Rumen fluid collection
At 3 h after the morning feeding, approximately 3 L of rumen fluid was collected from two ruminally fistulated, nonlactating Holstein cows (880 kg average body weight). The cows were maintained on a daily diet of Orchard grass (Dactylis glomerata) hay (organic matter (OM), 980 g/kg; crude protein (CP), 132 g/kg; neutral detergent fiber (NDF), 701 g/kg; acid detergent fiber (ADF), 354 g/kg; acid detergent lignin (ADL), 40 g/kg; on dry matter (DM) basis) with free access to clean drinking water and mineral blocks (KOEN® E250 TZ, Nippon Zenyaku Kogyo Co., Fukushima, Japan). The rumen fluid from each cow was collected from four different locations in the rumen. The collected rumen fluid was strained through four layers of surgical gauze into a thermos flask that was prewarmed to 39 °C and then immediately transferred to the laboratory within 15 minutes. The animal management and sampling procedures were approved by the Obihiro University of Agriculture and Veterinary Medicine, Animal Care and Use Committee (Approval number, 20–119).
Experimental treatments and in vitro incubation technique
Prior to the in vitro incubation, ten experimental groups, with six replicates each, were prepared with approximately 500 mg of ground substrate comprised of Kleingrass (Panicum coloratum) hay and commercial concentrate mixture at different ratios with and without MT inclusion. The MT mixture is composed of 90% garlic granules and 10% citrus extract powder. The garlic powder used for MT preparation was sourced from cultivated and carefully processed and dried non-GMO garlic of Chinese origin. The dried garlic granules were standardized to contain 1% (w/w) Allicin potential (S-Prop-2-en-1-yl prop-2-ene-1-sulfinothioate). Allicin concentration was determined by High Performance Liquid Chromatography (HPLC) as described in details by Eger et al. [15]. The citrus components for the MT mixture (Naringin, Naringenin, Neohesperidin, Rhoifolin, and Neoeriocitrin) were developed from commercially available citrus extracts (Khush Ingredients, Oxford, United Kingdom) mainly extracted from bitter oranges (Citrus aurantium). The total polyphenol content of the citrus extract was standardized to 45% (w/w) by the Folin-Ciocalteau method [17]. Flavonoid concentrations were analyzed by HPLC using standards from (Sigma-Aldrich Ltd., Dorset, United Kingdom). Further information on the MT preparation was described in details by Eger et al. [15]. This mixture was provided by a Swiss company (Mootral SA, Rolle, Switzerland).
The experimental diets were as follows: 1- 1000 g grass/kg ration (10GRS), 2- 10GRS + 200 g MT/kg of substrate (2MT), 3- 8GRS + 200 g concentrate/kg ration (2CON), 4- 8GRS + 2CON + 2MT, 5- 6GRS + 4CON, 6- 6GRS + 4CON + 2MT, 7- 4GRS + 6CON, 8- 4GRS + 6CON + 2MT, 9- 2GRS + 8CON, and 10- 2GRS + 8CON + 2MT. Five hundred milligrams of each of the experimental substrates (GRS and CON) was added to preweighed ANKOM filter bags (F57, ANKOM Technology, Macedon, NY, USA), which were heat-sealed and placed in 120 mL glass bottles, while the MT feed supplement was added directly to the bottles one day before incubation. The MT dose used in the current study was based on the most effective dose in our previous study [16]. The chemical composition of the substrates and the MT are described in Table 1.
Table 1
Chemical composition of ration and Mootral (g/kg of dry matter) used in 24 h in vitro incubation.
(g/kg dry matter) | Kleingrass hay | Concentrate | Mootral |
Dry matter (g/kg fresh matter) | 844.9 | 843.0 | 871.5 |
Organic matter | 904.7 | 934.2 | 955.5 |
Crude ash | 95.3 | 65.8 | 44.5 |
Crude protein | 134.8 | 223.1 | 210.7 |
Ether extract | 38.4 | 38.0 | 17.1 |
Neutral detergent fiber | 662.5 | 232.6 | 35.5 |
Acid detergent fiber | 362.6 | 109.1 | 33.7 |
Acid detergent lignin | 52.2 | 30.1 | 1.8 |
The procedure of in vitro batch culture was performed as described by Menke and Steingass [18]. In the laboratory, the collected rumen fluids from the two cows were mixed together in one beaker under a constant stream of CO2. Forty milliliters of fresh buffer solution at a pH of 6.8 prepared according to McDougall [19] with twenty mL of rumen fluid was added to each 120 mL bottle under continuous CO2 flushing to maintain anaerobic conditions. Thereafter, the fermentation bottles were flushed with CO2 before sealing with butyl rubber stoppers and aluminum caps (Maruemu Co., Ltd, Osaka, Japan). All bottles were incubated for 24 h at 39 °C. This batch culture procedure was repeated in three consecutive runs on three different days. In each run, two blanks without substrate (empty filter bag plus 60 mL of buffered rumen fluid) were included to be used for digestibility and gas production correction. In total, 180 bottles plus 6 blank bottles were examined in this study.
