Nutritional evaluation of sorghum silage forage AGRI 002E in natura and ensiled with additives in the Tropics

Abstract

This study evaluated the impact of bacterial inoculant and urea on the chemical composition of AGRI 002E forage sorghum silage over different storage periods. Four treatments were tested: control (WS), bacterial inoculant (SBI), urea (US) and both urea and bacterial inoculant (USBI), with samples evaluated after 0, 28, and 56 days of ensiling. Parameters analyzed included organic acid, soluble carbohydrates (SC), condensed tannins (CT), ammonia nitrogen (NH3-N) dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), non-fibrous carbohydrates (NFC), lignin, <italic>in vitro</italic> digestibility (<italic>IV</italic>D), CP fractions, and total carbohydrate fractions. Results showed increased in DM, OM, total carbohydrates, NFC and digestibility over time, while CT, NDF, ADF, and SC decreased. Urea supplementation notably increased CP and <italic>IV</italic>DCP, and reduced fiber concentrations compared to the control and bacterial inoculant treatments. Silage stability was achieved after 28 days, but a 56-day period is recommended for optimal fermentation and quality. Urea alone improves digestibility and non-protein nitrogen availability, and combining it with bacterial inoculants may enhance aerobic stability.

Key words
bacterial inoculant; fermentation; organic acids; silage; urea

INTRODUCTION

Dairy farming has intensified its production processes over the years, resulting in an increasing demand for high-quality and sufficient feed to meet the nutritional needs of animals (Machado et al. 2012). In this context, the use of preserved feeds, especially silage, has become an essential strategy for production systems with high energy demands.

The ensiling technique aims to preserve the nutritional value of forages over extended periods while maintaining their original characteristics, provided that the forage exhibits traits conducive to proper fermentation, such as dry matter content around 30%, low buffering capacity and high soluble carbohydrate concentrations (Lucena et al. 2021, Pinho et al. 2017). Forage sorghum (Sorghum bicolor) is particularly valuable for silage production due to its high energy content, high digestibility, and its adaptability to low fertility and drought conditions (Getachew et al. 2016).

During the ensiling process, the presence of lactic acid bacteria (LAB) in the anaerobic environment is critical for fermentation) as they utilize sugars and soluble nitrogen compounds as substrates producing organic acids (acetic, propionic, butyric and lactic) that lower pH and inhibit undesirable fermentations (Santos et al. 2010).

In recent years, the use of biological and chemical additives in silage has been the subject of numerous studies aimed at optimizing fermentation, reducing losses, and improving the nutritional value of the silage (Bernardes & Chizzotti 2012, Kung Jr. et al. 2018). Biological additives, such as homofermentative bacteria, (Lactobacillus plantarum and Pediococcus) accelerate the drop in pH and enhance aerobic stability, while chemical additives, such as urea, help preserve nutrients and control undesirable reactions, particularly under critical storage conditions (Arriola et al. 2021, Saylor et al. 2020).

Recent studies have highlighted the importance of stabilization time for the success of the fermentation process and the final quality of the silage. The combination of inoculants and chemical additives, such as urea, with longer stabilization periods has shown promising results in improving aerobic stability and reducing degradation by yeasts (Arriola et al. 2021, Kung Jr. et al. 2018). This study aimed to evaluate the effect of different combinations of biological additives (homofermentative bacteria) and chemical additives (urea) on the chemical composition of forage sorghum silage AGRI 002E and on the quality of the silage over the stabilization periods.

MATERIALS AND METHODS

The experiment was conducted under field conditions, in an area of 0.62 hectares, geographically located by the coordinates 24°32’49,7’’S latitude, 54°01’46,4’W of longitude and 392 m of altitude. The climate of the Region is Cfa (Humid Subtropical Climate), with hot summers, low frost frequency and tendency to defined dry season (Alvares et al. 2013). During the period of development of forage sorghum AGRI 002E (Sorghum bicolor) (September to January), average monthly rainfall of 130 mm, 22.7°C and 71% of IVR was observed. The soil in this region is classified as clayey red eutroferric latosol (Embrapa 2013).

Sowing was performed by the no-tillage system with a population density of 6 seeds per linear meter (row) following a row spacing of 0.7 meters. Chemical fertilization was 300 kg ha-1 of the formula 8:20:20 (NPK) and nitrogen fertilization 150 kg ha-1 after 40 days of planting.

Sorghum agronomic characteristics were evaluated after 110 days of planting. Final plant stand (123,484 plants ha-1), average height (358 cm), stem (78.7%) and leaves (21.3%) in plant green matter (GM) and green matter yield were determined (149 ton ha-1) and dry matter (27.3 ton ha-1).

Sorghum was harvested when the forage reached 111 days after planting, according to the technical recommendation, with the aid of a tractor-mounted forage harvester, cutting height at 10 cm from the soil and particle size from 1 to 2 cm. The shredded material was placed on a plastic tarpaulin for the incorporation of the additives by homogeneous mixing and then filling and compacting the experimental Polyvinyl chloride (PVC) silos (10 cm in diameter x 50 cm in length), closed with a PVC lid with a valve. Bunsen type and sealed with adhesive tapes.

