Experimental design

The CAL used in this study were purchased from a commercial source (Karya Herbal Nasional Ltd., company land-plot at Bogor, Indonesia 6°70′28″S; 106°90′90″E and 6°43′30.1″S; 107°05′09.2″E). The CAL were randomly collected after 2 to 3-month growth period and dried in an oven at 50–60 °C for 48 h. The leaves were ground and prepared for analyses.

In vitro experiment

The Exp. 1 was carried out using the long-term in vitro rumen simulation technique (RUSITEC) following the procedures described by Kozłowska et al. [8]. The RUSITEC experiment was designed with a completely randomized block design with four diets and two replicates in each run, and repeated three times. The four isonitrogenous and isoenergetic diets were prepared as follows: diet 1: a control diet CON based on grass silage (5.7 g DM) and concentrate (5.3 g DM); diet 2: 10% CAL (6.4 g DM of grass silage, 3.5 g DM of concentrate, and 1.1 g DM of CAL); diet 3: 15% CAL (6.5 g DM of grass silage, 2.85 g DM of concentrate, and 1.65 g DM of CAL diet), and diet 4: 20% CAL (6.5 g DM of grass silage, 2.3 g DM of concentrate, and 2.2 g DM of CAL). The diets for animals donating the rumen fluids as well as animals for in vivo experiments were formulated using the IZ INRA [9] system to meet the animals’ nutrient requirements (average for 20 kg of body weight and 150 g/d of growth: 0.72 unit for meat production per day and 69 g protein truly digestible in the small intestine per day). The chemical composition of the grass silage, concentrate, and CAL are presented in Table 1. Rumen fluid and solid digesta for the in vitro experiment diet were collected before morning feeding from six rumen-cannulated lambs (20 ± 3 kg) for microbial inocula. The lambs, donors of the rumen fluid, were fed the same diet as in the CON treatment.

Table 1 Chemical composition and fatty acids profile of dietary components and CAL

Samples of fermentation fluid were collected directly from each fermenter 3 h before replacing the bags with the diets. The pH, ammonia concentration, VFA profile, feed degradability, protozoa count, and populations of methanogen, total bacteria, and select bacteria were analyzed. To determine FA profile, samples of the fermenting fluid were directly collected from the effluent vessels while the bags were being replaced. Fermentation gases were collected over 24 h using gas-tight bags (Tecobag 81, Tesseraux Container, Bürstadt, Germany).

In vivo experiment

The Exp. 2 employed six rumen-cannulated lambs allocated into two treatments, i.e., the control diet (CON) and the experimental diet (CAL-containing diet) in a crossover design. The highest level of CAL (20%) was selected based on the in vitro results from RUSITEC experiment. Lambs on CON diet received 352 g DM/d of concentrate and 379 g DM/d of grass silage. The experimental lambs received concentrate, CAL and grass silage. During the first 14 d of the experiment, the concentrate (352 g/d) in the experimental diet was gradually replaced by CAL from 46 g DM/d to 173 g DM/d. From d 15, the experimental lambs received 176 g DM/d of concentrate, 173 g DM/d of CAL and 507 g DM/d of grass silage. The CAL dose of 173 g DM/d corresponded to 20% of CAL in daily diet intake (856 g DM/d). In Exp. 2, each period lasted 24 d, with a 21-day adaptation period and a 3-day sampling period. Rumen fluid from each lamb was collected daily for 3 d of the experimental period, before morning feeding (0 h), and then at 3 h and 6 h after morning feeding [8]. The pH, ammonia concentration, VFA profile, and numbers of protozoa, methanogens, and total bacteria were analyzed. Meanwhile, samples for quantification of total bacteria and methanogens using fluorescence in situ hybridization (FISH) were only collected at the 3 h timepoint.

