Effects of Adding Lactobacillus Inoculants on the Nutritional Value of Sesbania cannabina and Whole Corn Mixed Silage

Abstract

This study evaluated the potential of utilizing Sesbania cannabina, produced during saline–alkali soil improvement, as a high-quality feed resource for ruminants. Mixed silages were prepared by combining S. cannabina and whole corn at ratios of 1:1 and 1:3, with or without a compound Lactobacillus (LAB) inoculant, and were assessed for fermentation quality, nutrient composition, ruminal degradation, intestinal digestibility, and energy value. Results: The addition of Lactobacillus (LAB) inoculants increased lactic acid content, crude protein effective degradability (CPED), gross energy (GE), and dry matter apparent digestibility (DMAD), while decreasing ammonia nitrogen (NH₃-N), acetic acid (AA), propionic acid (PA), neutral detergent fiber (NDF), acid detergent fiber (ADF), rumen undegradable protein (RUP), intestinal crude protein degradability (ICPD), and intestinal digestible crude protein (IDCP). Increasing the proportion of whole corn increased dry matter (DM) and gross energy (GE), while reducing crude protein (CP), NDF, ADF, Ash, rumen degradable protein (RDP), RUP, IDCP, and the effective ruminal degradability of NDF (NDFED) and ADF (ADFED). Overall, a 1:1 mixing ratio maximized S. cannabina utilization without compromising feeding value, and LAB inoculation ensured successful ensiling while enhancing nutrient utilization.

1. Introduction

Protein is one of the essential components of livestock feed. Increasing the proportion of protein-rich forage in the ruminant diet is an effective way to enhance both the yield and quality of animal products [1,2]. As animal husbandry develops and the demand for meat, eggs, and milk continues to rise, the supply of protein-rich feedstuffs such as soybean meal remains insufficient. This shortage has led to rising prices and has become a major constraint on the sustainable growth of the livestock industry [3]. Therefore, the discovery and utilization of existing high-quality protein resources has become a hot topic in animal husbandry. In this context, exploring underutilized forage crops with the dual benefits of high protein yield and adaptability to marginal lands has become a strategic priority.

Sesbania cannabina, an annual legume with a high protein content, thrives in degraded environments such as saline–alkaline soils and waterlogged areas. It exhibits rapid growth under moderate-to-high salinity and provides dual benefits: nutritional value as livestock forage and soil remediation through ecological functions [4,5]. Field trials revealed that green manuring with S. cannabina significantly improved soil parameters: a pH reduction by 0.5 units (p < 0.05), a 22.68% decrease in electrical conductivity, and a 25.65% increase in organic carbon content [6]. The protein content is as high as 23.8% [7], which can be compared with alfalfa. Studies have shown that it can be used as green or hay for livestock, particularly small ruminants, because of easy digestion and low fiber fraction [8]. S. cannabina can be used in the diets of lambs (up to 40%) as alternatives to the commonly used alfalfa feed without detrimental effects on growth performance. Moreover, feeding of S. cannabina produced carcass characteristics similar to that of alfalfa, which can improve the meat quality of lambs. This might be because they reduce the content of fat and collagen, and increase the tenderness of the meat and the color of the fat [9,10,11]. Despite its high nutritional value, S. cannabina contains significant levels of tannins, which may pose limitations for its use in animal feed. Bhatta [12] reported a total tannin content of 13.2 g/kg DM in its leaves, primarily composed of hydrolyzable tannins. These compounds form complexes with proteins, reducing digestibility and causing astringency in the mouths of animals [13,14]. Therefore, strategies to mitigate tannin effects—such as ensiling—are essential to improve its feeding value. Studies have shown that tannins usually affect the digestibility of proteins by forming tannin–protein complex precipitates in the form of hydrogen bonds between the hydroxyl groups on tannins and the amide or peptide bonds on proteins, thereby reducing essential amino acids. Silage is not only a potential way to effectively degrade the anti-nutritional factors of forage, but can also preserve the nutritional components and active substances of forage. Zhang et al. [15] explored and compared the degradation effects of tannase and lactic acid bacteria on tannin in feed, and found that lactic acid bacteria M6 had a significant degradation effect on tannin in Anthocephalus chinensis Lam (p < 0.05). When the S. cannabina is silage alone, the fermentation often fails due to its low water-soluble carbohydrates (WSCs) content and strong buffering ability. A large number of studies have shown that mixing leguminous forage with WSC-rich grass and adding LAB are effective means of improving the fermentation quality of silage and improving the success rate of silage [16,17]. Tahir [16] revealed that inoculation with a compound LAB additive altered the consequences of aerobic exposure by increasing acetic acid production after ensiling, promoting diverse bacterial populations, and mitigating the negative effects of fungi on the aerobic stability of mixed silage of S. cannabina and whole corn.

During saline–alkali soil improvement through S. cannabina cultivation, substantial S. cannabina is yielded. Utilizing this biomass for livestock production would leverage its forage value to alleviate protein feed shortages, while fully exploiting land resources unsuitable for grain crops—thereby avoiding competition between livestock and staple food production. This study aimed to evaluate the effects of LAB and corn ratio—the ratio of S. cannabina to whole corn (1:1 or 1:3)—on the fermentation and nutritional value of mixed silage, and provide a theoretical basis for feeding with S. cannabina.