The treatments consisted of: silage without additives or control (WS), silage with inclusion of biological bacterial inoculant produced with strains of lactic bacteria (Lactobacillus plantarum and Pediococcus acidilactici) at a dose of 2 liters of solution containing two grams of inoculant for each ton (SBI) of green material, commercial-grade granulated (45N) urea-added silage at a dose of 5 g kg^-1 MV (US) and urea-added silage (5 g kg^-1 GM) + bacterial inoculant (2 g ton-1 of GM) (USBI). Samples were collected in natura and after 28 and 56 days of ensiling.

The silage samples were pre-dried in an oven with forced air ventilation at 55° C for 72 hours and then ground in a “Willey” type mill with a 1 mm sieve and submitted to bromatological analysis to characterize the product in natura and the silage times (28 and 56 days). Dry matter (DM - method 934.01), ashes (ASH - method 938.08) and crude protein (CP - method 981.10) were determined according to AOAC (2000). The determination of neutral detergent fiber (NDF) and acid detergent fiber (ADF) according to Van Soest et al. (1991). The organic matter (OM) content was estimated according to the equation, OM (%): 100 - % ASH.

Non-fibrous carbohydrate (NFC) levels were estimated according to the equations proposed by the NRC (2001). Soluble carbohydrates (SC) were determined according to the technique of Dubois et al. (1956). The lignin content (LIG) according to the methodology of Van Soest & Wine (1968). The concentration of condensed tannins according to Nozella (2001) methodology. Carbohydrate and protein fractionation were determined according to Sniffen et al. (1992) and Licitra et al. (1996), respectively.

Total nitrogen-based ammonia nitrogen (NH₃-N) was determined by sampling an aliquot of silage juice extraction using a hydraulic press (P15 ST) and further distillation with 2N potassium hydroxide (KOH) described by Fenner (1965) adapted by Vieira et al. (1980).

Organic acids (acetic, propionic, butyric and lactic) were determined by high-performance liquid chromatography (HPLC) with the ultraviolet detector (UV), according to the methodology of Lazaro (2009).

In vitro dry matter digestibility (IVDMD) was determined by the technique described by Tilley & Terry (1963) and adapted by Holden (1999). After weighing, the residue was burned in the muffle obtaining the in vitro organic matter digestibility (IVOMD). In vitro neutral detergent fiber digestibility (IVNDFD) was obtained according to the procedure of Goering & Van Soest (1975). In vitro crude protein digestibility (IVCPD) was performed according to the procedure described by Silva & Queiroz (2002).

The experimental design was completely randomized, with repeated measures over time (four treatments, four repetitions and three periods). Data normality was determined by the Shapiro-Wilk test and the results submitted to 5% significance level analysis of variance by the Dunnett test using the Sas PROC MIXED (mixed models) command (version 9.2, SAS Institute Inc. 2009).

RESULTS

The dry matter (DM) and organic matter (OM) contents were not influenced by the incorporation of additives (P>0.05) in the silage (Table I). Regarding silage times, DM and OM were higher (P≤0.05) at 28 and 56 days than in fresh material. The concentration of condensed tannins (TC) in forage sorghum silage was not influenced (P>0.05) by the use of additives, however, there was a decrease (P≤0.05) in the concentration of TC in silage concerning in natura material.

Table I
Dry Matter (DM), organic matter (OM), and condensed tannins (CT) of sorghum silage fresh with additives and after ensilage.

Treatment	Period (days)			SEM	Contrasts
	In natura	28	56		In natura vs 28	In natura vs 56	28 vs 56
Dry matter (g kg-1)
WS	133	173	170	1.96	<0.01	0.01	0.34
SBI	136	171	167	1.78	<0.01	<0.01	0.18
US	133	168	167	1.18	<0.01	<0.01	0.46
USBI	132	167	165	1.50	<0.01	<0.01	0.55
Organic Matter (g kg-1 DM)
WS	911	931	925	1.39	0.01	0.01	0.06
SBI	913	929	925	1.18	0.01	<0.01	0.13
US	909	930	924	1.09	<0.01	<0.01	0.01
USBI	910	927	929	1.72	0.01	<0.01	0.60
Condensed tannins (g kg-1 DM)
WS	3.44	2.23	0.88	0.33	0.05	0.01	0.04
SBI	3.47	2.17	0.84	0.70	0.14	0.03	0.15
US	3.58	1.51	0.60	0.71	0.15	0.02	0.49
USBI	3.31	1.59	0.79	0.28	0.01	<0.01	0.16

• Contrasts determined by Dunnett’s test (P≤0.05); SEM: standard error of mean; WS: witness silage; SBI: silage with bacterial inoculant; US: urea silage; USBI: Urea Silage + Bacterial Inoculant.

The neutral detergent fiber (NDF) of silage differed as a function of additives and changed its concentration as a function of silage times (P≤0.05) (Table II). The in natura material did not differ concerning the use of additives, however, at 28 days of ensiling the treatments with the addition of urea presented lower (P≤0.05) NDF concentration than the treatments WS and SBI, on the other hand, after 56 days of ensiling was the inverse (P≤0.05) in the NDF concentration in the urea silages concerning the treatments WS and SBI.

