Effects of Inoculation with Different Rhizobia on the Nutritional Value and Ruminal Fermentation of Desmodium intortum

Abstract

Rhizobia inoculation is widely used in legumes to improve the yield and quality of forage. However, the symbiotic interactions of different legumes are specific. The search for efficient strains plays an important role in legume forage and in practical production. In this study, Desmodium intortum was first inoculated with rhizobia from different regions, and then the production traits, nutritional quality, and fermentation of D. intortum in the rumen were evaluated according to the rhizobia strains. The results showed that, compared with the control, inoculation with different rhizobia strains significantly increased the number of nodules, shoot fresh weight, crude protein content, plant protein yield, and ether extract content of D. intortum, and inoculation with the R3 strain (Bradyrhizobium elkanii) increased these values by 61.0%, 29.6%, 16.1%, 62.9%, and 30.4%, respectively. In the basal diet of Pennisetum purpureum Rich. × P. americana King grass cv. Reyan No.4, D. intortum inoculated with different rhizobia was added (75:25), and the combination of the inoculated R3 and R5 strains increased the rumen’s ammonia-nitrogen (NH₃-N) levels and in vitro dry matter digestibility (IVDMD): R3 increased the contents of NH₃-N and IVDMD by 50.1% and 12.3%, respectively, while R5 increased them by 50.9% and 15.4%, respectively. Based on a comprehensive evaluation through principal component analysis, R3 was ranked first overall. These results support the development of competitive inoculants from indigenous strains as a strategy to improve the nutritional value of D. intortum and ruminal fermentation.

1. Introduction

Desmodium Desv. species are potential sources of high-protein feed in tropical and subtropical areas. Desmodium intortum is a creeping, non-climbing perennial legume that originated in Central and South America [1]. It has the characteristics of high crude protein (CP, 18–20%DM) content and yield, cold resistance and high temperature resistance, and fast coverage [1]. Rhizobium is a bacterial genus that has a symbiotic relationship with the roots of legumes. Fixing free nitrogen in the atmosphere provides nitrogen nourishment for plants, which has a good effect on the growth of legumes [2,3]. In the United States, Australia, and other countries, rhizobia inoculation before legume planting is widely used to improve crop performance [4]. Studies have shown that inoculation with rhizobia can promote the growth, yield, number of nodules (NON), and nutrient quality of leguminous plants, while also improving soil enzymes and nutrients [5,6]. Martínez et al. co-inoculated Medicago sativa L. with rhizobia, finding that it could improve the yield, nitrogen content, and total mineral, calcium, and magnesium contents of M. sativa L., while also increasing the antioxidant capacity of the plants [7]. Screening of efficient rhizobia strains for D. intortum shows their potential to be used as high-quality protein supplements.

It is generally accepted that forage nutrient composition is correlated with animal production and growth performance [8,9]. It has been reported that dietary energy and protein levels affect nitrogen metabolism and feed fermentation efficiency, respectively, in goats [10,11]. Song et al. reported that goats with a higher non-fiber carbohydrates/neutral detergent fiber ratio had enhanced feed intake [12]. The combination of whole-plant corn silage and wheat straw also exhibited promising impacts on intake and body weight gain in beef cattle [13]. Ammonia-nitrogen (NH₃-N) levels, pH, microbial protein (MCP), and volatile fatty acid (VFA) concentration are the main internal environmental indicators of rumen fermentation [14]. Phesatcha et al. noted that increasing the ratio of concentrate in the diet reduced the ruminal pH and NH₃-N concentration, while increasing the VFA concentration [15,16]. It has also been reported that adding yeast to the diet can change the feed utilization efficiency, promote rumen fermentation, alleviate pH fluctuations, and promote VFA production [17]. Researchers pay more attention to the ratio of diet and additives, but less attention is paid to how to improve the quality of roughage and its impact on rumen fermentation. Therefore, the purpose of this study was to determine the effects of inoculation with rhizobia on the nutritional quality of D. intortum, so as to explore its effects on rumen fermentation, gas production (GP), and digestibility.

2. Materials and Methods

2.1. Materials

The seeds of D. intortum were obtained from the backup library of the Chinese Academy of Tropical Agriculture Sciences (CATAS, Danzhou, China). Pennisetum purpureum Rich. × P. americana King grass cv. Reyan No.4 (RY4, trophic period) was collected from the germplasm resource nursery of CATAS. The sources and classification of tested rhizobia strains are shown in Table 1.

2.2. Methods

2.2.1. Experimental Design

The experiment was a single-factor randomized block design, and the method of sand cultivation was adopted. The non-inoculated strains were used as controls (CK), six rhizobia strains were inoculated (Table 1) as the treatment group, and four replicates were set, with a total of 28 pots (two seedlings per pot).

2.2.2. Preparation of Sterile Seeds

The seeds of D. intortum were immersed in boiled water at 75–80 °C for 6 min, sterilized in 95% ethanol for 6 min, sterilized in 30% hydrogen peroxide solution for 6 min, and then removed, washed 5 or 6 times with sterile water, and immersed in sterile water for 2 h.

