Strategic Supplementation of Flemingia Silage to Enhance Rumen Fermentation Efficiency, Microbial Protein Synthesis and Methane Mitigation in Beef Cattle

Abstract

Background: Good quality protein as an on-farm feed resources has been in great demand to support the productivity of ruminants. A digestion trial using beef cattle crossbreds was conducted to assess the four dietary treatments of Flemingia macrophylla silage (FMS) supplementation at 0, 0.2, 0.4 and 0.6 kg dry matter (DM)/day in a 4 × 4 Latin square design. Feed DM intakes were measured during the 14 days and sample of feeds, feces, urine, as well as rumen fluid, blood were collected during the 7 days while the animals were on metabolism crates.

Results: Based on this experiment revealed that strategic supplementation of FMS increased (P < 0.05) nutrients digestibility (organic matter, crude protein, and acid detergent fiber) enhanced rumen total volatile fatty acid production especially propionic acid (C₃), C₂:C₃ ratio while, remarkably promoted the microbial protein synthesis (MPS) by increasing N-balance and retention of purine derivatives.

Conclusion: Under this experiment, the results revealed the potential use of FMS as a good-quality feed to improve nutrients digestibility, rumen fermentation, microbial protein synthesis, and to mitigate methane production. FMS supplementation at 0.6 kg DM/day exhibited the best result.

Background

Feed resources for ruminants are important in the livestock feeding systems for small-scale tropical farmers; particularly in the dry season [1]. Flemingia is a multipurpose legume shrub that yields fresh biomass of about 55 tons/ha/year and thrives well in diverse conditions [2]. It contains high levels of crude protein (17–26%), condensed tannins (CT) 6–11% and saponins (SPN) [3–5]. The presence of these phytonutrients in feed resources has been shown to enhance the rumen fermentation efficiency and greatly reduce rumen methane (CH₄) production [6]. Fagundes et al. [7] also reported that supplementation of Flemingia at 125 g of dry matter intake in goats did not affect adversely the feed intake and milk production. In addition [8], reported that supplemented Flemingia hay meal at 150 g/head/day increased digestibility of nutrients, rumen fermentation and microbial protein synthesis. Moreover, Kang et al. [4] confirmed that Flemingia leaves supplementation improved rumen fermentation and reduced the CH₄ production. Conserving of feed in the form of silage has been a good practice especially for dry season feeding [9]. Silage quality can be enhanced by addition of urea and molasses in fodder crop silage [10–12].

However, there was limited information about the utilization of Flemingia macrophylla silage on rumen fermentation. Hence, the aim of this experiment was to investigate the impact of Flemingia macrophylla silage on nutrient digestibility, rumen fermentation and microbial protein synthesis in beef cattle.

Results

Rumen fermentation efficiency and blood urea nitrogen

FMS supplementation increased total VFA and propionic acid (C3) (P < 0.001), among treatments. While the concentrations of NH3–N, acetic acid (C3), butyric acid (C4), acetic acid to propionic acid (C2 to C3), CH4 production, and protozoal population were decreased (P < 0.001) among treatments, respectively. However, ruminal pH and BUN were not changed.

Nitrogen balance, excretion of purine derivatives and microbial nitrogen supply

Table 4. Shows nitrogen intake ranged from 34.81 ± 1.23 to 48.01 ± 2.12 g/d and was increased (P < 0.001), while N excretion was similar among treatments (P > 0.05). However, N absorbed and N retained were increased (P < 0.001), respectively. The FMS supplementation affected on allantoin, uric acid, PD, purine absorb, microbial nitrogen supply and EMNS (P < 0.001). Moreover, FMS affected on percent of MNS and EMNS were increased significantly among treatments (P < 0.001), respectively.

Table 4
Effects of Flemingia macrophylla silage (FMS) on nitrogen balance, excretion of purine derivatives and microbial nitrogen supply