Table II
Fibrous fractions of sorghum silage fresh with additives and after ensilage.

Treatment	Period (days)			SEM	Contrasts
	In natura	28	56		In natura vs 28	In natura vs 56	28 vs 56
Neutral Detergent Fiber (g kg-1 DM)
WS	728	663	659	4.34	<0.01	<0.01	0.17
SBI	732	668	656	4.67	<0.01	<0.01	0.11
US	720	643	672	5.14	<0.01	<0.01	<0.01
USBI	738	651	679	4.58	<0.01	<0.01	<0.01
Acid Detergent Fiber (g kg-1 DM)
WS	505	437	451	4.92	<0.01	<0.01	0.18
SBI	510	440	450	3.39	<0.01	<0.01	0.10
US	494	441	452	5.09	<0.01	<0.01	0.23
USBI	507	439	446	4.10	<0.01	<0.01	0.31
Non-Fibrous Carbohydrates (g kg-1 DM)
WS	84.6	160	172	5.10	<0.01	<0.01	0.09
SBI	82.7	160	175	4.29	<0.01	<0.01	0.06
US	84.7	140	105	3.99	<0.01	<0.01	<0.01
USBI	80.5	133	97.3	5.94	<0.01	0.04	0.02
Lignin (g kg-1 DM)
WS	79.3	85.2	80.1	1.91	0.06	0.77	0.09
SBI	82.2	82.1	80.6	2.65	0.98	0.65	0.68
US	85.5	82.5	80.5	1.49	0.20	0.08	0.64
USBI	84.7	79.7	80.0	1.69	0.13	0.10	0.93

• Contrasts determined by Dunnett’s test (P≤0.05); SEM: standard error of mean. WS: witness silage; SBI: silage with bacterial inoculant; US: urea silage; USBI: Urea Silage + Bacterial Inoculant.

The NDF content did not differ (P>0.05) between 28 and 56 days of ensiling of the WS and SBI treatments but reduced (P≤0.05) concerning the in natura material. However, for US and USBI treatments, NDF after 28 days of ensiling was less than 56 days (P≤0.05), which decreased compared to in natura material.

The acid detergent fiber content of silages did not differ (P>0.05) between the silages evaluated concerning the collection periods. However, it influenced of silage period on ADF concentration of silages (P≤0.05), and at 28 days (439 g kg^-1 DM) and 56 days (450 g kg^-1). ensilage reduction was observed in the ADF of all treatments concerning in natura material (504 g kg^-1 DM).

Lignin contents were not influenced (P>0.05) using additives and did not change the content when the in natura material was evaluated with the determined silage times.

In the concentration of non-fibrous carbohydrates (NFC) of sorghum silage at 28 and 56 days of ensiling, it was found that the treatments with the presence of urea showed a reduction in NFC (P≤0.05) compared to other treatments, due to mainly from the decrease in total carbohydrates.

The WS and SBI silages showed an increase (P≤0.05) of the NFC contents at 28 and 56 days of silage compared to fresh material. For urea-added silages (US and USBI), the highest NFC concentration was determined at 28 days of silage with subsequent reduction at 56 days, which were higher (P≤0.05) than in natura material.

Soluble carbohydrate (SC) content in natura sorghum material did not differ between treatments (P>0.05), however, after 28 and 56 days of ensiling, there was a reduction in treatments with the inclusion of additives concerning the control treatment (P≤0.05) (Table III). Soluble carbohydrate concentration for all treatments decreased (P≤0.05) during the ensiling period.

Table III
Soluble Carbohydrates (SC) and carbohydrate fractionation (A, B₁, B₂ and C) of sorghum silage fresh with additives and after ensilage.

Treatment	Period (days)			SEM	Contrasts
	In natura	28	56		In natura vs 28	In natura vs 56	28 vs 56
Soluble Carbohydrates (g kg-1 DM)
WS	121	89.8	63.0	3.22	<0.01	<0.01	<0.01
SBI	116	79.8	51.6	2.38	<0.01	<0.01	<0.01
US	116	77.9	51.7	3.09	<0.01	<0.01	<0.01
USBI	118	79.1	51.5	3.07	<0.01	<0.01	<0.01
Fraction A (g kg-1 TC)
WS	98.0	74.2	52.4	2.89	<0.01	<0.01	<0.01
SBI	95.9	70.1	42.9	2.30	<0.01	<0.01	<0.01
US	93.5	61.0	40.3	2.58	<0.01	<0.01	0.01
USBI	95.2	61.9	40.5	2.84	<0.01	<0.01	0.02
Fraction B1 (g kg-1 TC)
WS	46.4	143	174	2.43	<0.01	<0.01	0.02
SBI	47.9	150	187	2.96	<0.01	<0.01	<0.01
US	50.4	138	116	2.05	<0.01	<0.01	0.03
USBI	47.3	138	117	2.12	<0.01	<0.01	0.04
Fraction B2 (g kg-1 TC)
WS	675	618	616	7.21	<0.01	0.01	0.74
SBI	675	617	612	7.07	0.01	0.01	0.31
US	673	638	685	6.20	0.01	0.23	<0.01
USBI	674	638	684	6.25	0.01	0.28	<0.01
Fraction C (g kg-1 TC)
WS	180	165	157	3.53	0.03	<0.01	0.17
SBI	181	163	158	5.47	<0.01	<0.01	0.26
US	184	162	159	2.94	0.01	<0.01	0.37
USBI	183	162	158	4.04	0.01	<0.01	0.41

• Contrasts determined by Dunnett’s test (P≤0.05); SEM: standard error of mean. WS: witness silage; SBI: silage with bacterial inoculant; US: urea silage; USBI: Urea Silage + Bacterial Inoculant.