2. Materials and Methods

All animal management and experimental procedures followed the animal care protocols approved by the Animal Care and Use Ethics Committee of China Agricultural University (Approval No.: AW13305202-1-1).

The study employed a 2 × 2 factor design to evaluate the effects of Lactobacillus (LAB) inoculation and the mixing ratio of S. cannabina and whole corn on silage quality, nutrient composition, rumen degradation, intestinal digestibility, and energy value. The experimental procedure of this study is shown in Figure 1.

2.1. Silage Sample Preparation

S. cannabina (Yanjing 1, at the early flowering stage) and whole corn (Jingke 932, at the half milky stage) were collected from the experimental field of the Institute of Genetic Development, Tongliao City, Inner Mongolia, China on 30 August 2023.

Table 1. Chemical composition of S. cannabina and the whole corn (%, DM basis).

	Sesbania cannabina	Whole Corn
DM (%)	30.47	28.20
CP (%)	20.74	8.27
NDF (%)	46.43	49.63
ADF (%)	36.32	29.75
WSC (%)	6.13	18.00
Starch (%)	2.18	5.37

All abbreviations: DM (dry matter), CP (crude protein), EE (Ether extract), NDF (neutral detergent fiber), ADF (acid detergent fiber), WSCs (water-soluble carbohydrates).

shows the chemical compositions of S. cannabina and the whole corn. The harvested forages were processed by a forage chopper (92-2S, Sida Agri-Machine Co., Ltd., Luoyang, China) to achieve a particle size of approximately 1 to 2.0 cm. The chopped S. cannabina and whole corn materials were then mixed at the following ratios: 1:1 and 1:3. Two primary groups were treated with and without the inoculant, resulting in four treatments (1:1−, 1:1+, 1:3−, 1:3+), each replicated three times. Lactobacillus inoculants included Lactobacillus plantarum B90 (CGMCC No. 13318), Lactobacillus farciminis GMX4 (CGMCC No. 19434), Lactobacillus buchneri NX205 (CGMCC No. 16534, currently known as Lentilactobacillus buchneri NX205), and Lactobacillus hilgardii 60TS-2 (CGMCC No. 19435). The LAB inoculants were applied to the forage of mixed S. cannabina and whole corn at a rate of 10⁶ CFU/g fresh weight (FW), while for the control group without adding LAB inoculants, an equal amount of sterilized water was applied to the forage of mixed S. cannabina and whole corn. The mixture was packed using a mechanical packing machine (9YDB-55, Kaichuang Machinery Manufacturing Co., Ltd., Qingdao, China), with each package weighing approximately 75 kg. Silage bags were stored in a dry, dark environment at room temperature (25–30 °C) and were opened for sampling according to their respective treatments.

2.2. Sensory Evaluation

The sensory indicators such as color, odor, and texture of the mixed silage of S. cannabina and whole corn plants were comprehensively evaluated by using the scoring system of the German Agricultural Association (DLG).

2.3. Analysis of Fermentation Quality and Nutrient Composition of Mixed Silage

Fresh silage (10 g) was homogenized with 90 mL of deionized water and subjected to aqueous extraction at 4 °C for 24 h. After filtering through qualitative filter paper, the pH value was immediately determined. The remaining filtrate was stored at −20 °C. Ammonia nitrogen was determined using the phenol–sodium hypochlorite colorimetric method [18], and organic acids were determined using high-performance liquid chromatography [19]. Silage samples were weighed and oven-dried at 65 °C for 48 h to achieve a constant weight. The dried samples were then ground to pass through a 40-mesh screen and stored in sealed, light-protected bags for further analysis. The obtained powder samples were stored for later analysis of crude Ash, ADF, Ether extract (EE), NDF, and total nitrogen (TN). TN × 6.25 was used to calculate CP [20]. After reaction with an anthrone reagent, Van Soest et al.’s procedures were used to determine ADF and NDF contents [21].

2.4. In Vitro Ruminal Degradability and Gastro-Intestinal Digestibility Analysis

• Step 1. Ruminal degradation

The nylon bag experiment was carried out in January 2024. A total of four three-and-a-half-year-old Zhaowuda mutton sheep with a body weight of 54.75 ± 1.43 kg, similar constitutions, good health, normal deworming and immunization, and permanent rumen fistulas were selected as test animals. The pre-feeding period of this experiment was seven days, and the formal experiment began after the test sheep had acclimated to the metabolic cage and the test diets. The dietary composition and nutrient levels are as follows (

Table 2. Dietary composition and nutrient levels (air-dry basis).

