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.
Statistical analysis
All the obtained data were subjected to the General Linear Model (GLM) [30]. Differences among treatment means were compared by the Tukey’s multiple comparison test [31]. Comparison between roughage to concentrate ratio, FMS supplementation and interactions were tested by orthogonal contrast.