WITHDRAWN: Rumen fermentation kinetics as impacted by red amaranth (Amaranthus cruentus, L) leaf powder using in vitro gas production technique

Abstract

Background

A need for research searching for alternative rumen enhancers warrants immediate attention. The in vitro fermentation experiment was conducted using factorial arrangement of two factors of roughage to concentrate and seven level of red amaranth leaf powder percentage of total substrate in a Completely randomized design (CRD). Two factors, namely Factor A was two ratio of roughage (R) to concentrate (C) at 60:40 and 40:60 and Factor B was level of red amaranth (Amaranthus cruentus, L) leaf powder (RALP) supplementation at 0, 2, 4, 6, 8, 10, and 12% of total dietary substrate.

Results

Red amaranth leaf powder (RALP) contained phytonutrients both condensed tannins and saponins in addition with high macro minerals (Ca, K, and Mg). This experiment revealed innovations of the RALP supplementation by enhancing rumen propionate (C₃) production, reducing acetate (C₂) to (C₃) ratio, reducing protozoal population and mitigating methane (CH₄) production. Furthermore, rumen dry matter degradation percentages were remarkably enhanced (P < 0.001) by increasing RALP supplementation.

Conclusion

Plants rich in phytonutrients and minerals such as red amaranth leaf powder (RALP) have a vital and promising role in modulating rumen fermentation, mitigating methane production, as well as increasing substrate degradability.

Background

“Feeding the bugs, feeding the cows” has been stated for a long time in order to provide the rumen to perform the anaerobic fermentation process yielding the fermentation end-products degraded by residing microbiomes [1]. Wolin [2] and Murphy et al. [3] profoundly reported the stoichiometric balance in the rumen, showing the occurrence of rumen methane production via the metabolic pathway of rumen acetic production while capturing it in the propionate production randomization pathway. Using chemicals and/or antibiotics as rumen buffers or regulators have resulted improvement of rumen fermentation efficiency by increasing propionate while mitigating methane production [4]. Rumen pH has been demonstrated to greatly influence rumen microorganisms and their fermentation end-products [5, 6]. By shifting levels of concentrate ratio, as well as supplementation, the rumen pH was dramatically reduced. Buffering rumen pH is therefore a necessity when the ruminants receiving high level of concentrate and supplementation. Herremans et al. [7], currently reviewed using a meta-analysis, confirming the beneficial effect of dietary tannins especially condensed tannins on improving the nitrogen utilization in ruminants by decreasing rumen protein degradation, ammonia-nitrogen concentration, blood urea-nitrogen, milk urea-nitrogen, whilst digestibility of protein was significantly lowered.

Feed resources and plant extracts containing phytonutrients (PTN) have been increasingly important, as they exerted impacts on rumen microorganisms and fermentation especially the effects of condensed tannins (CT) and saponins (SP) [8]. Many kinds of plants and fruit wastes, containing PTN have been shown with their impacts on modulating the rumen fermentation especially mitigating methane production [9–11].

Amaranth, namely red amaranth (Amaranthus cruentus, L.) have been revealed to contain high concentration of phytonutrients, in which they could exert significant effect on human health [12]. Amaranth (Amaranth spp.) is a nutritious crop containing high levels of crude protein, minerals, vitamins, as well as polyphenols and flavonoids especially in the leaf and seeds [13, 14]. The main phenolic compounds reported in both leaf and seeds were gallic acid, vanillic acid and p-coumaric acid etc. [15]. Earlier study by Chairatanayuth [16] who found A. Spp. leaf especially in A. cruentus L., contained higher CP (16.50%) lower NDF (41.50%), when harvested at 45 days of growth. Khandaker et al. [17] reported the efficacy of polyphenols of red amaranth (A. trocor L.) on the antioxidant activity and have found that high level of total polyphenols in leaf was closely correlated (P < 0.05, R² = 0.82) with the antioxidant activity. Nevertheless, no research work using red amaranth leaf on its influence on rumen fermentation characteristics and nutrients digestibility in ruminants. Therefore, this experiment aimed it investigating the effect of red amaranth (Amaranthus cruentus, L.) on in vitro fermentation gas production experiment.