In Exp. 3, sixteen growing lambs (20 ± 3 kg live weight) were used for the final production performance test. Lambs were randomly allocated into CON or CAL dietary treatments based on their live weight (n = 8 per group). Lambs were kept individually during the whole experiment, except during the period when respiratory chambers were used. In order to reduce stress associated with isolation, two animals were always kept together in each cage placed in the respiratory chamber. The experiment lasted 30 d, a 21-day adaptation stage and an 8-day sampling period, with 1-day for the slaughtering process. During the adaptation period, the lambs were adapted to the CAL diet, as in Exp. 2. The CON lambs were fed the control diet comprised of grass silage (379 g DM/d) and concentrate (352 g DM/d). The CAL lambs were fed grass silage (507 kg DM/d), concentrate (176 g DM/d), and CAL (173 g DM/d). The diets were formulated following the IZ INRA [9] system to meet animals’ major nutritional requirements. All animals had free access to fresh water. The CON and CAL diets were fed in equal proportion at 8:00 h and 20:00 h daily. Feed intake, feed residue, and amount of feces were recorded daily. Animal weights were recorded weekly. During the sampling period (from d 22 to d 28 of the experiment), each cage was transferred into a respiratory chamber by daily rotation in order to determine the direct CH4 emission for 24 h consecutively. Two respiratory chambers were used. Each cage was tested twice but in order to obtain individual lamb’s gasses production, obtained results were divided by two.

On the last day of experiment (d 30), the animals were slaughtered 3 h after morning feeding. After slaughtering, the rumen digesta were taken from the top, bottom, and middle of the rumen and squeezed through a four-layer cheese-cloth for analysis of pH, ammonia concentration, and VFA profile in ruminal fluid, FA profile, and populations of protozoa, methanogens, total bacteria, and select bacteria in digesta. Samples of muscle from the right side of each carcass and drawn at the level of the thirteenth thoracic rib was immediately collected. Approximately 5 g of longissimus thoracis (LT) muscle was shock-frozen in liquid nitrogen for gene expression analysis. The LT muscle (ca. 50 g) was cooled and transferred in a cool (4 °C) atmosphere to the laboratory for FA analysis. All collected samples were stored at − 80 °C until analysis.

Meat quality traits

The LT (100 g) from the right-half carcass was used for meat quality analysis that was performed at the laboratory of the Institute of Agricultural and Food Biotechnology (Poland). The pH was measured in triplicates 24 h post-mortem on samples of LT muscle using a pH meter with an integrated electrode (pH meter 1140, Mettler-Toledo, USA) following ISO 2917 (2001) instructions. For the instrumental evaluation of meat color, 10 mm thick steaks of LT muscle were cut towards the direction of muscle fibers and exposed to electric light for 15 min. The values of L* (lightness), a* (redness), and b* (yellowness) were determined in triplicates using a Minolta Chroma Meter CR- 400 (Konica-Minolta, Japan). Compositional analysis of LT muscle (water, intramuscular fat (IMF) and total protein content) was performed using minced samples according to the methods described in ISO 1442 (2000) for water, ISO 1444 (2000) for fat (using a Soxtherm device, Gerhardt Analytical System, Germany), and PN-75/A-04018 (2000) for protein (using a Kjeltec System 1002 Distilling Unit, FOSS Analytical, Denmark). The water-holding capacity (WHC) of minced LT muscle samples was determined as described by Grau and Hamm [10], with later modifications introduced by Pohja and Ninivaara [11]. Visual evaluations of meat color and marbling of LT muscle samples were performed by a panel of four assessors using a 1–8 point Soicarni scale for meat color, with 1 being the lightest and 8 the darkest color, and a 1–4 point scale for marbling (developed by the Institute of Agricultural and Food Biotechnology, Poland), with 1 being related to minor and 4 to the greatest marbling. The taste panel of four professional assessors, trained in rating lamb for meat-eating quantity, was used to assess aroma, juiciness, tenderness, and flavor on boiled LT samples. Assessors scored the samples for each trait separately on a 1–5 point scale, where 1 was related to bad and 5 to a very good level of the traits according to the methodology of Barylko-Pikielna [12]. Concerning the above mentioned visual and sensory evaluations of meat, the mean values of the scores given by four assessors were taken for further calculations.