Discussion

Rumen fermentation efficiency and blood urea nitrogen

Ruminal pH and BUN were not significantly shifted differently among treatments. This result agreed with Phesatcha et al. [8] who showed that pH and BUN did not change by Flemingia leaf supplementation. The normal rumen pH was reported to be 6.3–6.8 which can support cellulolytic bacteria's normal activity [20]. These values indicated that all levels of FMS feeding were in favorable condition. While, ruminal NH₃-N concentration was decreased as a result of the CT in FMS which could protect protein in the diet by forming tannin-protein complexes to by-pass to lower-gut [21, 22] reported that CT in the diets may also reduce NH₃ concentration in the rumen by reducing protein degradation in the rumen. The optimal ruminal NH₃-N concentration for rumen fermentation activities, ruminal microbial protein synthesis efficiency, voluntary dry matter intake, and digestibility of feeds were in the range of 15–30 mg/ml [23]. In this study, the ruminal NH₃-N values (16.91 ± 0.08 to 19.31 ± 0.12 mg/ml) were found and could improve for rumen ecology in cattle crossbreds. With increased levels of FMS supplementation, the total VFA and C₃ were increased (P < 0.05), and the highest impact was found in the group fed with of 0.6 kg/day, while C₂, C₂ to C₃ ratio were reduced. Phesatcha et al. [8] reported that, FM supplementation in dairy steers decreased C₂ whilst C₃ was increased. Makkar et al. [24] found that C₃ could be improved by supplementing CT and decreased concentration of C₂. Furthermore, ruminal CH₄ production was decreased by CT and can inhibit methanogens growth in the rumen fermentation directly. CT inhibition of methanogenesis may also lead to a decrease in the ratio of C₂ to C₃, as a result of increased hydrogen transfer to the synthesis of propionate [25]. Another possible influence of rumen CH₄ depression could be influenced by the suppression of protozoal population by FMS supplementation. This may be attributed to CT in FMS which affected the protozoa cell membrane, thus interfering with ion exchanges [26]. Poungchompu et al. [27] earlier reported that dairy heifer crossbreds supplemented with feeds containing phytonutrients had reduced protozoal count. The population of rumen protozoal and methane emission were significantly reduced [28].

Nitrogen balance, excretion of purine derivatives and microbial protein synthesis

FMS supplementation affected N-balance as shown in Table 4. The N-absorbed and retention were linearly increased with FMS supplementation due to N-intake and CP digestibility, however N-excretion of fecal and urine were similar among treatments. Agreed with, Viennasay et al. [12] who reported that increased digestibility of the CP, consequently affected on the increase in retained N. The high intake of CP and CT increases the flow of protein from the rumen to the small intestine [29]. The urine PD excretion is considered to be an important determinant to predict the productivity of microbial proteins in the rumen. The synthesis of ruminal microbial proteins plays an important role in ruminants because it mainly provides high-quality protein resources for the host animals. Essentially, microbial protein synthesis and efficiency depend on many factors such as DM intake, nitrogen and carbohydrate ruminal degradability rate, rumen dilution rate, mineral and other factors [30]. The ammonia is the main N source of the rumen microbes, but the diet availability of amino acids, peptides, and both together increases the growth of cellulolytic and amylolytic bacteria [31]. In adequate doses, the efficiency in microbial synthesis and the microbial yield were increased by including saponins [32] or CT [33]. Under this study, supplementation of FMS containing both CT and SPN, could provide additional protein available at the lower-gut for the host ruminant.

Conclusion

Under this experiment, the results revealed the potential use of FMS as a good-quality feed to improve nutrients digestibility, rumen fermentation, microbial protein synthesis, and to mitigate methane production. FMS supplementation at 0.6 kg DM/day exhibited the best result. Making FMS should be encouraged to prepare for on-farm use especially during the long dry season. Furthermore, in vivo feeding trials should be conducted in both beef cattle and dairy cattle in order to obtain more relevant data.

Materials And Methods

This study was approved by the Institutional Animal Care and Use Committee of Khon Kaen University, Thailand. Record No. IACUC-KKU-94/61 and Reference No. 0201.2.11/73.

Preparation of Flemingia macrophylla Silage

Flemingia macrophylla was planted by stems on the experimental plots of Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand with close supervision of the advisory Professor. All plant parts were kept and stored at TROFREC. Flemingia macrophylla (FM) whole top plant was harvested from the shrub after three months of regrowth. Young stem, leaf and branch were chopped with a machine to about 3 cm in length. Molasses: urea: water at 2:1:10 were mixed well with 100 kg chopped FM and stored in the plastic barrel after pressing well (approximately 60 kg/barrel), and tightly covered for 21 days. Flemingia macrophylla silage (FMS) was sampled from various barrels, oven-dried at 60 °C for 48 hours and then ground, stored in bottles for chemical analysis. Standard methods were employed to analyze for chemical compositions including dry matter (DM), ash, crude protein (CP), neutral-detergent fiber (NDF), acid-detergent fiber (ADF) [34, 35]. While condensed tannins (CT) was by the vanillin-HCl [36], method modified by [37] FMS sample was collected weighing 40 g soaked in 200 ml of cool distilled water for 12 hours. The mixture was then filtered and supernatant divided into 4 aliquots for determination of pH using a portable pH meter (HANNA Instruments HI 8424 microcomputer, Singapore). Apart from that, a FMS sample was washed with deionizing water for analyzed lactic acid and acetic acid analysis [38].