For carbohydrate fractionation, fraction A decreased over the ensiling periods. The silages with urea inclusion presented lower fraction A concerning WS and SBI silages at 28 days of ensiling. At 56 days of silage, the additives presented lower fraction A compared to the control treatment.

To fraction B₁, in natura material did not differ between treatments (P>0.05). For the ensiling times of 28 and 56 days, there was an increase (P≤0.05) in the WS and SBI silages compared to the urea addition silages. Also, WS and SBI silages increased (P≤0.05) the content of fraction B₁ throughout the fermentation process, while US and USBI silages increased up to 28 days of silage and decreased in the following period (56 days).

Fraction B₂ was influenced (P≤0.05) by the addition of urea after 28 days of fermentation, being superior to the other treatments (WS and SBI). Regarding the WS and SBI treatments, there was a reduction (P≤0.05) of fraction B₂ after ensiling, however, for treatments US and USBI the reduction of fraction B₂ occurred only at 28 days of ensiling, being similar between the in natura material and silage with 56 days of silage.

For the carbohydrate fraction C, there was no difference (P>0.05) between the treatments. For the ensiling times, there was a reduction (P≤0.05) of the fraction C content in the ensiled material (28 and 56 days) concerning the in natura material for all treatments.

Crude protein from urea-treated sorghum silages (US and USBI) differed (P≤0.05) from treatments without additives (WS) and from bacterial inoculant-treated silage (SBI), presenting higher crude protein concentration due to the inclusion of non-protein nitrogen (NPN) from urea (Table IV). By contrasting the treatments, by evaluating the silage times with in natura forage sorghum material, it was found that the addition of urea increased (P≤0.05) the crude protein concentration at 28 and 56 days of silage in the treatments US and USBI compared to in natura material. However, for WS and SBI silages there was no difference (P> 0.05) between the evaluated periods.

Table IV
Crude protein, ammonia nitrogen in relation to total nitrogen (NH₃-N) and protein fractionation (A, B₁, B₂, B₃ and C) of sorghum silage fresh with additives and after ensilage.

Treatment	Period (days)			SEM	Contrasts
	In natura	28	56		In natura vs 28	In natura vs 56	28 vs 56
Crude Protein (g kg-1 DM)
WS	75.4	74.8	73.8	1.36	0.78	0.44	0.64
SBI	73.7	72.5	72.2	1.73	0.60	0.16	0.87
US	81.6	122	129	3.24	<0.01	<0.01	0.19
USBI	82.4	124	121	2.53	<0.01	<0.01	0.45
NH3-N (g kg-1 DM)
WS	12.1	12.0	11.8	0.24	0.95	0.47	0.58
SBI	11.8	11.7	11.5	0.31	0.66	0.13	0.80
US	13.2	19.5	20.8	0.49	0.01	0.01	0.18
USBI	13.2	19.9	19.4	0.43	<0.01	0.01	0.44
Fraction A (g kg-1 CP)
WS	79.9	546	505	9.25	<0.01	<0.01	0.05
SBI	60.8	516	512	12.7	<0.01	<0.01	0.79
US	153	693	720	13.1	<0.01	<0.01	0.21
USBI	170	713	710	11.8	<0.01	<0.01	0.88
Fraction B1 (g kg-1 CP)
WS	20.5	23.6	19.0	3.00	0.56	0.71	0.40
SBI	16.7	24.2	26.2	2.01	0.06	0.01	0.57
US	7.5	20.2	25.4	2.60	0.02	0.01	0.20
USBI	7.3	14.7	17.0	1.75	0.02	0.01	0.28
Fraction B2 (g kg-1 CP)
WS	432	188	254	8.76	<0.01	<0.01	0.01
SBI	464	219	236	13.1	<0.01	<0.01	0.29
US	458	155	128	10.3	<0.01	<0.01	0.06
USBI	446	134	115	11.1	<0.01	<0.01	0.23
Fraction B3 (g kg-1 CP)
WS	281	123	102	9.74	<0.01	<0.01	0.10
SBI	261	118	111	7.24	<0.01	<0.01	0.56
US	238	60.0	59.7	6.72	<0.01	<0.01	0.98
USBI	233	66.9	86.0	3.50	<0.01	<0.01	0.02
Fraction C (g kg-1 CP)
WS	187	119	121	6.09	0.01	<0.01	0.91
SBI	197	122	114	4.14	<0.01	<0.01	0.13
US	143	72.3	66.9	3.35	<0.01	<0.01	0.30
USBI	144	71.2	71.1	4.49	<0.01	<0.01	0.99

• Contrasts determined by Dunnett’s test (P≤0.05); SEM: standard error of mean. WS: witness silage; SBI: silage with bacterial inoculant; US: urea silage; USBI: Urea Silage + Bacterial Inoculant.