2.2.3. Preparation of Rhizobia Liquid

The test strains were picked up and transferred to a single colony on YMA medium, and then cultured in an incubator (Keheng Co., Ltd, Hangzhou, China) at 28 °C for 3–5 days. The sterilized toothpick segment was dipped into the rhizobia colony, washed in the liquid YMA medium, and cultured in the shaker (Juchuang Group Co., Ltd, Qingdao, China) at 180 r/min for 4 days. After the detection of the bacterial solution, the OD₆₀₀ nm value was greater than 1 (if the OD₆₀₀ nm value was less than or equal to 1, it continued to be cultured in the shaker until the OD₆₀₀ nm value was greater than 1—generally for no more than 10 days) [18].

2.2.4. Preparation of Sand Culture System

The flowerpot (40 × 60 cm) needed for the test was soaked in 10 mL/L hydrochloric acid overnight, rinsed with deionized water, and dried for reserve use. The sand was rinsed 10–15 times with tap water (until the outflow water was not turbid), and then it was rinsed with deionized water, dried and cooled, and placed in the flowerpot, at a coarse sand: fine sand ratio of 1:2, for further use [18].

2.2.5. Inoculation and Management of Rhizobia

The treated seeds were seeded in seedling trays containing sterile culture soil with 2–4 seeds per cell. The seedlings of D. intortum were over 5 cm high in the seedling tray, and the strong seedlings were transplanted into the sand culture system, after which the low-nitrogen nutrient solution was poured evenly and thoroughly on them. Then, 5 mL of each YMA bacterial solution was poured on the roots of each seedling, thus ensuring that the number of bacteria inoculated on each seedling was over 10⁹. Subsequently, the low-nitrogen nutrient solution was applied 4–5 times per week. If the weather was excessively dry, sterile water was applied 1–2 times. After 4–5 months of cultivation, the whole plants were harvested. The shoots and the root system were separated, and the fresh matter was weighed and placed in mesh bags. The shoots were directly placed in the oven and degreened at 105 °C for 20 min, dried at 75 °C for 48 h, and then crushed through 40-mesh sieves to prepare the samples for testing.

2.3. In Vitro Fermentation

In this experiment, a ratio of 25:75 was adopted for in vitro fermentation [19]. On the basis of dry matter, RY4 was assigned to one group, and the remaining seven groups were mixed with RY4 at a ratio of 25:75 (RY4, 25%). A total of eight groups (RY4, CK + RY4, R1 + RY4, R2 + RY4, R3 + RY4, R4 + RY4, R5 + RY4, and R6 + RY4) were fermented in vitro, with three replicates in each group. In vitro fermentation was carried out according to the experimental method of Zhao et al. [20].

The buffer solution consisted of 9.8 g of NaHCO₃, 0.04 g of CaCO₃, 0.47 g of NaCl, 0.57 g of KCl, 9.3 g of Na₂HPO₄, and 0.12 g of MgSO₄·7H₂O dissolved in 600 mL of distilled water; finally, the volume was fixed to 1 L.

Three healthy adult Hainan black goats of similar age and body weight were selected for the extraction of ruminal fluid before morning feeding (08:00), and 300 mL of ruminal fluid was filtered through four layers of gauze. Ruminal fluid and buffer at a ratio of 1:2 were added to 600 mL of buffer that was pre-prepared and pre-heated in a 38 °C water bath to produce a mixture, and CO₂ was continuously injected into the mixture for reserve use. Next, 0.3 g of dried grass powder samples from the different groups were weighed in fermentation bottles, into which 30 mL of the above mixtures was poured, and then CO₂ was added for 5–10 s to exhaust the air and ensure the mixtures were in a vacuum state. They were then cultured in a shaker bath at 38 °C. This experiment was designed to read the GP at 2, 4, 8, 12, 24, 36, 48, and 72 h after fermentation. Then, the supernatant was divided into centrifugal tubes and stored at −20 °C for testing.

2.4. Index Measurement and Method

Four plants were selected for each treatment, and their shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), root dry weight (RDW), nodule fresh weight (NFW), nodule dry weight (NDW), NON, fresh weight root-to-shoot ratio (FWRSR), and dry weight root-to-shoot ratio (DWRSR) were measured. The protein production (PP) was the product of the dry weight of each plant and its CP content. On the basis of dry matter, CP was determined by the Kjeldahl method, while ether extract (EE) was determined by Soxhlet extraction. The Chinese national standard was used to determine neutral detergent fiber (NDF, GB/T 20806-2006), acid detergent fiber (ADF, NY/T 1459-2007), ash (GB/T 6438-2007), and tannin (GB/T 27985-2011) contents. Trace minerals were determined through inductively coupled plasma emission spectrometry (iCAP PRO X, Waltham, MA, USA). The levels of VFA were determined through high-performance liquid chromatography (Agilent 1260LC Co., Santa Clara, CA, USA). The supernatant was extracted after 72 h and the pH was determined with a pHS-2F pH meter (Rex Electric Chemical, Shanghai, China). Microbial protein (MCP) was determined by the trichloroacetate precipitation method. NH₃-N was determined using the method of Broderick and Kang [21].

