid (µmol/g)	Butyric Acid (µmol/g)
Control	18.94 c	8.58	1.54	0.56
Chinook	19.56 bc	11.85	1.74	0.52
Galena	20.73 a	8.56	1.63	0.54
β-acids	21.73 ab	9.014	1.74	0.67
SEM	0.25	0.56	0.03	0.02
Time
0	19.31 b	20.03 a	1.91 a	0.94 a
6	20.62 a	6.25 b	2.11 a	0.44 b
24	20.80 a	2.22 b	1.07 b	0.33 b
SEM	0.17	3.30	0.20	0.11

^abc Means (µmol/mL) in columns and with different letters are significantly different (p < 0.05).

).

The β-acid hops treatment showed the highest (p < 0.05) ammonia concentration compared to Control and Chinook. No differences (p > 0.05) were found between the Galena and β-acid treatments or between the Control and Chinook treatments. The Control treatment showed the lowest (p < 0.05) value compared to the Galena and β-acid treatments. For the time factor, the lowest (p < 0.05) concentration was observed for the treatment at 0 h. Differences (p < 0.05) were found between treatments at 0 h compared to 6 and 24 h (Table 4).

3.2.4. Acetic, Propionic, and Butyric Acid Concentrations

For acetic, propionic, and butyric acid, no interaction was found between the evaluated hop treatments and time (p = 0.5432, p = 0.2742 and p = 0.2477), respectively. It can be seen that there were no significant differences in the concentration of acetic (Table 4) or propionic acids (Table 4) for the evaluated treatments (p > 0.05).

For the time factor in the acetic acids, it was determined that there were significant differences between 0 and 6 h (p < 0.05) (Table 4). Acetic acid concentration decreased fourfold over time, between 0 and 6 h (p > 0.05). At 24 h, the lowest value of acetic acid was obtained, statistically different from the other time points (p < 0.05). On the other hand, propionic acid concentration showed a marked increase at 6 h compared with 0 h, which subsequently declined at 24 h (p < 0.05). At 24 h, it showed the lowest concentration and was statistically different from the other time points (p < 0.05).

Butyric acid concentrations at different times in the evaluated silages also show the effect of the interaction between the evaluated treatments and the sampling times (Table 4). The treatment with Chinook hops showed a decrease (p < 0.05) in butyric acid concentration at 6 h, which persisted until 24 h.

Chinook and Galena treatments exhibited the lowest butyric acid concentrations compared to the Control and β-acid treatments (p > 0.05); however, these differences were not statistically significant. The β-acid treatment showed the highest butyric acid concentration, although this difference was not significant when compared with the other treatments.

For the time factor, the differences among values were significant (p < 0.05), showing a decrease in concentration over time, as presented in Table 4. The values at 6 and 24 h were not statistically different (p > 0.05). However, the value at 0 h was higher and significantly different compared with the other sampling times (p < 0.05).

3.3. In Vitro Ruminal Fermentation Results

pH and Total Gas, Hydrogen, Methane, Ammonia Gas, Carbon Dioxide Production, and VFAs

The in vitro ruminal fermentation characteristics of the studied silages at 24 h regarding pH, total gas production, hydrogen (H₂) production, methane (CH₄) production, and ammonia gas concentrations (

Table 5. Influence of Chinook hops, Galena hops, and β-acid extract on in vitro ruminal fermentation characteristics.

	Treatments				SEM	p-Value
	Control	Chinook Hops	Galena Hops	β-Acid Extract
Hydrogen 1, µmol/mL	ND	ND	0.011	ND	0.009	0.44
Methane 1, µmol/mL	15.0	15.4	14.7	15.7	2.54	0.77
Carbon dioxide 1, µmol/mL	31.9 ab	33.6 a	28.4 b	26.9 b	3.26	0.05
Ammonia, µmol/mL	7.58	7.83	8.69	8.29	2.82	0.09
pH	7.39	7.35	7.44	7.45	0.07	0.36
Total gas production, mL	11.5	11.1	10.2	9.69	1.12	0.21
Acetate, µmol/mL	9.64	8.82	16.9	30.7	5.43	0.09
Propionate, µmol/mL	0.702 b	1.04 b	1.76 b	6.02 a	1.07	0.02
Butyrate, µmol/mL	0.261	0.485	0.791	1.88	0.19	0.07
Acetate/Propionate ratio	7.16	8.27	13.2	5.10	16.9	0.36

Control, without additive. ¹ Headspace gases means in each row and with different letters are statistically different (p < 0.05). ND, not detected.

) did not show differences (p > 0.05) under the evaluated treatments. The treatment with the highest (Table 5) CO₂ concentration was the Chinook hops; however, there were no differences (p > 0.05) compared to the Control.