Ingredients	Content
Concentrate feed 1 (%)	28.57
Compound grass particles 2 (%)	71.43
Total (%)	100
Nutrient composition (%)
DM (%)	94.02
CP (%)	10.03
EE (%)	2.13
NDF (%)	48.71
ADF (%)	32.46
Ca (%)	1.41
P (%)	0.86
Ca/P (%)	1.64

¹ The main components of the concentrate are corn, sugarcane molasses, soybean meal, puffed soybeans, barley, wheat bran, corn germ meal, soybean husk, NaCl (sodium chloride), CaHPO₄ (superphosphate), CaCO₃ (calcium carbonate), MgO (magnesium oxide), and sodium chloride. ² The main components of the compound grass pellets are oat hay and corn stalks. Supplementation of trace elements, minerals, and vitamins is provided in the form of licks. The main components of lick blocks are Cu (copper), Fe (iron), Zn (zinc), Mn (manganese), Se (selenium), Co (cobalt), I (iodine), vitamin E, and vitamin A. All abbreviations: DM (dry matter), CP (crude protein), EE (Ether extract), NDF (neutral detergent fiber), ADF (acid detergent fiber), Ca (Calcium), P (phosphorus), Ca/P (calcium/phosphorus).

).

The in situ degradability of DM, CP, ADF and NDF in the four silage treatments were determined according to the procedure described by Mehrez and Ørskov [22]. A secondary-use nylon bag with a diameter of 48 μm and a specification of 6 × 10 cm was selected. The silage samples to be tested were grounded and passed through an 8-mesh sieve, then dried at 65 °C for 8 h before use and moistened for 24 h. After weighing the mass of each nylon bag, the silage samples were loaded into each bag in portions of approximately 3 g and sealed with a rubber band. The nylon bag was attached to the bottom of a semi-plastic hose with an inner diameter of 3 mm and a length of 25 cm. Each set of four bags served as a parallel, and each group of four sheep constituted four replicates. A total of 320 bags were analyzed. The experiment adopted the principle of simultaneous injection and sequential extraction. The nylon bags were placed into the rumen abdominal sac before morning feeding at 7 a.m., and removed from the rumen fistula at 4, 8, 16, 48, and 72 h, respectively [23]. They were then immediately washed with water until the water from the final rinse was clear. The evaluation of each silage treatment was conducted in sequence. A 48 h rest period followed each 72 h test cycle. The cleaned nylon bag was placed in the oven at 65 °C for 48 h to achieve a constant weight. Subsequently, the total mass of the nylon bag and residue was measured, and the residue was collected into a No. 4 zip-lock bag for the subsequent determination of DM, CP, ADF, and NDF content.

• Step 2. Gastric digestion of mixed silage residue after rumen incubation

After 16 h of rumen incubation, the nylon bags were removed, washed until the water was clear, and then placed in 0.1% methylcellulose solution and incubated at 37 °C with shaking for 30 min. Then, the nylon bags were removed and rinsed, and dried in an oven at 65 °C until constant weight was achieved (48 h).

1 g of rumen non-degraded residue prepared was placed into a nylon bag (5 cm × 10 cm), sealed, and placed into a culture bottle with thirty nylon bags per bottle in the Daisy II incubation system (Ankom Technology Corp., Macedon, NY, USA). The culture vial contained 2 L of a preheated hydrochloric acid solution with 1 g/L of pepsin (P-7000, Sigma, Kawasaki, Japan) and had a pH of 1.9. The culture vial was placed in an ANKOM Daisy II simulated fermentation incubator in vitro and cultured at 39 °C for one hour [24].

• Step 3. Intestinal digestion of mixed silage residue after rumen and gastric incubation

After one hour of culture, the nylon bag was removed and cleaned, and two liters of a preheated 0.5 mol/L phosphate (KH₂PO₄-K₂HPO₄) buffer solution containing 3 g/L of trypsin (P-7545, Sigma) and 0.05 g/L of thymol was added. The pH of the solution was adjusted to 7.75. The culture vial was placed in an ANKOM Daisy II simulated fermentation incubator in vitro and cultured at 39 °C for 24 h [24].

2.5. In Vitro Ruminal Degradability and Gastro-Intestinal Digestibility Calculation

After all the analysis, the values of ruminal degradability and gastro-intestinal digestibility were calculated as follows:

XD = (X_{silage before ruminal incubation} − X_{silage after ruminal incubation})/X_{silage before ruminal incubation}

(1)

ICPD = (CP_{silage before Intestinal degradation} − CP_{silage after Intestinal degradation})/CP_{silage before Intestinal degradation}

(2)

IDCP = (CP − RDP) × ICPD + RDP × 0.9 × 0.7

(3)

where

▪

XD is the rumen degradation rate of X;

▪

ICPD is the intestinal degradation rate of crude protein;

▪

IDCP is the intestinal digestible protein, CP is the crude protein content of silage, RUP is rumen nondegradable protein, RDP is rumen degradable protein, 0.9 is the efficiency of conversion of rumen-degraded protein to microbial protein, 0.7 is the digestibility of microbial protein in the small intestine.

2.6. Calculations of Degradation Kinetics Parameters

The kinetic parameters of in situ degradation were calculated based on the measured degradabilities at all five time points. The data of instant degradability were fitted using the following exponential equation:

Y = a + b(1 − e^−ct)

(4)

where Y is the nutrient disappearance at time point t, a is the rapidly degradable fraction, b is the potentially degradable fraction, c is the degradation rate of fraction b (%/h), and t is the time (h) of incubation.

ED = a + bc/(c + k)

(5)

where a, b, and c are the same as those in Equation (1), and k is the rumen outflow rate. In this study, the rumen outflow rate was set by referring to previous studies, roughages 3.14%/h [25].