Animal ethical statement: The research project has been reviewed and approved by the Chinese Academy of Tropical Agriculture Sciences (Approval number：PZSYYSP-202303208), in line with the welfare and ethical principles of laboratory animals. Here by certify.

2.5. Statistical Analyses

Statistical analysis of one-way ANOVA was performed using SPSS 23.0 (IBM Inc., Armonk, NY, USA). Duncan’s test was employed to determine the differences in the treatments’ means. The results were expressed as the mean ± SEM. The differences were deemed to be significant when p < 0.05. Pearson’s correlation analysis and principal component analysis were performed for SDW, RDW, NDW, NON, CP, PP, EE, NDF, ADF, ash, IVDMD, NH₃-N, MCP, acetate, propionate, and TVFA. The linear equations of Y₁ and Y₂ were obtained using the coefficients of the principal component analysis, as follows:

Y1 = 0.388X₁ + 0.261X₂ + 0.241X₃ + 0.360X₄ + 0.361X₅ + 0.404X₆ + 0.240X₇ − 0.236X₈ + 0.053X₉ − 0.157X₁₀ − 0.188X₁₁ + 0.248X₁₂ + 0.199X₁₃ + 0.078X₁₄ + 0.009X₁₅ + 0.131X₁₆

Y₂ = −0.068X₁ − 0.205X₂ − 0.319X₃ − 0.150X₄ − 0.012X₅ − 0.072X₆ + 0.345X₇ + 0.132X₈ + 0.298X₉ − 0.101X₁₀ − 0.227X₁₁ + 0.145X₁₂ + 0.001X₁₃ + 0.431X₁₄ + 0.467X₁₅ + 0.336X₁₆

The gas production parameters were calculated according to the exponential model of Ørskov and McDonald, as follows [22]:

GP_t = a + b × (1 − e^−ct)

where GP_t (mL) is the gas production at time t, a (mL) is the gas production of the rapidly fermented fraction, b (mL) is the gas production of the slowly fermented fraction, c (mL/h) is the rate constant of gas production of the slowly fermented fraction, and t (h) is the fermentation time.

3. Results and Analysis

3.1. Root Traits of D. intortum Inoculated with Rhizobia

After D. intortum was inoculated with rhizobia, the effects of inoculation with different strains were significantly different (Table 2). Compared with CK, the RFW, RDW, NFW, NDW, and NON of the D. intortum inoculated with the R3 strain were significantly increased (p < 0.05), by 47.2%, 43.7%, 18.1%, 26.1%, and 60.9%, respectively. The RDW, NFW, and NDW did not significantly differ among strains R1, R2, R4, R5, and R6 (p > 0.05).

Compared with CK, the SFW and SDW of the six strains were significantly increased after inoculation (p < 0.05), and the SFW and SDW of the sample inoculated with strain R3 showed the largest increases, with yield increases of 42.1% and 40.8%, respectively. The inoculation effect of strains R4, R5, and R6 was also better, and SFW increased by 29.5%, 23.3%, and 30.7%, respectively, while SDW increased by 24.9%, 16.1%, and 27.1%, respectively. The FWRSR of the samples inoculated with strains R1, R2, R4, R5, and R6 was significantly lower than that of CK (p < 0.05), but DWRSR showed no significant differences (p > 0.05).

3.2. Nutritional Quality

The CP and EE concentrations and PP differed significantly between the different strains (p < 0.05, Table 3). The CP content of the R3 group was significantly higher than that of the CK, R2, and R6 groups; PP was also significantly higher than in the other groups, increasing by 15.9% and 62.9% compared with CK, and the EE content of groups R5 and R6 was significantly higher than that of the other groups. However, the NDF content in the R3 group was significantly lower than in the CK and R6 groups. The contents of ADF, ash, and tannins in D. intortum inoculated with different strains were not significantly different from those of the CK group (p > 0.05).

3.3. Trace Minerals

The contents of Fe and Mn were not significantly different between the different strains (p > 0.05, Table 4). The Cu content in the R5 group was significantly higher than that in the R3 and R2 groups. Compared with CK, the Zn content of D. intortum inoculated with strain R1 was significantly increased (p < 0.05), while that of groups R2, R3, R4, R5, and R6 was significantly decreased (p < 0.05). The Mo content of D. intortum in the groups inoculated with strains R3, R4, and R5 was significantly reduced (p < 0.05).

3.4. In Vitro Fermentation Gas Production

Among the different groups, there were no significant differences in the a and c parameters (p > 0.05, Table 5). Compared with CK + RY4, the a, b, a + b, and c were not significantly different (p > 0.05). Additionally, the gas production of the slow fermentation was consistent with the potential gas production.

3.5. In Vitro Fermentation Parameters

Compared with CK + RY4, the other treatment groups showed no significant effects on the MCP content and pH value in the ruminal culture fluid (p > 0.05), and the pH value of all groups was between 6.68 and 6.74 (Table 6). Compared with CK + RY4, the NH₃-N content of the R3 + RY4 and R5 + RY4 groups was significantly increased (p < 0.05); the differences among the other groups were not significant. The IVDMD of each group ranged from 47.9% to 55.3%, and the IVDMD of all inoculation treatments was significantly increased (p < 0.05).