Determination of phenolic acid, flavonoid, and diterpenoid contents

The CAL preparation and extraction were caried out as described previously [7]. All the analyses were performed in triplicate for three independent samples that were stored in a freezer at − 20 °C before analysis. The CAL bioactive compounds (phenolic acid, flavonoid, and diterpenoid) were analyzed by ultra-high-resolution mass spectrometry (UHRMS) on Dionex UltiMate 3000RS system (Thermo Scientific, Darmstadt, Germany) with a charged aerosol detector interfaced with a high-resolution quadrupole time-of-flight mass spectrometer (HR/QTOF/MS, Compact, Bruker Daltonik, Bremen, Germany) according to the procedure of Ślusarczyk et al. [7].

Determination of the chemical composition of feeds

Samples of grass silage, concentrate, CAL, and feces were analyzed according to AOAC [13] for DM (method no. 934.01), ash (method no. 942.05), crude protein (CP; using a Kjel-Foss Automatic 16,210 analyzer; method no. 976.05), and ether extract (EE, using a Soxhlet System HT analyzer; method no. 973.18). The organic matter (OM) content was calculated by subtracting ash concentration from DM content. The aNDF was determined following the method of Van Soest et al. [14], with the addition of amylase and sodium sulfite without residual ash.

Basic rumen fermentation analysis and CH4 measurement

The pH of ruminal samples from all experiments was measured immediately after samples collection using a pH meter (CP-104; Elmetron, Zabrze, Poland). The ammonia concentration was analyzed using the colorimetric Nessler method described earlier by Bryszak et al. [5]. The VFA profile was determined by gas chromatography (GC Varian CP 3380, Sugarland, TX, USA) following the protocol of Varadyova et al. [15]. The in vitro CH4 concentration was measured using a gas chromatography in SRI PeakSimple model 310 (Alltech, PA, USA) following the procedure described by Kozłowska et al. [8]. Methane production in the in vivo experiment was measured using two respiration chambers (SPA System, Wrocław, Poland). The total chamber volume (8.2 m3) was ventilated by recirculating fans set at 40 m3/h giving approximately 5 air changes per hour. The temperature and relative humidity were set at 16 °C and 60%, respectively. The concentrations of CH4 and CO2 were measured using two nondispersive infrared spectroscopy detectors operating in the near-infrared spectrum (Servomex 4100, Servomex, UK; 1210 Gfx detector). Measurements were taken at two-second intervals. Two measuring channels were used: the concentration of CO2 in the range of 0–2.5% (0–48, 450 mg/m3) and the CH4 concentration in the range of 0–1000 ppm (0–706 mg/m3). The sample was collected and then ducted to the analyzer via a polyethylene tube with a diameter of 8 mm. The sampling rate was 0.6 L/min. Before starting the experiment, the analyzers were calibrated using as calibration gases (99.999% nitrogen gas by volume, 1210 ppm CH4 in nitrogen, and 4680 ppm CO2 in nitrogen). The analyzer was equipped with a 0.17 L cuvette with an optical track of 540 mm for CH4 and a 0.012 L cuvette with an optical track length of 154 mm for CO2.

Microbial quantification

The protozoa population was quantified following the method described by Michalowski et al. [16]. Methanogen numbers were quantified by fluorescence in situ hybridization (FISH) technique according to the procedure of Yanza et al. [6]. For bacteria quantification, total DNA was extracted from fermented fluid using QIAamp DNA Stool mini kit (Qiagen GmbH, Hilden, Germany) according to Yanza et al. [6]. Sequences of primers specific to the particular bacterial species or genera are presented in Table 2 [17,18,19,20,21,22,23,24]. Bacteria quantification was performed with a QuantStudio 12 Flex PCR system (Life Technologies, Grand Island, NY, USA).