2.7. Determination of Nutrient Digestion Metabolism

The digestion and metabolism test was carried out in March 2024. The composition of raw materials in the experiment was formulated using the same batch of raw materials to ensure the consistency of raw materials. On the dry matter basis, the basal diet was replaced by silage in the experimental group, and the replacement ratio of silage was 50%.

There were four replicates, with one sheep per replicate. The experiment lasted for 8 days, including 5 days of pre-test feeding and 3 days of formal experiment. All sheep were dewormed before the trial, weighed before morning feeding, and acclimated to metabolic cages. The feed intake of the sheep with the lowest feed intake in the pre-trial period was taken as the feeding amount in each group during the trial period.

The sheep were fed at 07:00 and 16:00 and could drink water freely. During the formal trial period, feces and urine were collected using the total collection method, the feces and urine volume of each sheep were weighed and recorded every day, and the feces samples were sampled at the rate of 10%. Finally, the feces samples of each sheep for 3 days were mixed and frozen. The urine was collected in a plastic bucket containing 100 mL 10% H₂SO₄ to prevent the precipitation of uric acid and the loss of protein during storage. After the urine was fully mixed every day, it was filtered with gauze, and then 1% was sampled. Finally, the urine sample of each sheep for 3 days was mixed and frozen at −20 °C for the determination of urine energy.

The content of nutrients in feed and feces was determined by the same method as in Section 2.3. The total energy of feed, fecal energy and urine energy were determined by using the oxygen bomb tester recommended in ISO9831:1998 [26].

2.8. Calculation of Apparent Digestibility and Effective Energy Value

After all the analyses, the values of apparent digestibility and the apparent digestibility and effective energy of the test diet were determined by substitution methods and calculated as follows [27,28]:

XAD_diet(%) = (Daily feed intake × X content in the diet − Daily excretion volume × X content in feces)/(Daily feed intake × X content in the diet)

(6)

XAD_silage(%) = (X of intake − X in feces − dietary intake of X × XAD_diet)/(Intake of silage × X content in silage)

(7)

DCP_silage(g/kg) = (Total dietary protein intake − Total fecal protein − basal dietary protein intake × apparent digestibility of protein in the base diet)/silage intake × 1000

(8)

CH4(g/d) = 2.5325 − 0.0122CP(g/d) + 0.0016DE(kJ/d) + 0.0175CF(g/d) − 0.0489EE(g/d)

(9)

CH4-E(MJ/d) = CH4(L) × 0.03954(MJ/L)

(10)

DE(MJ/d) = GE(MJ/d) − FE(MJ/d)

(11)

ME(MJ/d) = DE(MJ/d) − UE(MJ/d) − CH4 − E(MJ/d)

(12)

Silage XE(MJ/kg) = (Base diet XE value − (base diet E value − replacement group diet E value))/replacement ratio

(13)

where

▪

XAD_diet is apparent digestibility rate of X in the diet;

▪

XAD_silage is apparent digestibility rate of X in the silage;

▪

DCP_silage is digestible crude protein in the silage;

▪

Silage XE is X energy in the silage.

2.9. Statistical Analyses

Excel 2019 was used to collect data, SPSS 21 was used to conduct variance analysis, Duncan’s test was used to conduct multiple comparisons of the mean values of each group, and Python 3.9 was used to model and calculate the kinetic parameters of rumen degradation parameters: fast degradation rate a, slow degradation rate b, and slow degradation rate c. In the statistical results, p < 0.05 showed a significant difference.

3. Results

3.1. Sensory Quality

Table 3. Effects of adding LAB on sensory quality of Sesbania cannanbina and whole corn mixed silage.

Sensory Quality
Item	1:1−	1:1+	1:3−	1:3+
Smell	14	14	14	14
Texture	4	4	4	4
Color	1	1	2	2
Score	19	19	20	20
Rank	excellent	excellent	excellent	excellent

“1:1” and “1:3” are the mixing ratios of sesbania and whole corn plants, and “+” and “−” indicate whether LAB is added or not, the same applies below.

shows that all silage groups had pH values below 4.0 and exhibited a brown-green coloration without butyric acid odor. The silages displayed pleasant aromatic characteristics, indicating excellent fermentation quality.

3.2. Fermentation Quality

The effects of adding LAB on the fermentation quality of S. cannabina and whole corn mixed silage are summarized in

Table 4. Effects of adding LAB on fermentation quality of Sesbania cannanbina and whole corn mixed silage.