3.6. Volatile Fatty Acid Content of In Vitro Fermentation

Compared with CK + RY4, the other treatment groups showed no significant effects on the contents of acetate, propionate, acetate/propionate, and TVFA in the ruminal culture fluid (p > 0.05) (Table 7). The isobutyrate contents of R1 + RY4 and R6 + RY4 were significantly increased, but the butyrate contents of the R1 + RY4 and R6 + RY4 groups were significantly lower than that of the CK + RY4 and R5 + RY4 group. The contents of acetate, propionate, and TVFA in the R6 + RY4 group were higher than those in other groups, but not significantly.

3.7. Correlation and Principal Component Analysis

Correlation and factor analyses were carried out on indicators such as D. intortum production traits, nutritional components, and in vitro fermentation status. Among these indicators, NON showed a significant positive correlation with IVDMD and MCP, while CP showed an extremely significant positive correlation with NH₃-N and MCP (Table 8). Two common factors (Table S2) were obtained from the loaded matrix after rotation, summarizing the main variable information. The first common factor (Y₁) was composed of SDW, RDW, NON, CP, and PP, representing growth and nutrient quality traits, while the second common factor (Y₂) was composed of acetate, propionate, TVFA, EE, NDF, ADF, and NH₃-N, representing fermentation quality characteristics. In the comprehensive scoring of the principal components, R3 ranked first, with the best inoculation effect; CK ranked seventh, with the worst comprehensive effect (Table 9).

4. Discussion

4.1. Effects of Inoculation with Rhizobium on the Nutritional Quality of D. intortum

The effects produced by different rhizobacterial inoculations on different legume forages also differ [23]. Hernández et al. inoculated Vicia sativa with 16 rhizobia agents; only UFSC-M8 and UFSC-M9 were able to form nodules, and the number of nodules differed significantly [21]. D. intortum showed different results after inoculation with different rhizobacterial agents. Inoculation with R3, belonging to the Bradyrhizobium elkanii strain, significantly increased the growth and yield of D. intortum, with significantly better results than the other rhizobacterial treatments. Célica et al. also found that Bradyrhizobium elkanii has a good symbiotic relationship with soybeans. Co-inoculation of B. elkanii and Delftia sp. strain JD2 promoted soybeans’ growth, nodulation rate, and yield [24]. Verma et al. reported that inoculation with Bradyrhizobium BHURC03 increased the dry weight of the aboveground and underground parts of chickpeas by 26.0% and 44.0%, respectively [25]. However, R4, which also belongs to Bradyrhizobium elkanii, reduced the RDW and RFW of D. intortum. The inoculated rhizobial agent competed with the indigenous rhizobia in the soil, resulting in a poor inoculation effect [26]. Pilar et al. also found that, in terms of nodule occupancy between the inoculum and the local strains, the native rhizobia with lower efficiency had an advantage, reducing the symbiotic efficiency [23].

The nutrient contents of forage determine its quality, and the CP in forage is an important source of nutrition for the growth and reproduction of ruminants [27]. RY4 is commonly used for feeding black goats in Hainan, China, but its protein content is only 8–11%. According to the National Research Council of the United States, the protein content required for ruminant production is 12–25% [28]. Souza et al. found that the VC28 strain screened locally could increase the N content of common beans by 17% [29]. In this experiment, the CP content of D. intortum inoculated with six different rhizobia reached 20%, representing a high-quality protein supplement within the range required for ruminant production. Trace minerals are indispensable mineral elements in forage grasses. As the components of biologically active substances in plants, they participate in a series of substance metabolism processes in the body, and they have very important physiological and biochemical effects [30]. Studies have shown that inoculation with rhizobia promotes nitrogenase activity. Molybdenum, iron, protein, and ferritin are important components of nitrogenase, but the Mo contents of D. intortum inoculated with strains R3, R4, and R5 decreased instead. Bishop et al. found that a second nitrogenase system consisting of vanadium and ferritin was more efficient [31]. In the absence of Mo, this nitrogenase-fixing enzyme containing vanadin-ferritin is expressed and has higher activity in catalytic reduction. Therefore, strains R3, R4, and R5 may play additional roles in the second nitrogenase system. Some studies have shown that rhizobia are not only able to stimulate plant growth, but also enhance resistance to heavy metals and adsorption accumulation by increasing hormone secretion (indole-3-acetic acid, cytokinins, and auxins) [32,33]. Liu et al. found that rhizobia isolated from heavy metal soil showed different tolerances to Cu, Zn, and Ni [34]. R5 strains may be more resistant to Cu and Zn.