Results

Table 1, presents details of feeds and their nutritive values. Rice straw composited of 2.80% CP, 72.70% NDF and 47.50% ADF, respectively. Concentrate was formulated and analyzed to contain 14.40% CP, 75% TDN, 28.90% NDF, and 17.20% ADF. The nutritive value of red amaranth leaf power were 15.80% CP, 40.30% NDF, 29.40% ADF and 2.37% Ca, 2.33% K, and with 0.90% condensed tannins, 0.50% saponins, respectively.

The gas production kinetics parameters are presented in Table 2. Ratio of roughage to concentrate and percentage of red amaranth leaf powder (RALP) had impacted on the fermentation process and subsequently on the gas production kinetics (b, c, a + b). The c and a + b values for both R:C ratio and RALP were highly significant. As level of RALP supplementation increased, regardless of R:C, the values were enhanced. For in vitro DM degradabilities percentages for both at 12 and 24 h the data were also incremental and were highly significant for both R:C and level of RALP supplementation.

Table 3, presents the findings of pH, NH₃-N and volatile fatty acid production. The rumen pH data remained similar, although slightly dropped when ratio of R:C were lowered (6.69 to 6.56). Notable changes on rumen NH₃-N when level of RALP increased for both R:C at 60:40 and 40:60. Remarkable changes on rumen propionate production were obtained when level of RALP increased for both level of R:C, thus, lowering the C₂:C₃ ratio.

Table 4, shows rumen protozoal population and methane production. The rumen protozoal population (× 10⁵cell/ml) and methane production (mol/L) were greatly influenced by the R:C and RALP supplementation and there were significant interactions for both factors. The methane (CH₄) production resulted in remarkable decline of CH₄ for both R:C and RALP supplementation but more dramatically for R:C at 40:60, respectively.

Discussion

In Vitro Gas Production And DM Degradability (%)

Under Table 2, the treatment combinations of two ratio of R:C, 60:40 and 40:60, along with level of RALP supplementation from 0, 2, 4, 6, 8, 10, and 12% total substrate and the fermentation gas kinetics and DM degradability (%) are fully presented. The R:C ratio and RALP supplementation level did not significantly impact on a and b, although there was an increasing trend for the b values, when RALP supplementation level increased for both R:C ratio. The R:C ratio at 40:60 yielded higher c constant than R:C at 60:40. The a + b data for both R:C ratio were increased (P < 0.01) by the RALP supplementation level and there were interactions (P < 0.01). The accumulated total production tended to be enhanced for both R:C ratio but there were significant difference among treatments.

In vitro DM degradability (%) were measured both at 12 and 24 h of incubation. Higher RALP supplementation level remarkably increased the DM degradabilities for both R:C more pronounced for R:C at 40:60. The significant for both R:C and RALP interactions supplementation level were occurred (P < 0.01). This occurrence could be due to more available carbohydrate for R:C at 40:60 and from the incremental RALP supplementation with combined phytonutrients (condensed tannins and saponins) could enrich the fermentation process. Furthermore, higher concentration of minerals could help buffer pH especially for R:C at 40:60. Aslam et al. [35] pointed out that the use of rumen buffer such as NaHCO₃ could be beneficial when more concentration was fed. It was indicative that RALP supplementation could enhance the DM degradability, nevertheless, suitable level of supplementation be further investigated in in vivo trials.