Table 5 presents the concentrations of volatile fatty acids (VFAs) from the in vitro ruminal fermentation of the silages. Acetate and butyrate concentrations did not differ significantly between treatments (p > 0.05). However, the β-acid treatment exhibited a higher propionate concentration compared to the other treatments (p < 0.05), while the Control, Chinook, and Galena treatments showed similar propionate levels (p > 0.05). No differences (p > 0.05) were found regarding the acetate/propionate ratios for the different evaluated treatments.

4. Discussion

4.1. Preliminary Silages and Chemical Composition

The maralfalfa plants were harvested at 90 days of growth at the beginning of spike formation (with less than 10%), showing a high water content in its chemical composition, which enhances the silage fermentation stage but also increases the proliferation of undesirable microorganisms, reducing the nutritional value of the feed [23]. Consistent with results from a study by Herrera et al. [20], an increase in nitrogen was observed along with an increase in urea concentrations in the preliminary Silage 3 and 4 treatments. These results suggest that once the feed is consumed by the animal, there is the potential for greater availability of urea for the ruminal microbiota and protein synthesis. Crude protein concentrations in silage should range between 5% and 7%, with higher values between 10% and 15% only expected in silages with forages such as alfalfa, due to the high availability of soluble protein [26]. Maralfalfa grass also has a protein content above 7% [27]. The production of lactic acid by LAB allows the ensiled plant material to preserve nutrients for a longer period, preventing the growth of microorganisms that degrade these nutrients [8]. The observed pH increase in the treatments with added urea can be explained by the addition of urea itself, as it reacts with water to produce ammonium hydroxide, raising the pH [28].

Silages 3 and 4 exhibited lower acid detergent fiber (ADF) values, indicating higher levels of soluble carbohydrates available for microbial metabolism, thereby enhancing feed quality. For neutral detergent fiber, the results were not significant, but they ranged between 44.0% to 71.9%, values established for this type of silage containing sorghum [29]. The addition of urea did not modify DM, ash, or EE, with results similar to those obtained by Santos da Silva et al. [5]. For silages with maralfalfa grass, the acid detergent fiber content is usually around 46% [30]. The values obtained in our study revealed greater fermentation of the feed, which could be due to the action of bacteria and fungi capable of degrading the structural carbohydrates of the cell wall [31].

4.2. Experimental Silages: Microbial Growth and Fermentation

The pH increase observed at 24 h in the Chinook and Galena treatments may be due to the β-acid concentrations in these varieties, which vary between hops varieties and are always lower than the β-acid extract used in this study. It is worth noting that this increase remains within acidic values, ensuring the antimicrobial activity of lupulones (one of the four β-acids found in hops, the others being adlupulone, colupulone, and prelupulone). It is known that lupulones are more effective under acidic conditions compared to alkaline conditions [32]. The pH increase is also associated with lower yeast growth in the β-acid extract treatment, as described by Bocquet et al. [33]. However, no established concentrations for yeast exist, differing from Siragusa et al. [34], who reported that hops do not have antifungal activity.

All experimental silages showed a slight increase in ammonia concentrations compared to the Control. These results indicate that all hop treatments slightly increased proteolysis, without exceeding the values described in the literature, in which a silage with ammonium levels below 10% is considered to be of good quality [31]. However, proteolysis results in inefficient nitrogen utilization. This is because the dissolved ammonia is absorbed through the ruminal epithelium, transported to the liver, and converted into urea, which represents a loss of utilizable nitrogen and increases the environmental footprint associated with ruminant production [32]. It also reduces the efficiency of microbial protein synthesis due to a lower concentration of peptides and free amino acids available for microbial activity. Special attention must be paid if ammonia levels are too high, as they may cause toxicity due to accumulation. This may lead to an imbalance in the ruminal microbial populations, favoring hyper-ammonia-producing bacteria [35,36].

Butyric acid production in silage is primarily driven by spore-forming bacteria, including the genera Clostridium spp., Bacillus spp., and Paenibacillus spp. [37,38]. This short-chain volatile fatty acid is not commonly found in high-quality silages. The literature reports a high likelihood of maralfalfa grass silage being degraded by Clostridium spp. [30]. High butyric acid concentrations lead to feed rejection by the animal, and the silage is considered to have low energy and protein content due to the degradation of these nutrients originally present in the forage [39], with it being established that 0.5%/MS is the maximum butyric acid concentration for considering a silage as good quality [26,28]. The butyrate concentrations obtained in this study did not exceed this value for any of the evaluated treatments, suggesting the absence of appreciable numbers of bacteria capable of producing butyric acid [32]. It is recognized that microorganisms that produce butyric acid are often spore-formers, so the drying process carried out in our treatments would be expected to be ineffective against them [40]. However, the effect of this parameter on spore-forming bacteria is still unclear.