4.2. In Vitro Fermentation of the Combination of D. intortum and RY4 Inoculated with Different Rhizobia

GP is strongly related to the contents of fermentable substances in the feed [35]. It was reported that, with the increase in the NDF content, the rumen fermentation rate and GP also increased [15]. Kang et al. found that as the proportion of concentrate in the diet increased, the cumulative GP increased [36]. Moreover, increasing the CP content in the diet improved the digestibility of NDF [37]. Yang et al. also showed that higher CP content in the diet provides more amino acids, peptides, and branched-chain fatty acids, consequently increasing the fiber’s digestibility [38]. Therefore, we hypothesized that inoculation of rhizobia increases GP by increasing the NDF and CP content. However, this was not the case: D. intortum inoculation with rhizobia had no effect on GP. The NDF content in the R3 group was significantly reduced. The proportion of lignin in NDF also affects GP, and lignin is resistant to microorganisms in the rumen and thus affects digestibility. Although inoculation with rhizobia increased the CP content of D. intortum, it had no effect on VFA. The lack of change in GP may also have been due to the lack of energy support, preventing the ruminal fibers from being effectively decomposed in time. Non-structural carbohydrates are also an important source of GP, but the main role of rhizobia is to carry out symbiotic nitrogen fixation, which has no significant effect on NSC. When fermentable carbohydrates such as starch are broken down, structural carbohydrates such as cellulose begin to slowly ferment. The slow fermentation of cellulose may be one of the reasons why the GP content did not significantly change.

The availability of fermentable carbohydrates and nitrogen sources in the diet—i.e., the degree of nitrogen balance—determines the digestibility of feed in the rumen [39]. It has been reported that increasing the levels of concentrates (fermentable carbohydrates, organic matter, CP, etc.) in the diet has a positive effect on the digestibility of organic matter and CP [40]. The study by Ribeiro et al. also found that the digestibility of DM and CP was significantly increased in dairy cows fed a diet with increasing proportions of concentrate to fiber [41]. Correlation analysis showed that IVDMD was significantly positively correlated with CP. Inoculation with rhizobia increased the CP content of D. intortum, induced the growth and activity of ruminal microorganisms, and improved the subsequent digestibility. The increase in NH₃-N content can also indicate the increase in the protein degradation rate.

The rumen is a digestive organ that is unique to ruminants, and its main function is to digest and decompose organic matter such as starch, protein, and fiber in forage grass with the help of ruminal microorganisms. The pH value of ruminal fluid can affect the activity of ruminal microorganisms and the stability of microbial flora. The pH range suitable for the survival and fermentation of ruminal microorganisms is 6.0–7.0 [42]. After adding D. intortum inoculated with rhizobia to the feed, the pH value was within the appropriate range. As an intermediate product of protein degradation and MCP synthesis, NH₃-N is an important index to measure the nitrogen metabolism of ruminal microorganisms [43]. Adding D. intortum inoculated with different rhizobia to RY4 increased the NH₃-N content in the ruminal fluid. It has been reported that NH₃-N concentration increases with increasing dietary nitrogen content [44]. Paula et al. showed that higher CP in the diet may cause microorganisms to decompose into higher concentrations of NH₃-N. NH₃-N and CP showed a significant positive correlation, and inoculation with rhizobia increased the CP and IVDMD of D. intortum, resulting in enhanced carbohydrate and protein degradation, thereby increasing the ruminal NH₃-N content, which also confirmed this. The NH₃-N content in the rumen was 50–250 mg/L, which is most suitable for the growth and reproduction of ruminal microorganisms [45]. Some studies have noted that increasing the proportion of concentrates in the diet can increase the concentration of bacteria and microorganisms in the rumen [46,47]. Furthermore, due to the limitations of the in vitro gas-producing fermentation device, the contents of the container could not be moved outside, resulting in the accumulation of fermentation end products and the growth of ruminal microorganisms. Changes in the living environment led to the death of microorganisms, bacterial lysis, ciliate autolysis, etc., releasing ammonia. The lack of absorption sites in vitro culture can also lead to an increase in NH3-N content. MCP can provide 40–80% of the absorbable protein for the small intestine and is the main protein source for ruminants [48]. However, in the absence of fermentable carbohydrates, the microbial uptake of NH₃-N was reduced, resulting in no change in MCP. This may also be one of the reasons for the high NH₃-N content.

The VFA produced by the fermentation of carbohydrates by ruminal microorganisms is the source of the energy required for the life activities of ruminants and the raw materials for the synthesis of milk fat and body fat [49]. VFA in the rumen is mainly composed of acetate, propionate, butyrate, etc., and acetate accounts for ~70–75% of TVFA production. Different fermentation substrates produce different VFA components. Studies have shown that the fermentation substrate has high cellulose content and produces high amounts of acetate [50]. Chen et al. also found that adding alfalfa to sweet sorghum increased the contents of acetate and TVFA in the ruminal fluid [19]. However, different rhizobia had no significant effects on the NDF and ADF of D. intortum; as a result, adding D. intortum to RY4 caused no changes in the content of acetate, TVFA, or the levels of acetate/propionate. Due to the slow utilization and fermentation of NDF and other structural carbohydrate microorganisms, the rumen lacks fermentable carbohydrates, resulting in the accumulation of NH₃-N and no changes in TVFA and MCP. Therefore, in addition to protein supplements in the diet, increasing the intake of carbohydrates is also particularly important.