Rumen Fermentation Process

Rumen fermentation end-products namely volatile fatty acids (VFAs) and ammonia-nitrogen (NH₃-N) were synthesized by rumen microbiome. Russell abd Rychlik [36]; Wallace et al [37] and Huws et al. [38] have reiterated the close relationship of rumen microbiome and their fermentation efficiency. These fermentation end-products will serve as important substrates for the host-ruminants. Under this work, rumen pH not changed but maintained higher for R:C at 60:40 as compared to 40:60 as the roughage could have attributed. It was notable that rumen NH₃-N concentrations were higher for R:C at 40:60 and were declined when RALP supplementation level were incremental for both R:C at 60:40 and 40:60, respectively and there were no significant interactions. Total VFAs C₂, C_3, C₄ and C₂:C₃ ratio were shown in Table 3. The C₃ concentrations were elaborately shown to be enhanced by both R:C and RALP supplementation level, being more explicit for R:C at 40:60 and with increasing supplementation level of RALP, whilst C₂:C₃ were greatly lowered (P < 0.01). Dietary source of RALP under this experiment has enormously supported the fermentation in which the beneficial outcomes were obtained as shown in Table 3. It was further speculated that level of RALP supplementation should be evaluated as they exhibited their supplementation effects.

Fermentation gas such as methane (CH₄) has been produced during anaerobic fermentation in the rumen. Johnson and Johnson [39] reported the loss of metabolizable energy in the form of CH₄ up to 15% gross energy. Furthermore, CH₄ is the one of the greenhouse gases and is 23 times more potent then CO₂ equivalent. Hence, the mitigation of CH₄ via rumen fermentation has been the major concern. Dietary manipulation in the rumen has been receiving more interests [40]. Under Table 4, the protozoal population enumerated at 4 and 8 h of incubation; were dramatically reduced for both R:C ratio and by higher RALP supplementation level and ther were greatly interative (P < 0.01). As shown by many researchers that the reduction of protozoal population in the rumen has a direct effect on methane production since some methanogens adhead on protozoa. In addition, the presence of phytonutrients contained in the feeds could also attribute to the mitigation of rumen methane. As presented under this work the rumen methane production were profoundly mitigated by increasing level of RALP supplementation for both R:C ratio at 60:40 and 40:60, respectively. This result could very well support that RALP could be highly promising to be employed in feeding to ruminant to possible improve rumen fermentation efficiency and mitigate methane production.

Conclusion

Under this investigation, plants rich in phytonutrients and minerals such as red amaranth leaf powder (RALP) have a vital and promising rode to modulate rumen fermentation in maintaining pH enhancing propionate production mitigating methane production, as well as increasing DM degradability. RALP exhibits its potential role and deserves further in vivo trials investigation.

Methods

This study was approved by the Animal Care and Use Committee of Khon Kaen University.

Experimental Design And Dietary Treatments

The red amaranth seeds (Chia Tai seed company, Bangkok, Thailand) were bought from local market under the supervision and approval by corresponding author. The experiment was randomly assigned in factorial arrangement of two factors of roughage to concentrate and seven level of red amaranth leaf powder percentage of total substrate in a Completely randomized design (CRD). There were two factors, Factor A was two levels of roughage to concentrate ratio (R:C) at 60:40 and 40:60 of dietary substrate at 0.20 g, while Factor B was level of red amaranth (Amaranthus cruentus, L) leaf powder (RALP) supplementation at 0, 2, 4, 6, 8, 10, and 12% of total dietary substrate.

Red amaranth leaf was collected from the plant after 25 days of growth. The red amaranth was planted on our experimental farm Khon Kaen University, Khon Kaen, Thailand. It was sun-dried, chopped and ground to achieve the 1 mm length. Standard chemical analyzed were employed to analyze for (DM), (OM), (CP) [18], neutral-detergent fiber (NDF), acid-detergent fiber (ADF) [19]. Additional chemical procedures on condensed tannins (CT) [20, 21] as modified by Wanapat et al. [22] and saponins (SP) [23] were used. Macro minerals were determined using wet digestion (nitric-perchloric digestion), atomic absorption spectrophotometry (total Ca, K, Mg, Zn, Fe) (model: analytic jena nova 350).