Sample collection
After 24 h of incubation, the total gas production was measured, and gas samples (3 mL) were collected from the headspace of the glass bottles into vacutainer tubes (BD Vacutainer®, Becton Drive, USA). The tubes were stored at room temperature until CH4 and CO2 determination. Thereafter, the bottles’ caps were removed, and the pH of each tube was recorded using a pH meter (LAQUA F-72, HORIBA Scientific, Kyoto, Japan). Then, aliquots of the culture fluid were transferred into 1.5 ml Eppendorf tubes and centrifuged at 16,000×g and 4 °C for 5 minutes. The supernatant was collected and transferred into a new Eppendorf tube® (Eppendorf AG, Hamburg, Germany), which was stored at − 20 °C until use for volatile fatty acid (VFA) and ammonia nitrogen (NH3-N) analysis. The bags were removed from the bottles, washed under running tap water until the draining fluid became clear, and then dried at 60 °C for 48 h to determine the in vitro dry matter digestibility (IVDMD). After IVDMD determination, the bags were used for the determination of in vitro organic matter digestibility (IVOMD), in vitro neutral detergent fiber digestibility (IVNDFD), and in vitro acid detergent fiber digestibility (IVADFD). The residues in the fermentation bottles were discarded.
Chemical analysis
The chemical composition of the GRS, CON, MT and remaining substrate in the bags was determined following the standard procedure of AOAC [20]. DM content was measured by drying the samples in an air-forced oven at 135 °C for 2 h (930.15). OM and ash were measured by placing the samples into a muffle furnace at 500 °C for 3 h (942.05). Nitrogen (N) was measured according to the method of Kjeldahl (984.13) using an electrical heating digester (DK 20, VELP Scientifica, Usmate (MB), Italy) and an automatic distillation apparatus (UDK 129 VELP Scientifica, Usmate (MB), Italy), and then CP was estimated as N × 6.25. aNDF, ADF, and ADL were measured and expressed as inclusive residual ash using an ANKOM200 Fiber Analyzer (Ankom Technology Methods 6, 5 and 8, respectively; ANKOM Technology Corp., Macedon, NY, USA). aNDF was measured using sodium sulfite with heat-stable α-amylase.
Gas composition analysis
The concentrations of CH4 and CO2 in the gas samples were determined by injection of 1 mL using a Hamilton gastight syringe (Hamilton Company, Reno, Nevada, USA) into a gas chromatograph (GC-8A, Shimadzu Corp., Kyoto, Japan). The carrier gas was helium. The temperatures of the infuser port, column, and detector were 70 °C, 150 °C, and 150 °C, respectively. The identification of CH4 and CO2 was based on the retention time.
Volatile fatty acids and NH3-N analysis
The concentration of VFA was determined using high-performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) after diluting the supernatant 3 times with distilled water. Briefly, the analytical specifications were as follows: column, Shim-pak SCR-102H (7 mm, i.d. 8.0 mm×300 mm, Shimadzu Corp., Kyoto, Japan); eluent flow rate and mobile phase for organic acid analysis (Shimadzu Corp., Kyoto, Japan) at 0.8 mL/min; column temperature, 40 °C; reaction reagent and flow rate, pH buffer for organic acid analysis (Shimadzu Corp., Kyoto, Japan) at 0.8 mL/min; conductivity detector (CDD-10AVP, Shimadzu Corp., Kyoto, Japan). Quantification of the VFA concentration was performed using an external standard quantitation method [16].
The NH3-N concentration was measured by diluting samples 50 times with 0.1 M phosphate buffer (pH 5.5) and then they were analyzed following the procedure of the modified Fujii-Okuda method [21] using an NH3 kit (FUJIFILM Wako Pure Chemical Corp, Osaka, Japan). The plate was read by a microplate reader (SH-1000 Lab, Corona Electric Co., Ltd., Japan) at an optical density of 630 nm.
Statistical analysis
All variables were analyzed using PROC MIXED by SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). The model included the treatment (diet) effect, MT effect, and their interaction as fixed effects, while the experimental runs were considered random effects. Least square means and standard error (SEM) were calculated, and the differences of means were estimated by pairwise t-tests (PDIFF option of PROC MIXED). Significance was declared at P < 0.05, and a tendency toward significance was declared when the P value was between 0.05 and 0.10.