5. Conclusions

After D. intortum was inoculated, the NDW and NON increased. Nodules can promote the growth and development of roots and shoots through nitrogen fixation, thereby significantly increasing the yield of D. intortum. CP was also significantly positively correlated with NH₃-N and MCP. The increase in NON after inoculation with R3 promoted the nitrogen absorption of D. intortum and increased its CP. When D. intortum with a high CP content was added to RY4, the nitrogen source was supplemented, and the contents of NH₃-N were increased, improving the nutritional value of D. intortum. In conclusion, inoculation of rhizobia can improve rumen fermentation by affecting the content of CP, NDF, etc. of D. intortum. Among them, the R3 strain showed a better inoculation effect and had a high promotion value.

Author Contributions

Conceptualization, X.-X.H. and R.-S.D.; software, data curation, X.-X.H. and Y.-C.Z.; investigation, resources, methodology, S.-Q.H. and W.-J.X.; visualization, writing—review and editing, X.-X.H. and A.H.; validation, M.L. and Y.-C.Z.; supervision, project administration, and funding acquisition, R.-S.D. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Animal ethical statement: The research project has been reviewed and approved by the Chinese Academy of Tropical Agriculture Sciences (Approval number: PZSYYSP-202303208), in line with the welfare and ethical principles of laboratory animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the first author. The data are not publicly available due to restrictions by the research group.

Acknowledgments

Thanks to Sheng Wei for his guidance in writing, Chun-Liu Yang and Yu-Shu Zhang for their help in the experiment operation.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Tables

Table 1

Tested rhizobia strains, along with their source and classification status.

Serial Number	Strains	Source
R1	Bradyrhizobium pachyrhizi	Wenshan, Yunnan
R2	Bradyrhizobium pachyrhizi	Wenshan, Yunnan
R3	Bradyrhizobium elkanii	Ledong, Hainan
R4	Bradyrhizobium elkanii	Fusui, Guangxi
R5	Bradyrhizobium embrapense	Xingyi, Guizhou
R6	Bradyrhizobium embrapense	Danzhou, Hainan

Table 2

Effects of rhizobia inoculation on the root system of D. intortum.

	CK	R1	R2	R3	R4	R5	R6	p-Value
SFW(g)	46.6 ± 2.8 d	58.4 ± 1.6 b	52.2 ± 2.5 c	66.3 ± 0.67 a	60.4 ± 2.8 b	57.5 ± 0.5 bc	60.9 ± 1.0 ab	<0.001
SDW(g)	9.9 ± 0.5 d	11.6 ± 0.4 bc	10.5 ± 0.4 cd	13.9 ± 0.4 a	12.3 ± 0.8 b	11.5 ± 0.2 bc	12.6 ± 0.3 b	<0.001
RFW(g)	9.8 ± 0.5 bc	10.6 ± 0.7 b	9.5 ± 1.0 bc	14.4 ± 1.1 a	7.7 ± 0.2 d	9.2 ± 0.2 bc	11.0 ± 0.2 bc	<0.001
RDW(g)	1.35 ± 0.07 b	1.39 ± 0.05 b	1.26 ± 0.15 b	1.94 ± 0.19 a	1.33 ± 0.05 b	1.28 ± 0.11 b	1.47 ± 0.07 b	<0.001
NFW(g)	2.82 ± 0.39 abc	2.52 ± 0.05 bc	2.16 ± 0.24 c	3.33 ± 0.14 a	2.88 ± 0.07 ab	2.71 ± 0.22 abc	2.80 ± 0.17 abc	0.037
NDW(g)	0.46 ± 0.06 b	0.45 ± 0.02 b	0.42 ± 0.05 b	0.58 ± 0.03 a	0.48 ± 0.01 ab	0.45 ± 0.04 b	0.47 ± 0.03 ab	0.027
NON	344.3 ± 16.5 c	418.5 ± 24.9 b	385.3 ± 13.6 bc	554.0 ± 28.7 a	441.5 ± 15.4 b	396.0 ± 22.2 bc	407.0 ± 6.3 b	<0.001
FWRSR (%)	21.09 ± 1.09 a	16.11 ± 1.08 b	14.77 ± 1.11 b	21.71 ± 2.75 a	12.21 ± 0.73 b	15.10 ± 0.43 b	15.75 ± 1.12 b	0.035
DWRSR (%)	13.12 ± 0.60 a	12.03 ± 0.68 a	11.93 ± 1.30 a	12.87 ± 1.28 a	10.69 ± 0.82 a	11.13 ± 1.02 a	11.72 ± 0.61 a	0.001

Different superscript letters of data in the same row indicate significant differences between different treatments (p < 0.05). SFW, shoot fresh weight; SDW, shoot dry weight; RFW, root fresh weight; RDW, root dry weight; NFW, nodule fresh weight; NDW, nodule dry weight; NON, number of nodules; FWRSR, fresh weight root-to-shoot ratio; DWRSR, dry weight root-to-shoot ratio.

Table 3

Effects of rhizobia inoculation on the nutritional components of D. intortum.