Fermentation Quality
Item	LAB	1:1	1:3	T (LAB)	M (Ratio)	T × M
pH	−	3.77 ± 0.04	3.69 ± 0.02	0.18	0.18	0.22
	+	3.69 ± 0.01 b	3.69 ± 0.09 b
NH3-N (mg/kg DM)	−	4.57 ± 0.55 Aa	3.73 ± 0.31 b	<0.05	<0.05	0.17
	+	3.73 ± 0.32 Bb	3.51 ± 0.13 b
LA (mg/kg DM)	−	55.88 ± 9.96 Bb	61.27 ± 7.20 Ba	<0.05	<0.05	0.83
	+	62.31 ± 27.55 Ab	75.86 ± 10.81 Aa
AA (mg/kg DM)	−	10.07 ± 0.01 Aa	9.87 ± 0.01 Ab	<0.05	<0.05	<0.05
	+	9.65 ± 0.01 Ba	8.80 ± 0.01 Bb
PA (mg/kg DM)	−	10.01 ± 0.05	9.99 ± 0.09	<0.05	<0.05	<0.05
	+	10.13 ± 0.08 a	8.91 ± 0.01 b

NH₃-N, ammonia nitrogen content; LA, lactic acid content: AA acetic acid content; PA, propionic acid content. ‘1:1′ and ‘1:3′ represent the mixing ratios of S. cannabina to whole corn, and ‘+’ and ‘−’ indicate the presence or absence of LAB, respectively. The data in the table is ’mean ± SD’. In the same line, different lowercase letters indicate significant differences (p < 0.05). In the same column and with the same indicator, different capital letters indicate significant differences (p < 0.05).

. In mixed silage, the addition of LAB and increasing whole corn increased the content of LA (p < 0.05), reduced the content of NH3-N, AA, and PA (p < 0.05), and had no significant effect on pH (p > 0.05). Moreover, the addition of LAB and increasing whole corn had a synergistic effect on the reduction in PA and AA (p < 0.05). Adding LAB and increasing the whole corn ratio significantly enhanced the reduction effect of PA and AA. Therefore, the fermentation quality of 1:3+ is the best.

3.3. Nutrient Composition

The effects of adding LAB on the nutrient composition of S. cannabina and whole corn mixed silage are summarized in

Table 5. Effects of adding LAB on nutrient composition of Sesbania cannanbina and whole corn mixed silage.

Nutrient Composition
Item	LAB	1:1	1:3	T (LAB)	M (Ratio)	T × M
DM	−	34.33 ± 1.86	33.52 ± 1.06	0.06	<0.05	<0.05
	+	33.05 ± 1.13 b	37.59 ± 0.59 a
CP (%DM)	−	12.10 ± 0.71 a	10.87 ± 0.29 b	0.07	<0.05	0.73
	+	11.71 ± 0.19 a	10.31 ± 0.18 b
NDF (%DM)	−	57.18 ± 3.98 a	46.91 ± 3.37 Ab	<0.05	<0.05	<0.05
	+	51.56 ± 1.61 a	43.83 ± 9.64 Bb
ADF (%DM)	−	45.58 ± 1.11 a	41.27 ± 5.26 b	0.83	<0.05	0.95
	+	45.00 ± 2.78	44.32 ± 3.00
EE (%DM)	−	2.18 ± 0.14	2.16 ± 0.08	0.69	0.9979	0.86
	+	2.13 ± 0.06 a	2.15 ± 0.02 a
Ash	−	5.79 ± 0.23 a	4.63 ± 0.24 b	0.57	<0.05	0.76
	+	5.89 ± 0.56 b	4.56 ± 0.36 c

DM, dry matter content; CP, crude protein content; NDF, neutral detergent fiber content; ADF acid detergent fiber content; EE, crude fat content; Ash, ash content. ‘1:1’ and ‘1:3’ represent the mixing ratios of S. cannabina to whole corn, and ‘+’ and ‘−’ indicate the presence or absence of LAB, respectively. The data in the table is ’mean ± SD’. In the same line, different lowercase letters indicate significant differences (p < 0.05); In the same column and with the same indicator, different capital letters indicate significant differences (p < 0.05).

. In mixed silage, the addition of LAB can significantly reduce the content of NDF (p < 0.05), and there is a tendency to increase the content of DM (p = 0.06) and reduce the content of CP (p = 0.07). Increasing the proportion of whole corn can significantly increase the DM content (p < 0.05) and reduce the contents of CP, NDF, ADF and Ash (p < 0.05). Moreover, adding LAB and increasing the proportion of whole corn had a synergistic effect on increasing the DM content of mixed silage and reducing the NDF content (p < 0.05).

3.4. Effects of Adding LAB Inoculant S. cannabina and Whole Corn Mixed Silage on Rumen Effective Degradation Rate of Nutrients

The effects of adding LAB on the rumen effective degradation rate of nutrients of S. cannabina and whole corn mixed silage are summarized in

Table 6. Effects of adding LAB on rumen effective degradation rate of Sesbania cannanbina and whole corn mixed silage.

Ruminal Effective Degradability
Item	LAB	1:1	1:3	T (LAB)	M (Ratio)	T × M
DMED (%)	−	55.66 ± 3.30 Bb	62.01 ± 0.04 a	0.12	0.74	0.61
	+	63.33 ± 2.50 A	63.15 ± 1.47
CPED (%)	−	76.05 ± 1.47 B	74.62 ± 1.22 B	<0.05	0.10	0.08
	+	80.00 ± 0.64 A	80.21 ± 0.93 A
NDFED (%)	−	50.58 ± 2.04 a	42.96 ± 4.97 b	0.13	<0.05	0.81
	+	47.72 ± 0.80 a	40.56 ± 1.38 b
ADFED (%)	−	25.43 ± 3.40 a	17.45 ± 0.68 Bb	0.71	<0.05	<0.05
	+	22.60 ± 4.60	20.25 ± 2.72 A

DMED, dry matter rumen effective degradation rate; CPED, rumen effective degradation rate of crude protein; NDFED, neutral detergent fibrinol rumen effective degradation rate; ADFED, effective rumen degradation rate of acid detergent fiber. ‘1:1′ and ‘1:3′ represent the mixing ratios of S. cannabina to whole corn, and ‘+’ and ‘−’ indicate the presence or absence of LAB, respectively. The data in the table is ’mean ± SD’. In the same line, different lowercase letters indicate significant differences (p < 0.05); In the same column and with the same indicator, different capital letters indicate significant differences (p < 0.05).