4.3. In Vitro Ruminal Fermentation of Silage Treated with Hops and β-Acid Extract

The parameters of in vitro ruminal fermentation were not affected by the addition of hops or the β-acid extract, an important factor to consider regarding the use of these compounds in silages. However, there are inconsistencies among different studies conducted regarding the effect of hops as a modifier of ruminal fermentation. These inconsistencies are largely due to the plant variety, plant age, cultivation region, storage conditions, and other factors that must be considered when extracting the essential oil from the plant or when processing it [41]. It is known that β-acids can control certain populations of Gram-positive bacteria such as species belonging to Bacillus, Clostridium, Enterococcus, Listeria, Propionibacterium, Staphylococcus, and Streptococcus [38], with the latter containing lactic acid-producing bacteria that cause acidosis in animals on high-carbohydrate diets [34]. Additionally, Flythe et al. [42] reported that doses of 30 to 60 ppm of hops β-acids were inhibitory to amino acid-degrading Gram-positive involved in rumen hyper-ammonia-producing bacterial populations that could cause acidosis in animals on high-carbohydrate diets [43].

Thus, in practice, the application of β-acids may therefore present a strategy to prevent metabolic problems in animals due to the ability of these acids to disrupt bacterial membrane integrity, similarly to the action of ionophores [14]. In 2008, García-González et al. [44] added 0.7 g of hops to in vitro ruminal fermentation, with a substrate composed of alfalfa hay, grass hay, and barley grain, and did not observe any changes in gas production. On the other hand, Lavrenčič et al. [45], when using a totally mixed ration, observed decreases in gas production, as did Staerfl et al. [46], who found only a slight decrease in 24 h gas production when hops cones were added to a diet consisting of corn silage, soybean meal, and wheat grain. Differences in results between studies may be due to the use of different amounts of hops.

The variations that can be found in VFAs when using secondary hops metabolites as rumen modulators are not fully clarified in the literature [45]. The significant increase in propionic acid is linked to the greater antimicrobial effect of hops on Gram-positive bacteria, favoring the growth of Gram-negative genera such as Prevotella spp. This genus is highly prevalent in the rumen and has great versatility in metabolizing carbohydrates, producing large amounts of propionic acid that can be used by the animal for gluconeogenesis, increasing productive indicators such as meat and milk [43,47]. Moreover, the addition of 30 ppm of hops extracted with propylene to mixed cultures of rumen microbes or to pure culture of a Gram-negative, lactate-consuming ruminal bacterium, Megasphaera elsdenii, was not inhibitory to growth or metabolic output [42]. When grown under appropriate conditions, M. elsdenii can be an important producer of butyric or propionic acid [48]. As alluded to earlier, however, growth of an important lactic acid-producing bacterium, Streptococcus bovis, was inhibited by 30 ppm hops extract when likewise grown in pure culture [49]. In the Control treatment, propionic acid levels were quite low and very similar to those obtained in the Chinook and Galena treatments. However, in the β-acid treatment, the amount of propionic acid increased sixfold compared to the other treatments. This finding also suggests a potential enrichment of the lactic acid-consuming M. elsdenii, which is able to also produce propionic acid as a fermentation end product [49].

The explanation for this result could be that the concentration of β-acids in the pelleted Chinook and Galena varieties was lower than in the extract. While high concentrations of butyric acid in feedstuffs such as silage can cause negative palatability issues and even contribute to clinical ketosis within the host [50], an increase in butyric acid production in the rumen may represent greater availability of this volatile fatty acid (VFA) for the animal. Accordingly, the butyric acid produced in situ can be utilized in processes such as de novo synthesis of fatty acids in the mammary gland, potentially increasing milk fat content. Additionally, butyrate enhances ruminal mucosa absorption, leading to more efficient nutrient utilization by the animal. This difference in β-acid content could be used as a strategy to increase productive indicators while reducing the availability of hydrogen for methane synthesis [34,38].

5. Conclusions

The results suggested that the use of β-acid extracts could be effective in reducing the deterioration of silage once exposed to air. The addition of urea at the tested concentrations increased the nitrogen content in the silages, and the increases in sorghum grain content reduced pH significantly in the silages. This study showed that β-acids in treated silages could have a residual effect, favoring propionic acid production in ruminal fermentation.

In addition, butyric acid concentrations remained within an appropriate range to prevent silage deterioration. It was concluded that β-acids could help reduce silage deterioration during the aerobic phase, reducing the butyric acid and yeast populations, and their residual effect could improve ruminal fermentation, increasing propionate and acetate concentrations. However, further studies are needed to determine the optimal dose of β-acid extract to achieve the best results.