	CK	R1	R2	R3	R4	R5	R6	p-Value
CP (%)	18.0 ± 0.3 c	19.7 ± 0.4 ab	19.4 ± 0.5 b	20.9 ± 0.3 a	19.9 ± 0.3 ab	20.0 ± 0.4 ab	19.5 ± 0.4 b	0.001
EE (%)	4.41 ± 0.14 e	5.54 ± 0.07 cd	5.13 ± 0.36 d	5.75 ± 0.07 bc	5.39 ± 0.12 cd	6.18 ± 0.09 ab	6.42 ± 0.15 a	0.001
NDF (%)	54.5 ± 0.8 ab	54.7 ± 1.0 ab	56.6 ± 1.5 a	50.1 ± 0.9 c	52.7 ± 1.6 bc	51.7 ± 0.9 bc	57.5 ± 1.5 a	0.002
ADF (%)	36.6 ± 2.5 ab	34.5 ± 0.7 b	36.4 ± 1.1 ab	36.4 ± 0.5 ab	39.6 ± 1.9 a	39.6 ± 1.0 a	39.6 ± 1.5 a	0.043
Ash (%)	7.34 ± 0.02 ab	7.69 ± 0.05 a	7.72 ± 0.03 a	7.16 ± 0.26 b	7.29 ± 0.20 ab	7.46 ± 0.19 ab	6.98 ± 0.13 b	0.024
Tannins (%)	1.46 ± 0.06	1.74 ± 0.12	1.50 ± 0.22	1.42 ± 0.07	1.49 ± 0.05	1.59 ± 0.05	1.30 ± 0.03	0.214
PP (g/plant)	1.78 ± 0.13 d	2.29 ± 0.10 bc	2.05 ± 0.12 cd	2.90 ± 0.09 a	2.45 ± 0.17 b	2.30 ± 0.03 bc	2.45 ± 0.08 b	0.001

Different superscript letters of data in the same row indicate significant differences between different treatments (p < 0.05). CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; PP, plant protein yield.

Table 4

Effects of rhizobia inoculation on the trace minerals in D. intortum (mg/kg).

	CK	R1	R2	R3	R4	R5	R6	p-Value
Fe	1513.9 ± 16.2	1565.5 ± 75.9	1358.3 ± 122.5	1560.1 ± 79.3	1594.2 ± 26.7	1610.8 ± 50.5	1477.7 ± 43.2	0.222
Mn	263.3 ± 9.2	287.7 ± 38.5	256.1 ± 36.5	335.9 ± 31.2	283.5 ± 12.2	290.3 ± 18.8	289.1 ± 13.4	0.492
Cu	112.8 ± 4.9 ab	114.3 ± 2.9 ab	100.5 ± 2.9 b	100.4 ± 2.1 b	110.3 ± 3.2 ab	118.3 ± 11.1 a	103.9 ± 1.3 ab	0.036
Zn	828.9 ± 76.4 b	998.3 ± 34.2 a	568.8 ± 40.9 c	595.3 ± 51.2 c	566.4 ± 7.4 c	685.2 ± 24.9 c	628.8 ± 24.6 c	<0.001
Mo	115.9 ± 6.9 ab	123.8 ± 4.5 a	110.1 ± 2.2 bc	88.1 ± 4.2 d	92.9 ± 4.7 d	101.4 ± 3.6 cd	107.4 ± 3.0 bc	<0.001

Different superscript letters of data in the same row indicate significant differences between different treatments (p < 0.05).

Table 5

In vitro fermentation gas production.

	RY4	CK + RY4	R1 + RY4	R2 + RY4	R3 + RY4	R4 + RY4	R5 + RY4	R6 + RY4	p-Value
a (mL)	14.45 ± 4.99	11.42 ± 1.35	13.61 ± 0.26	12.47 ± 0.39	13.5 ± 1.77	8.1 ± 0.04	7.58 ± 0.75	9.68 ± 1.44	0.239
b (mL)	117.1 ± 2.4 B	164.6 ± 16.1 A	145.8 ± 0.8 AB	146.2 ± 4.3 AB	146.9 ± 1.7 AB	165.3 ± 20.7 A	140.7 ± 7.2 AB	145.6 ± 2.4 AB	0.042
a + b (mL)	131.5 ± 4.8 B	176.0 ± 6.3 A	159.5 ± 1.1 AB	158.7 ± 3.9 AB	160.4 ± 3.5 AB	173.4 ± 9.4 A	148.9 ± 8.0 AB	155.3 ± 3.8 AB	0.035
c (mL/h)	0.015 ± 0.003	0.016 ± 0.001	0.017 ± 0.002	0.015 ± 0.001	0.019 ± 0.003	0.019 ± 0.005	0.019 ± 0.002	0.020 ± 0.001	0.080

Different superscript letters of data in the same row indicate significant differences between different treatments (p < 0.05). a, gas production of the rapidly fermented fraction; b, gas production of the slowly fermented fraction; a + b, potential gas production; c, rate constant of gas production of the slowly fermented fraction.

Table 6

In vitro fermentation parameters.