. In mixed silage, the addition of LAB can significantly increase CPED (p < 0.05). Increasing the proportion of whole corn can significantly reduce NDFED and ADFED (p < 0.05). Moreover, when silage is mixed in a ratio of 1:1, the addition of LAB has no significant effect on ADFED, while at a 1:3 ratio, the addition of LAB can significantly increase ADFED.

3.5. Intestinal Digestible Protein

The effects of adding LAB on the intestinal digestible protein of S. cannabina and whole corn mixed silage are summarized in

Table 7. Effects of adding LAB on intestinal digestibility and intestinal degradation characteristics of Sesbania cannanbina and whole corn mixed silage.

Intestinal Digestible Protein
Item	LAB	1:1	1:3	T (LAB)	M (Ratio)	T × M
RDP (g/kg)	−	92.02 ± 4.79 a	81.11 ± 2.47 b	0.40	<0.05	0.98
	+	93.68 ± 2.61 a	82.70 ± 1.63 b
RUP (g/kg)	−	28.98 ± 1.51 A	27.59 ± 0.84 A	<0.05	<0.05	0.18
	+	23.42 ± 0.65 Ba	20.40 ± 0.40 Bb
ICPD (%)	−	72.75 ± 7.88 A	70.13 ± 2.98 A	<0.05	0.5095	0.79
	+	59.73 ± 6.14 B	59.47 ± 5.23 B
IDCP (g/kg)	−	79.06 ± 4.12	70.45 ± 2.15 A	<0.05	<0.05	0.95
	+	73.01 ± 2.03 a	64.23 ± 1.27 Bb

RDP, rumen degradable protein; RUP, rumen non-degradable protein; ICPD, small intestine degradation rate of crude protein; IDCP, small intestine digestible crude protein. ‘1:1′ and ‘1:3′ represent the mixing ratios of S. cannabina to whole corn, and ‘+’ and ‘−’ indicate the presence or absence of LAB, respectively. The data in the table is ’mean ± SD’. In the same line, different lowercase letters indicate significant differences (p < 0.05). In the same column and with the same indicator, different capital letters indicate significant differences (p < 0.05).

. In mixed silage, the addition of LAB significantly reduced the RUP, ICPD, and IDCP (p < 0.05). Increasing the proportion of whole corn significantly reduced the RDP, RUP, and IDCP (p < 0.05). The IDCP of 1:1− is higher than that of the other groups, and the digestible protein in the small intestine is a key index to evaluate the quality of feed, so it is considered that 1:1− is better.

3.6. Effective Energy and Apparent Digestibility

The effects of adding LAB on the effective energy and apparent digestibility of S. cannabina and whole corn mixed silage are summarized in

Table 8. Effects of adding LAB on nutrient apparent digestibility and effective energy of Sesbania cannanbina and whole corn mixed silage.

Effective Energy and Apparent Digestibility
Item	LAB	1:1	1:3	T (LAB)	M (Ratio)	T × M
GE (MJ/kg)	−	16.47 ± 0.20	16.73 ± 0.06	<0.05	<0.05	0.45
	+	16.65 ± 0.23	17.13 ± 0.14
DE (MJ/kg)	−	13.96 ± 1.18	12.50 ± 0.98	0.52	0.11	0.30
	+	13.07 ± 1.00	13.19 ± 0.76
ME (MJ/kg)	−	11.78 ± 0.55	10.29 ± 1.98	0.58	0.42	0.20
	+	10.87 ± 0.42	10.99 ± 0.27
DMAD	−	68.25 ± 11.58 B	71.22 ± 6.72 B	<0.05	0.82	0.51
	+	78.48 ± 3.02 A	76.17 ± 6.45 A
CPAD	−	80.66 ± 11.68	82.76 ± 8.02	0.35	0.17	0.45
	+	87.29 ± 7.26	86.41 ± 7.27
NDFAD	−	61.96 ± 9.44	65.28 ± 10.45	0.47	0.94	0.58
	+	68.73 ± 6.60	66.18 ± 12.05
ADFAD	−	62.12 ± 3.00	68.98 ± 8.12	0.17	0.13	0.98
	+	68.38 ± 6.19	71.99 ± 12.08
DCP (g/kg)	−	97.60 ± 14.13	82.76 ± 8.02	0.58	<0.05	0.95
	+	102.22 ± 8.50	86.41 ± 7.27

GE, gross energy; DE, digestive energy; ME; metabolic energy; DMAD, dry matter apparent digestibility; CPAD, crude protein apparent digestibility; NDFAD, neutral detergent fiber apparent digestibility; ADFAD, acid detergent fiber apparent digestibility; DCP, can digest crude protein. ‘1:1′ and ‘1:3′ represent the mixing ratios of S. cannabina to whole corn, and ‘+’ and ‘−’ indicate the presence or absence of LAB, respectively. The data in the table is ’mean ± SD’. In the same column and with the same indicator, different capital letters indicate significant differences (p < 0.05).