Rumen And Substrate Inocula

As a source of rumen inocula, two rumen-fistulated Holstein-Friesian dairy steer crossbreds (75% Holstein-Friesian and 25% Thai native breed, about 370 kg body weight) were used. Before the morning feeding, 1000 ml of rumen fluid was collected from each animal and combined. The rumen fluid donors were fed with concentrate (14% DM of CP) at 0.5% of body weight (BW) to maintain normal rumen ecology and rice straw was offered on ad libitum. The method used in this study was according to Menke et al. [24], as modified and described by Kang et al. [25].

In Vitro Fermentation And Gas Production

The in vitro fermentation kinetics and gas production of all treatment samples were run intervally starting from 1, 2, 4, 6, 8, 12, 24, 48, 72, to 96 h post-incubation. Measurement of fermentation gas production was recorded at each time, pH was measured at 4, 8, and 12 h while the fluid was collected at 4 and 8 h, and was divided into two parts. The first part of rumen fluid (18 mL) was collected and kept in a plastic bottle to which 2 mL of 1 M H₂SO₄ was added to discontinue fermentation process for later analyses of NH₃-N by Kjeltec Auto 1030 Analyzer [18], volatile fatty acids (C₂, C₃ and C₄) using HPLC, Instruments by Water and Nova Pak model 600E; water mode l484 UV detector; column nova Pak C18; column size 3.9 mm × 300 mm; mobile phase 10 mMH₂PO₄ [pH 2.5] according to Samuel et al. [26] and the second portion of 1 ml rumen fluid was collected and kept in a plastic bottle to which 9 ml of 10% formalin solution for measuring of protozoal population using total direct count method by haemocytometer [27]. Methane production was determined from samples collected starting from 4, 8, to 12 h post-incubation internally using gas chromatography (Instruments by GC-17A System, Shimadzu; TCD detector; column shin carbon; column size 3 m × 3 mm, activated charcoal 60/80 mesh) [28]. The fermentation kinetic: y = a + b (1-e^(−ct)); where a = gas production from immediately soluble fraction, b = production of gas from the insoluble fraction, c = constant gas production rate for the insoluble part (b), t = time for incubation, (a + b) = the potential scope of gas production. y = gas produced at the time “t.” The in vitro dry matter degradability (%) were calculated at both 12 and 24 h post-incubation were performed based on the Orskov and McDonal [29]. The in vitro dry matter degradability (%) were calculated for both 12 and 24 h post-incubation.

Abbreviations

ADF: acid detergent fiber; BUN: blood urea nitrogen; BW: body weight; C: concentrate; CP: crude protein; C₂: acetate; C₃: propionate; C₄: butyrate; CP: condensed tannins; DM: dry matter; NDF: neutral detergent fiber; NH₃-N: ammonia-nitrogen; N: nitrogen; OM: organic matter; RALP: red amaranth leaf powder; R: roughage; SP: saponins; TDN: total digestibility nutrient; VFA: volatile fatty acid

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 donor did not have role in the design of the study; collection, analysis, and interpretation of data; and in writing the manuscript.

Authors’ contributions

MW designed the experiments; BV and TA. Conducted the animal experiments: BV and TA performed the analyses: MW, BV and TA. 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 Tropical Feed Resources Research and Development Centre (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand were gratefully acknowledged. Special gratitude are also extended to Thailand Research Fund under International Research Network (TRF-IRN), KKU Scholarship for ASEAN and GMS Countries’ Personnel, TRF-IRN57W0002 and TRF-IRG598001, respectively. All graduate students especially Burarat Phesatcha, Maharach Matra, Pajaree Totakul are thankful for their assistances with samplings.

Author information

¹Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal  Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand

² Department of Animal  Science, Faculty of Agriculture and Technology Rajamangala University of Technology Isan Surin Campus, Surin 32000, Thailand

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