	RY4	CK + RY4	R1 + RY4	R2 + RY4	R3 + RY4	R4 + RY4	R5 + RY4	R6 + RY4	p-Value
NH3-N (mg/L)	345.3 ± 5.0 c	457.4 ± 13.7 b	456.3 ± 25.3 b	459.4 ± 14.7 b	518.4 ± 9.5 a	470.8 ± 6.0 ab	521.2 ± 2.3 a	467.0 ± 26.6 b	0.001
MCP (mg/mL)	3.44 ± 0.17	3.16 ± 0.28	3.48 ± 0.31	3.57 ± 0.15	3.76 ± 0.18	3.37 ± 0.17	3.40 ± 0.17	3.50 ± 0.15	0.077
IVDMD (%)	47.9 ± 0.7 d	49.6 ± 0.8 d	52.1 ± 0.8 c	51.9 ± 0.6 c	53.8 ± 0.4 abc	52.3 ± 0.6 bc	55.3 ± 0.4 a	54.4 ± 0.5 ab	0.027
pH	6.72 ± 0.02	6.68 ± 0.01	6.73 ± 0.02	6.69 ± 0.02	6.73 ± 0.03	6.72 ± 0.01	6.74 ± 0.02	6.73 ± 0.01	0.499

Different superscript letters of data in the same row indicate significant differences between different treatments (p < 0.05). NH₃-N, ammoniacal nitrogen; MCP, microbial protein; IVDMD, in vitro dry matter digestibility.

Table 7

Volatile fatty acid content of in vitro fermentation (mol/L).

	RY4	CK + RY4	R1 + RY4	R2 + RY4	R3 + RY4	R4 + RY4	R5 + RY4	R6 + RY4	p-Value
Acetate	0.49 ± 0.10 b	0.79 ± 0.10 a	0.86 ± 0.02 a	0.80 ± 0.01 a	0.87 ± 0.01 a	0.84 ± 0.01 a	0.85 ± 0.03 a	0.89 ± 0.02 a	0.001
Propionate	0.18 ± 0.02 b	0.25 ± 0.03 a	0.27 ± 0.01 a	0.24 ± 0.01 a	0.26 ± 0.01 a	0.24 ± 0.01 a	0.26 ± 0.01 a	0.29 ± 0.02 a	0.008
Isobutyrate	0.01 ± 0.03 d	0.09 ± 0.03 c	0.16 ± 0.01 ab	0.12 ± 0.01 bc	0.13 ± 0.02 bc	0.11 ± 0.01 bc	0.14 ± 0.01 bc	0.20 ± 0.02 a	<0.001
Butyrate	0.10 ± 0.01 d	0.15 ± 0.02 a	0.11 ± 0.01 cd	0.14 ± 0.01 abc	0.13 ± 0.01 abc	0.14 ± 0.01 ab	0.15 ± 0.01 a	0.12 ± 0.01 bc	0.002
TVFA	0.78 ± 0.15 b	1.28 ± 0.17 a	1.41 ± 0.04 a	1.29 ± 0.02 a	1.39 ± 0.03 a	1.34 ± 0.03 a	1.40 ± 0.06 a	1.50 ± 0.06 a	0.001
Acetate/Propionate	2.73 ± 0.27 c	3.14 ± 0.02 ab	3.17 ± 0.08 ab	3.34 ± 0.04 ab	3.40 ± 0.04 ab	3.49 ± 0.03 a	3.28 ± 0.06 ab	3.07 ± 0.18 bc	0.015

Different superscript letters of data in the same row indicate significant differences between different treatments (p < 0.05). TVFA, total volatile fatty acids.

Table 8

Correlation analysis of production performance, nutritional composition, and in vitro fermentation.

	SDW	RDW	NDW	NON	CP	PP	EE	NDF	ADF	Ash
IVDMD	0.32	0.10	−0.05	0.42 *	0.43 *	0.35	0.15	0.09	0.19	0.07
NH3-N	0.37	0.31	0.27	0.35	0.49 **	0.43 *	0.37	−0.27	0.20	−0.28
MCP	0.32	0.18	0.14	0.51 **	0.50 **	0.39 *	0.23	−0.06	0.04	0.07
Acetate	0.01	−0.09	−0.27	0.06	0.16	0.04	0.34	−0.04	0.21	−0.14
Propionate	−0.18	−0.18	−0.26	−0.18	−0.01	−0.15	0.34	−0.03	0.38 *	−0.08
TVFA	0.22	0.07	−0.10	0.12	0.11	0.18	0.54 **	−0.03	0.17	−0.15

In correlation analysis, ** represents extremely significant correlation (p < 0.01), while * represents significant correlation (p < 0.05). SDW, shoot dry weight; RDW, root dry weight; NDW, nodule dry weight; NON, number of nodules; CP, crude protein; PP, plant protein yield; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; NH₃-N, ammoniacal nitrogen; MCP, microbial protein; IVDMD, in vitro dry matter digestibility; TVFA, total volatile fatty acids.

Table 9

Principal component scores and comprehensive scores of D. intortum inoculated with rhizobia.

Treatment	Y1 Score	Y2 Score	Overall Ratings	Ranking
CK	−3.35	−1.33	−1.39	7
R1	−0.39	−0.99	−0.33	5
R2	−2.06	0.10	−0.68	6
R3	3.98	−1.53	1.04	1
R4	0.30	−0.27	0.05	4
R5	0.64	2.01	0.61	3
R6	0.88	2.01	0.69	2

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9040316/s1, Table S1. Nutritional composition of RY4. Table S2. Principal Factor Loading Matrix. Table S3. In vitro fermentation gas production.

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