. In mixed silage, the addition of LAB significantly increased GE and DMAD (p < 0.05). Increasing the proportion of whole corn significantly increased GE (p < 0.05) and reduced DCP (p < 0.05). Moreover, no interaction was observed between the two treatments on the effective performance and apparent digestibility of mixed silage.

4. Discussion

4.1. Effect of Adding LAB and Increasing the Proportion of Whole Corn on the Fermentation Quality of the Mixed Silage

Legumes are not suitable for silage alone, mainly because of their low sugar content, which makes it difficult to support a good lactic acid fermentation; at the same time, having a high buffering capacity, they tend to maintain a high pH, which inhibits the activity of LAB [29]. Moreover, due to the lack of WSCs, they are susceptible to spoilage during silage, which affects the quality of silage [30]. Therefore, the addition of LAB and mixing legume forage with grass forage for silage are both effective means to improve the fermentation quality of legume forage [31,32].

The pH of silage is considered to be a key indicator for evaluating silage quality [33], The addition of LAB had no significant effect on the pH of the mixed silage, probably because the mixed silage contained higher soluble sugars, which were sufficient to support the growth and fermentation of LAB [34]. Therefore, even without the addition of additional LAB, the natural LAB in the silage were able to function and convert the sugars into lactic acid, resulting in a decrease in pH to near the same level. The NH₃-N content of the 1:1− group was significantly higher than that of the rest of the groups (p < 0.05), which may be due to the lack of WSCs, and the activity of the LAB may not be strong enough to rapidly inhibit the activity of other microorganisms [35], which can cause the ammonia production from proteolysis, which was alleviated by the addition of LAB.

4.2. Effect of Adding LAB and Increasing the Proportion of Whole Corn on the Nutrient Composition of the Mixed Silage

The evaluation of the nutritional components of silage is one of the methods to assess the quality of silage, which is of great significance for guiding the application of silage in livestock production.

DM is an indicator for measuring organic matter accumulation and feed value. The addition of LAB shows a trend of increasing the DM content of mixed silage (p = 0.06). Increasing the proportion of whole corn can significantly increase the DM content of mixed silage (p < 0.05), and the addition of LAB and the increase in the proportion of whole corn plants had a synergistic effect on increasing the DM content (p < 0.05). This might be because the DM content of the whole corn plant is higher than that of S. cannabina. Increasing the proportion of the whole corn improves the DM content of the silage raw materials. The addition of LAB improved the fermentation quality and reduced the loss of DM during silage [35].

CP content is an important index reflecting the nutritional quality of silage; the higher the CP content, the better the nutritional quality. In this study, the Kjeldahl method was mainly used to determine the CP content of silage, so the difference in CP content of silage mainly comes from the difference in CP content of raw materials and NH₃-N produced by the decomposition of proteins and amino acids during the silage process. The CP content of S. cannabina is higher than that of whole corn, which is the main source of CP in the mixed silage, and the difference in the NH₃-N content between 1:1 and 1:3 is not significant [36,37], so when the raw materials are mixed at a ratio of 1:1, the CP content of S. cannabina is higher than that of whole corn, which is the main source of CP for mixed silage. The CP content when the mixing ratio is 1:1 is higher than that of 1:3.

NDF and ADF can reflect the feeding value of silage, where the lower the content of ADF and the higher the CP content, the better the nutritional value and fermentation quality of silage, and the higher the digestibility of silage by ruminants. In this study, the addition of LAB significantly reduced the contents of NDF and ADF (p < 0.05), and the increase in the proportion of whole corn significantly reduced the NDF content of mixed silage (p < 0.05). During the process of silage fermentation, NDF and ADF are digested and degraded by microorganisms into nutrients that can be utilized by animals, which improves the digestibility of the nutrients in the feed [38].

4.3. Effect of Adding LAB and Increasing the Proportion of Whole Corn on the Effective Rumen Degradation Rate of the Mixed Silage

Increasing the proportion of whole corn significantly increased DMED, which may be associated with improved quality of mixed silage when whole corn is added. Adding whole corn helps disrupt the structurally confined cell wall components that are difficult to utilize during the ensiling process. These changes promoted the degradation of DM, ultimately further enhancing the effective degradation of the feed [39]. The addition of Lactobacillus inoculants improved DMED significantly, which might be due to the fact that Lactobacillus degraded part of the lignin and released hemicellulose and cellulose, which were then decomposed by rumen microorganisms into monosaccharides more easily absorbed by ruminants and the degradation of mixed silage in rumen was accelerated [40].