Ammonia nitrogen concerning total nitrogen (NH₃-N) was higher (P≤0.05) in US and USBI silages, due to the application of urea to the ensiled material. Throughout the evaluated periods, the WS and SBI silages did not present alterations (P>0.05) in the concentration, however, the urea contributed to the increase (P≤0.05) of NH₃-N from 28 days of silage.

In evaluating protein fractionation, fraction A is influenced by the addition of urea in silage, since the treatments with the inclusion of this additive presented higher (P≤0.05) concentration than the other silages tested. Also, this fraction showed great variation (P≤0.05) along with the fermentative profile, increasing its concentration at 28 and 56 days concerning the material evaluated on the day of ensiling.

Considering that the addition of urea caused an increase in fraction A levels concerning WS and SBI treatments, for fraction B₁ the opposite occurred, with the treatments treated with urea there was lower (P≤0.05) concentration of fraction B₁. As with the most soluble fraction, there was an increase (P≤0.05) of fraction B₁ at 28 and 56 days of ensiling for treatments with additives.

For fractions B₂, B₃ and C, higher levels of certain fractions in WS and SBI silages were found, and all treatments showed similar reduction patterns (P≤0.05) of these fractions at 28 and 56 days of silage differing from in natura material.

Total organic acids from sorghum silage differed between treatments (P≤0.05), presenting higher concentration of organic acids in the treatment with the inclusion of bacterial inoculant when determined in the in natura material and higher concentrations in treatments WS and USBI at 28 days’ ensilage, not different between the ensilage treatments at 56 days. The same effect previously mentioned between treatments was observed for lactic acid concentration (Table V).

Table V
Organic acids (mmol L-1) of sorghum silage fresh with additives and after ensilage.

Treatment	Period (days)			SEM	Contrasts
	In natura	28	56		In natura vs 28	In natura vs 56	28 vs 56
Total Organic Acids (mmol L-1)
WS	190	84.6	70.4	2.54	<0.01	<0.01	0.01
SBI	27.5	77.2	67.6	1.41	0.01	<0.01	0.02
US	17.9	73.0	69.8	2.44	<0.01	<0.01	0.45
USBI	20.5	83.2	68.5	1.65	<0.01	<0.01	0.01
Lactic Acid (mmol L-1)
WS	17.1	60.7	51.0	1.51	<0.01	<0.01	0.01
SBI	24.7	50.6	47.9	1.19	<0.01	<0.01	0.43
US	16.5	52.5	50.7	1.89	<0.01	<0.01	0.60
USBI	16.7	56.9	51.0	1.76	<0.01	0.01	0.09
Acetic Acid (mmol L-1)
WS	ND	20.7	16.9	1.26	<0.01	<0.01	0.07
SBI	0.53	22.8	16.3	0.65	<0.01	<0.01	0.01
US	ND	18.0	14.7	0.75	<0.01	<0.01	0.04
USBI	1.13	22.4	13.7	0.95	<0.01	<0.01	0.01
Propionic Acid (mmol L-1)
WS	1.91	3.18	2.45	0.21	0.01	0.11	0.08
SBI	2.19	3.77	3.09	1.06	0.37	0.07	0.69
US	1.48	2.55	2.94	0.24	0.04	0.01	0.32
USBI	1.71	3.09	2.70	0.12	0.01	0.01	0.16
Butyric Acid (mmol L-1)
WS	ND	ND	ND	-	-	-	-
SBI	ND	ND	0.22	0.13	1.00	0.36	0.33
US	ND	ND	1.45	0.23	1.00	0.01	0.02
USBI	0.93	0.81	1.11	0.49	0.82	0.79	0.57

• Contrasts determined by Dunnett’s test (P≤0.05); SEM: standard error of mean. WS: witness silage; SBI: silage with bacterial inoculant; US: urea silage; USBI: Urea Silage + Bacterial Inoculant.

The concentrations of acetic acid and propionic acid did not differ between treatments (P>0.05), however for the butyric acid content only at 56 days of ensiling the treatments with urea inclusion were higher (P≤0.05) than the treatments. control and inoculant treatments.

Regarding the ensiling times, the behavior was similar for the concentration of lactic, acetic and propionic acid, where there were higher values (P≤0.05) of the contents at the 28 days of ensiling compared to the 56 days, which was above in natura material of sorghum silage, reflecting in the same effect for the concentration of total organic acids. For the butyric acid concentration, there was the only difference in the treatment with urea inclusion (P≤0.05), in which after 56 days of ensiling there was an increase concerning the other days evaluated.

In vitro dry matter digestibility (IVDMD) of sorghum silage with bacterial inoculant was lower (P≤0.05) than WS, US, and USBI when determined from in natura material (Table VI). At 28 days of silage there was no difference (P>0.05) between treatments, however, at 56 days the silages treated with additives presented lower IVDMD than silage without additives. The WS and SBI silages increased IVDMD at 28 and 56 days compared to in natura material. However, in the silages with urea inclusion (US and USBI) at 28 days of ensiling presented higher digestibility (P≤0.05) compared to the other evaluation times of the ensiled material.