Animals and Design

These experimental beef crossbreds belonged to TROFREC, Khon Kaen University and were provided for use to support Ph.D. students for research. After the termination the experiment, they have been maintained in good health and were used for another experiment by a student. Four, beef cattle about two year old with 172 ± 43 kg liveweight, were randomly assigned to in a 4 × 4 Latin square design. Concentrate was offered at 0.5 kg of body weight (BW)/day and rice straw offered ad libitum with supplementation of FMS at 0, 0.2, 0.4 and 0.6 kg DM/head/day. The trial was conducted for four periods each was consisted of 21 days, during the first 14 days was for matter feed intake measurement, while during the last 7 days was for sample collection using total collection method. All animals were in individual pens, clean water and mineral-salt blocks were available at all times. The diet was offered to the animals twice in the morning (07:00a.m.) and afternoon (04.00p.m.). The liveweight of each cattle was weighed at the beginning and the end of each period to calculate feed intake. Feed provided and refusals were measured daily throughout the experimental period. Feed samples were collected twice a week for DM analysis [34]. Samples of feeds including concentrate, FMS, rice straw, feces were collected randomly daily during the last 7 days of each period. A daily sample of feces of each animal (about 100 g) was collected to be analyses [34]. Urine samples were acidified immediately after collection by diluting one volume of urine with four volumes of 0.1 N H₂SO₄ and stored at − 20 ºC urine samples were analyzed for total Nitrogen content [34] total purine derivatives (PD) excretion per day was calculated as the sum of allantoin and uric acid the daily absorbed exogenous purines estimated, and microbial N supply (MNS) predicted [39, 40], respectively. In the last day of each period, rumen fluid samples were collected at 0 and 4 hour post-feeding. Details of sampling procedures of rumen fluid for animal of volatile fatty acids (VFA) [38], protozoal population count [41], and estimated methane (CH₄) production [42] are presented in details in Wanapat et al. [43]. Blood samples (about 10 ml) were collected from the jugular vein at each rumen sampling time and kept into the tubes to which 0.1 g EDTA was added for analysis of blood urea-nitrogen (BUN) [44].

Data Management and Statistical Analysis

All data were subjected to ANOVA according to a 4 × 4 Latin square design using the General Linear Models (GLM) procedures [45] (24). The results were presented as mean values with the standard error of the means. Difference among means with P < 0.05 was accepted as statistical differences while 0.05 < P < 0.10 was accepted as a tendency. Treatment means were statistically compared by Duncan’s New Multiple Range Test [46] (25).

Abbreviations

Declarations

Ethics approval and consent to participate

The experiment was officially agreed and approved by the Khon Kaen University Committee of Animal Care and Use for Research. The experimental cattle were provided by our research farm (TROFREC, KKU).

Consent for publication

Not applicable.

Availability of data and materials

All experimental data are responsible and available under the holding of the corresponding author.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University. Thailand Research Fund and International Research Network (TRF-IRN) TRF-IRN57W0002. KKU Scholarship for ASEAN and GMS Countries’ Personnel. The Funding donors did not have roles in the design of the study; research conduct, samples collection, analysis, and interpretation of data; and in writing the manuscript.

Ethics approval

The experiment was officially agreed and approved by the Khon Kaen University Committee of Animal Care and Use for Research. Record No. IACUC-KKU-94/61 and Reference No. 0201.2.11/73.

Author contributions

MW: BV designed the experiments. BV: Conducted the animal experiments. BV: performed the analyses. MW, BV: Wrote the manuscript. All authors reviewed and contributed to the manuscript. MW revised the final draft of manuscript. All authors read and approved the final manuscript.

Acknowledgments

The technical support rendered by Animal Nutrition Research Institute, Department of Livestock Development-Thapra, Dairy Production Organization of Thailand, Northeast (DPO–NE), are greatly acknowledged.

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