The degree of digestibility of feed proteins affects rumen microbial protein synthesis and nitrogen deposition in ruminants, and the degradation rate is related to the protein content and composition of the feed itself, the fibrous structure of the plant cell wall, and the retention time in the rumen [41]. Addition of lactobacilli inoculants were both able to increase the ability to significantly increase CPED (p < 0.05). CPED tended to increase when the proportion of whole corn was increased. This may be due to the fact that feed proteins were mainly present in the cytoplasm. The increase in fiber content and lignification may slow down the rate of protein breakdown and affect its release. Addition of whole corn reduced the NDF content of mixed silage, which suggests that plant cell wall fibrosis and lignification are reduced and CP in the cytoplasm is more readily released to improve rumen CP degradation. The ability of tannins to complex with proteins affects the activity of digestive enzymes such as protease, lipase and amylase in animals, reducing the digestibility of nutrients in forage [42]. Wang et al. [43] showed that LAB may be involved in tannin degradation by modulating catechol element removal, aromatic compound degradation, and hydrolyzed tannin enzymatic degradation pathways, thus improving the rumen degradation rate of mixed silage CP.

NDF and ADF digestibility is an important parameter for assessing and optimizing the quality of ruminant feeds, which plays an important role in ensuring animal health, improving productivity, and promoting the sustainable development of the livestock industry [44]. A high NDF digestibility means that more fiber components can be decomposed by rumen microorganisms to provide energy for the animals. Hristov et al. [45] showed that, with the prolongation of the silage time, the maize silage ADF/NDF increased and the rumen degradation rate of NDF decreased, which indicated that structural polysaccharides, such as hemicellulose, were gradually degraded during the silage process, and that the addition of whole corn with the addition of Lactobacillus inoculant improved the fermentation quality of the silage, contributed to the colonization of Lactobacillus, and facilitated the disruption of the cell wall structure, which in turn increased the rate of degradation of the fibers. The NDFED was higher when the silage mix of Sesbania cannanbina and whole corn was mixed as 1:1 than when the mixing ratio was 1:3, which may be caused by the different CP content of the silage. The CP in the feed can be used as the N source of microorganisms to promote the growth activity of microorganisms, and the higher the CP content of the feed, the more it is conducive to the growth and reproduction of beneficial bacteria, thus increasing the degradation rate of fibers [46].

4.4. Effect of Adding LAB and Increasing the Proportion of Whole Corn on the Degradation Characteristics of the Small Intestine of the Mixed Silage

The total amount of protein that can be absorbed by the animal after two processes of rumen fermentation and small intestinal digestion in the ruminant body is the amount of protein that can be provided by the feed. The small intestine has good absorption of feed over rumen protein and is also the main source of protein for the animal body [47]. In this study, in mixed silage, increasing the proportion of whole corn significantly reduced the RDP, RUP, and IDCP (p < 0.05). The addition of whole corn to mixed silage can increase the rumen degradation rate of mixed silage CP with the effective degradation rate of ED, which leads to a significant decrease in the protein content remaining after rumen degradation and a decrease in the IDCP. S. cannabina contains a certain amount of antinutritional factors such as tannins and other anti-nutritional factors, which can combine with protein to form complexes and thus inhibit digestion, and microorganisms such as LAB can utilize tannins as a carbon source for metabolism during the silage process [48]. In this study, the addition of LAB decreased the RUP, ICPD, and IDCP (p < 0.05), which may be due to the fact that the proteins entering the small intestine are mainly bacterial proteins synthesized by rumen microorganisms, proteins in the feed that have not yet been degraded by the rumen, and protein complexes bound to antinutritional factors that are difficult to degrade [49], and that the addition of LAB group had a higher rate of degradation of CP in rumen, which resulted in the majority of the easy-to-digest proteins being degraded in rumen. Thus, the RUP, IDG, and IDCP decreased [50].

4.5. Effect of Adding LAB and Increasing the Proportion of Whole Corn on the Nutrient Apparent Digestibility and Effective Energy of the Mixed Silage

In mixed silage, the addition of LAB significantly increased GE and DMAD (p < 0.05). Adding LAB reduced DM loss during the silage process, which improved GE and DMAD. Increasing the proportion of whole corn significantly increased GE (p < 0.05) and reduced DCP (p < 0.05). Increasing the proportion of whole corn increased the DM content and GE of mixed silage. Moreover, increasing the proportion of whole corn plants reduced the content of CP without changing its CPAD; thus, the DCP decreased. The addition of LAB and the increase in the proportion of whole corn had no significant effect on DE, ME, CPAD, NDFAD, and ADFAD (p < 0.05), indicating that although the addition of LAB and the increase in the proportion of whole corn changed the fermentation quality and nutritional components of mixed silage, their improvement in feed value was limited.

5. Conclusions

In summary, the effects of adding Lactobacillus inoculants and whole corn ratio on fermentation quality, nutrient composition, rumen degradation characteristics, small intestine degradation characteristics, whole digestive tract digestibility and available energy of S. cannabina silage were studied in this paper. The results showed that increasing whole corn ratio and adding Lactobacillus inoculant were effective means to improve the quality of S. cannabina silage. Silage mixed at a ratio of 1:1 can maximize the input of S. cannabina into production without affecting its feeding value. When mixing 1:1 silage, Lactobacillus inoculant can be added in the production to ensure the success of ensiling. Utilizing non-grain land to meet the demands of livestock production is a feasible strategy.