Table VI
In vitro digestibility of dry matter and nutrients of sorghum silage fresh with additives and after ensilage.

Treatment	Period (days)			SEM	Contrasts
	In natura	28	56		In natura vs 28	In natura vs 56	28 vs 56
In vitro dry matter digestibility (g kg-1DM)
WS	661	711	722	9.46	<0.01	<0.01	0.40
SBI	632	708	685	7.01	<0.01	<0.01	0.13
US	684	721	694	8.36	0.02	0.45	0.05
USBI	673	735	689	7.13	<0.01	0.12	<0.01
In vitro organic matter digestibility (g kg-1DM)
WS	671	728	747	10.9	0.01	<0.01	0.29
SBI	638	717	710	11.0	0.01	<0.01	0.74
US	701	739	722	8.83	0.02	0.15	0.22
USBI	684	762	722	5.87	<0.01	0.01	<0.01
In vitro crude protein digestibility (g kg-1DM)
WS	686	685	698	6.44	0.97	0.75	0.73
SBI	665	661	667	4.61	0.93	0.93	0.88
US	773	808	830	12.9	0.24	0.01	0.43
USBI	739	845	833	13.1	0.01	<0.01	0.62
In vitro neutral detergent fiber digestibility (g kg-1DM)
WS	681	669	676	5.59	0.10	0.55	0.29
SBI	677	653	654	7.31	0.03	0.02	0.89
US	698	664	686	8.68	0.10	0.17	0.22
USBI	663	674	677	5.45	0.27	0.11	0.72

• Contrasts determined by Dunnett’s test (P≤0.05); SEM: standard error of mean. WS: witness silage; SBI: silage with bacterial inoculant; US: urea silage; USBI: Urea Silage + Bacterial Inoculant.

The addition of urea in silage caused higher IVOMD in the in natura and 28 days of ensilage compared silage to inoculated and untreated silage. At 56 days there was no effect (P>0.05) of the additives on sorghum silage IVOMD. As with IVDMD, the IVOMD of ST and IBS increased after silage (28 and 56 days) concerning in natura silage. For silage with exclusively urea inclusion, in natura silage presented lower IVOMD than the material fermented for 28 days but did not differ from silage with 56 days of fermentation. However, for silage with urea inclusion associated with a bacterial inoculant, the IVOMD at 28 days of ensiling was higher than the other periods evaluated.

The treatment of sorghum silage with urea resulting from the increase (P≤0.05) of crude protein digestibility (IVCPD) in all ensilage levels and no in natura material concerning other damages. Developing the fermentation effect on the ensiled material may also cause an increase (P≤0.05) of IVCPD concerning the in natura material. Inclusion of bacterial inoculant alone does not interfere (P>0.05) in digestibility, as does silage without additives.

Regarding the in vitro neutral detergent fiber digestibility (IVNDFD), it was found that the addition of bacterial inoculant (SBI and USBI) presented lower IVNDFD in the in natura material (P≤0.05) than urea silage. After 28 days of silage, no differences were observed between silages, but with the advance of fermentation time (56 days), the exclusive bacterial inoculant reduced the IVNDFD compared to the other treatments.

DISCUSSION

The DM of sorghum silage AGRI 002E, considered low for the recommended standards for a good silage fermentation that should be above 250 g kg^-1 (Nussio et al. 2011), however, its high concentration of soluble carbohydrates and low buffer capacity contributed to a good fermentation of the ensiled material.

According to Herrera et al. (2024) of the fractions that make up the structure of sorghum plants, the panicle is mainly responsible for the increase in the DM content of the ensiled material followed by the leaves and stem. However, as forage sorghum AGRI 002E did not contain panicles and the stem represented the largest portion of the plant (>70%), it may have contributed to the higher moisture content of the ensiled material.

The dry matter content increased in the silage material compared to the fresh material in all silages tested, due to losses associated with the production of water, gas, and heat during the silage process, as well as the production of effluents during the fermentation (Oliveira et al. 2024). These losses are caused by microbial activity and chemical reactions that convert sugars into acids, CO₂, and water during fermentation, and are exacerbated by the initial aerobic phase of ensiling, which results in the production of CO₂, water and heat. Although these losses are inevitable, they can be minimized with proper silage management (Wróbel et al. 2023).

Forage sorghum AGRI 002E, being a low grain variety, belongs to the class of low-tannin type II sorghum (Bullard & York 1996), as determined in the present work, where the levels were less than 4 g kg^-1 MS. The reduction in TC during ensiling process occurs independently of the use of additives, as determined by Osman (2004), who reported reductions of 15 to 35% in CT concentration after the fermentation period. Kondo (2004) suggests that the microbial activity responsible for fermentation contributes to the degradation of condensed tannins into low molecular weight phenolic compounds, which may enhance the palatability and nutritional value of the silage.

The fermentation process reduced the NDF content in the silage compared to the fresh material, mainly due to the consumption and degradation of the more soluble fractions (SC) and potentially degradable components (hemicellulose and cellulose) by microorganisms (Ávila et al. 2014). However, the inclusion of bacterial inoculants in the control silages did not significantly alter the NDF concentration after 28 and 56 days of ensiling, as (28 also observed by Pereira et al. 2008). This could be explained by the greater adherence and preference of lactic acid bacteria (LAB) for the soluble substrates available in the silage.