Table 2 The sequences of primers specific to the analyzed bacteria species

Analysis of fatty acid in feed and meat samples

The FA profiles of the grass silage, concentrate, CAL, rumen fluid, and LT muscle were analyzed following the procedure of Bryszak et al. [5]. Sample hydrolysis was carried out in a closed system using screw-cap Teflon-stoppered tubes (Pyrex, 15 mL). Three milliliters of 2 mol/L NaOH was added to 100, 2500, 100, 10, and 500 mg of grass silage, concentrate, CAL, rumen fluid, and meat samples, respectively A gas chromatograph (GC Bruker 456-GC, USA) fitted with a flame ionization detector and a 100 m fused-silica capillary column (0.25 mm i.d.) coated with 0.25 μm Agilent HP (Chrompack CP7420) were used. The conjugated linoleic acid (CLA) peaks were identified via comparison with the retention times of the reference standard (conjugated linoleic acid methyl esters, and a mixture of cis- and trans-9, − 11 and − 10,12-octadecadienoic acid methyl esters; Sigma) using Galaxie Work Station 10.1 (Varian, CA, USA). The desaturase index, atherogenic index, and thrombogenic index were calculated as described by Bryszak et al. [5].

Analysis of mRNA expression in meat samples

Transcript analysis of FADS1, FASN, LPL, SCD, and ELOVL5 genes in the meat samples was performed using quantitative PCR (qPCR) analysis. Total RNA was isolated from 100 mg of LT muscle using Extrazol reagent. In brief, the meat samples were homogenized in 0.5 mL of Extrazol reagent using a TissueLyser II (Qiagen, USA). After 10 min incubation, 200 μL of chloroform was added and shaken vigorously for 15 s. The samples were then incubated for 10 min at room temperature and centrifuged for 15 min at 12,000 × g. Next, the upper aqueous phase was transferred to a new tube and 0.5 mL of isopropanol was added. The samples were again incubated and centrifuged as in the previous step. The resulting RNA pellet was washed with 1 mL of 75% EtOH and dissolved in RNAse free water (Sigma Aldrich). The quantity and quality of the isolated total RNA was checked using an NP80 NanoPhotometer (Implen, Germany). A reverse transcription reaction (RT) was carried out with 1 μg of total RNA and the Firescript RT cDNA Synthesis MIX with Oligo (dT) and Random primers (Solis BioDyne), following the manufacturer’s protocol. The mRNA expression was quantified using QuantStudio 12 Flex PCR system (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primer pairs used for RT-qPCR amplification are listed in Table 3 [25]. The specificity of reaction products was determined by the melting points (0.1 C/s transition rate). Two genes have been considered as reference, GAPDH and β-actin. Due to its higher stability, the β-actin gene was applied and relative mRNA expression was evaluated by delta-delta CT (ΔΔCT).

Table 3 The sequences of primers specific to the analyzed genes expression in the longissimus thoracis muscle of lambs

Statistical analysis

The data of the experiment 1 (RUSITEC) were analyzed using a mixed model procedure (PROC MIXED) in SAS (university edition, version 9.4; SAS Institute, Cary, NC, USA) with repeated measures of day and fermenter treated as the experimental unit. The dietary treatment was considered as the fixed effect, experimental run as the random effect, and the day (6 to 10 d) as the repeated factor. Differences among treatments were further determined using Tukey’s post hoc test and linear orthogonal contrast was used to ascertain the tendency of the dose effect of CAL. In experiment 2, data were analyzed using PROC MIXED of SAS with the model containing dietary group, hour, and their interaction (group × h) as the fixed effects and the animal and hour of sample collection as the random effect with repeated measures. When the significant value of the interaction occurred, Tukey’s post hoc test was used to estimate the differences between means. In experiment 3, data were analyzed using PROC TTEST procedure of SAS, and for all parameters each animal was considered as the experimental unit. Significance was accepted at P < 0.05 and tended to significance at 0.05 < P < 0.10. All the values are shown as group means with pooled standard errors of means.

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