The addition of urea to forage sorghum silage resulted in a reduction in NDF content after 28 days of ensiling, followed by an increase at 56 days. The initial reduction can be attributed to the action of urea on the cell wall, promoting the breakdown of intermolecular bonds, as seen in cellulose (Yahaya et al. 2001). Conversely, the increase in NDF at 56 days may be due to the formation of large volumes of effluent during the fermentation process, leading to the loss of soluble substrates and increasing the concentration of cell wall components, which have a slower degradation rate.

The fiber content of the forage is mainly determined by plant genotype (Di Marco et al. 2009) and the leaf blade ratio (Elseed et al. 2007). Neumann et al. (2004) reported NDF levels ranging from 650 to 742 g kg^-1 in forage sorghum silages, especially in larger plants with a higher stem proportion and little to no panicle development. This results in higher concentrations of hemicellulose, lignin and soluble carbohydrates, due to the greater accumulation of tissues responsible for forage support (Yosef et al. 2009).

ADF concentrations were not influenced by the additives and remained constant after the fermentation period (28 and 56 days). Tolentino et al. (2016) also reported no changes in ADF concentrations when evaluating the fermentative profile of sorghum hybrids.

The high concentration of soluble carbohydrates in the ensiled material promotes rapid microbial growth, organic acid production and a decrease pH, which enhances the exposure of fibrous components and consequently increases hemicellulose degradation (Halmemies-Beauchet-Filleau et al. 2013). However, urea has a buffering function, preventing a sharp pH drop in the presence of high soluble carbohydrate concentrations, as observed in the ensiled material.

The variation in CNF concentration in silage is primarily due to the reduction of structural carbohydrates throughout the fermentation process. The addition of urea led to lower CNF concentrations at 56 days compared to 28 days, likely due to the reduced hemicellulose degradation in urea treated silages and the higher crude protein content in these silages. NFC values are estimated based on the concentration of mineral matter, CP, NDF and EE.

Lignin concentrations remained unchanged during ensiling because lignin, a phenolic compound present in plant cell walls, is resistant to microbial degradation (Mnich et al. 2017). Higher lignin concentrations result in reduced digestibility, as they lower the availability of cellulose and hemicellulose, which are potentially degradable and limit feed utilization by the animal.

Carbohydrates are the main energy substrates for microbial fermentation. The lower concentration of total carbohydrates in urea-added silages is mainly attributed to the increase in crude protein content, as total carbohydrate concentration decreases when the mineral matter, CP and EE increase.

The concentration of soluble carbohydrates (SC) and starch in forage plants is crucial for the quality of the fermentation process. The reduction of SC in ensiled material compared to fresh material is due to plant enzymatic activity and microbial fermentation, resulting in the production of organic acids like lactic and acetic acid, which regulate the pH of the silage (Zhang et al. 2016).

Significant reductions in SC concentration were observed at 28 days is silages with bacterial inoculants and in all silages with additives by 56 days. This is likely due to the increased population of LAB, which rapidly consumed SC to promote anaerobic conditions. This result aligns with Naeini et al. (2014), who reported a reduction in SC concentration after 30 days of ensiling in saccharin sorghum.

In the present study, the use of additives improved the efficiency of SC degradation compared to untreated material, likely due to the increased LAB population (inoculation) and enhanced environmental conditions provided by urea. However, Kleinschmit & Kung Jr. (2006) did not observe differences in SC concentrations after the fermentation process in corn silages with and without bacterial inoculants.

Regarding carbohydrate fractionation (Sniffen et al. 1992), there was a reduction in fraction A, which corresponds to SC, throughout the fermentation process. This decrease is due to the high degradability of SC in this fraction. Fraction B₁, corresponding of starch, pectin and glucans, increased during the fermentation process due to the rise in non-fibrous carbohydrate content.

Fraction B₂, which is associated with the NDF content of the material, decreased as a result of hemicellulose and cellulose hydrolysis during ensiling. Fraction C also decreased throughout fermentation, likely due to the relative increase in fraction B₁.

The CP content of fresh forage sorghum was 78.3 g kg^-1 DM, which falls within the ideal range (60.0 to 90.0 g kg^-1) for ensiling (Pedreira et al. 2003). Analysis of the silages, both with and without bacterial inoculant, revealed minimal changes in CP content before and after ensiling. This stability suggests low proteolysis during the fermentation, indicating minimal dry matter losses during the preservation of the silage material.

The inoculation of lactic acid bacteria serves to enhance the population of these bacteria in the environment, increase organic acid production and reduce undesirable fermentation, as well as proteolysis to some extent (Ding et al. 2013). Simon et al. (2009) reported 80.30 g kg^-1 in fresh sorghum silage, which decreased to 76.1 g kg^-1 after 90 days of ensiling. Similarly, Ribeiro et al. (2007) observed CP levels of 73.2 g kg^-1 in fresh material, which decreased to 64.4 g kg^-1 by the 56th day of ensiling for five forage sorghum genotypes.

The use of urea as an additive in ensiled material increases the availability of non-protein nitrogen (NPN) for microorganisms during fermentation, thereby reducing true protein proteolysis (Pires et al. 2009). The addition of urea led to a significant increase in CP concentration, ranging from 82.0 g kg^-1 DM in fresh material to 126 g kg^-1 DM after 56 days of ensiling. Santos et al. (2018) also observed an increase in CP from 31.2 g kg^-1 to 123 g kg^-1 DM with urea addition. Vieira et al. (2004) demonstrated that the adding 0.5% urea to forage sorghum silage increased CP content by approximately 40% compared to untreated silage (105 versus 74.0 g kg^-1 DM).

Urea is an effective additive for improving the nutritional value of silage, especially in forage sorghum with a low grain-to-grain ratio (Neumann et al. 2010). The use of urea also increased NH₃-N concentration throughout the fermentation process, with notable increases at 28 and 56 days compared to fresh material.

The rise in ammonia nitrogen content is attributed to the buffering action of urea, which helps maintain pH levels and provides conditions conducive to proteolysis. N-NH₃-N can originate from various biochemical processes during fermentation, including decarboxylation, deamination, ureolysis and oxidation and reduction reactions (McDonald et al. 1991).

In materials with high SC concentrations, such as the sorghum evaluated, the addition of urea has a beneficial effect on the anaerobic environment. Urea helps control yeast populations responsible for ethanol production and undesirable fermentation by increasing ammonia levels (Lopes & Evangelista 2010). The NH₃-N concentrations observed were lower than those reported by Vieira et al. (2004), who measured 157 g kg^-1 of DM in sorghum silage treated with the same level (0.5%) of urea. However, these results are consistent with normal conditions, given that fresh sorghum plants typically contain around 10 g kg^-1 DM of ammonia nitrogen relative to total nitrogen.

During fermentation, NH₃-N values typically increase, with normal levels ranging from 70 to 100 g kg^-1 DM (Kleinschmit & Kung Jr. 2006). The values observed in this Study are lower than these ranges. It is important to note that high concentrations of NH₃-N do not necessarily indicate poor material quality, as they can result from the increased nitrogen availability due to urea addition (Gonçalves et al. 2009).

The increase in protein fractions A and B₁ during fermentation of forage sorghum is primarily due to the enhanced protein content and proteolysis converting sorghum protein to NPN (Andrade et al. 2010, Pires et al. 2009). In high-grain sorghum, protein hydrolysis is limited due to the dense protein matrix, whereas forage hybrids exhibit more intense proteolysis, especially with urea addition (Rooney et al. 2007). This result in a higher proportion of NPN in silage (Viana et al. 2012). Protein fractionation varies among sorghum cultivars due to climatic conditions and cultivar suitability (Behling Neto et al. 2017). High NPN levels are crucial for rumen function as ammonia supports microbial activity (Sá et al. 2010). Silages without urea showed higher levels of fractions B₁, B₂ and B₃ indicating greater protein availability for rumen microorganisms (Sá et al. 2010).

Organic acids, key indicators of fermentation quality, increased with ensiling time due to high soluble carbohydrate content (Schmidt et al. 2007, Oliveira et al. 2023).

Lactic acid concentrations were similar across silages with and without additives, likely due to sufficient SC and a high population of epiphyte bacteria (Carvalho et al. 2014). Low acetate and propionate levels were observed, likely due to the reduced activity of acetic and propionic bacteria (Santos et al. 2010, Güney et al. 2007). Butyric acid levels were low, indicating effective inhibition of clostridia, which is essential for maintaining silage quality (Machado et al. 2012).

The increase in IVDMD and IVOMD in silage is attributed to reduced fiber content and high soluble carbohydrate levels (Huhtanen et al. 2006, Ntaikou et al. 2008). Urea addition improved IVCPD by increasing NPN availability (Araki et al. 2017). The reduction IVNDFD with bacterial inoculants likely resulted from enhanced microbial action (Dehghani et al. 2012, Desta et al. 2016).

CONCLUSIONS

The addition of urea to the ensiling process of forage sorghum AGRI 002E significantly improved the nutritional value of the silage, increasing crude protein content, ammonia nitrogen concentration, and crude protein digestibility, while reducing neutral detergent fiber and non-fibrous carbohydrate contents. Urea-supplemented silages adequately stabilized after 28 days of storage, making this the minimum recommended period to obtain a stable and quality forage mass.

However, to maximize the benefits of additives and ensure long-term maintenance of nutritional quality, it is recommended that silage be stored for up to 56 days. This additional period guarantees complete fermentation and stabilization, especially in conditions where moisture content or the initial forage composition may not be ideal.

Therefore, we recommend the use of urea as an additive in forage sorghum silages, particularly in systems aiming to increase the availability of non-protein nitrogen and optimize nutrient digestibility. The combination of urea with bacterial inoculants, although showing more modest results compared to urea alone, can also be considered in situations where aerobic stability is a concern.

Acknowledgements

The present work was carried out with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Financing Code 001.

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Publication Dates

• Publication in this collection
  22 Sept 2025
• Date of issue
  2025

History

• Received
  31 Oct 2023
• Accepted
  27 May 2025