Effects of Gelidium amansii extracts on in vitro ruminal fermentation characteristics, methanogenesis, and microbial populations

Preparation of G. amansii extract

Air-dried G. amansii samples were obtained from the Jeju Biodiversity Research Institute (JBRI, Jeju, Korea), and were stored under dry and dark conditions. Washed samples were cut into small pieces, freeze dried and crushed. Crushed powder was extracted with 80% EtOH at room temperature, using an ultrasonic cleaner (Branson Ultrasonics corporation, Danbury, CT, USA) and the EtOH eluate solution was filtered through Whatman No. 1 filter paper and concentrated under vacuum.

In vitro fermentation design

One cannulated Holstein cows (450±30 kg) was used as rumen fluid donors and provided with ad libitum access to water and a mineral-vitamin block. Timothy and commercial concentrate ratio (60:40, w/w) were fed twice daily at 09:00 and 17:00, equivalent to 2% of body weight. The rumen fluid collected before morning feeding was strained through four layers of cheese gauze, and the rumen contents were diluted by addition of artificial saliva and maintained at 39°C. All experimental protocols used in this research were approved and maintained in accordance with the Guide for the Animal Care and Use Committee of Gyeongsang National University (Jinju, Korea).

Dietary treatments were as follows: control (CON), basal diet (without G. amansii extract); treatment 1 (TRT 1), CON+1% G. amansii extract; treatment 2 (TRT 2), CON+3% G. amansii extract; and treatment 3 (TRT 3), CON+5% G. amansii extract as basis of substrate (timothy). The chemical composition of timothy was measured to contain the following: amount of moisture, 8.87%; crude protein, 13.37% ether extracts, 2.25%; crude fiber, 21.87%; crude ash, 8.62%; neutral detergent fiber, 53.18%; and acid detergent fiber, 30.57% as percentage of dry matter (DM).

Fifteen mL of rumen fluid: artificial saliva mixture (McDougall, 1:2, v/v) was dispensed into 50 mL serum bottles, containing 300 mg of timothy for CON and G. amansii extract for TRTs (3 mg for TRT 1, 9 mg for TRT 2 and 15 mg for TRT 3). The serum bottles were sealed under anaerobic conditions and capped with a butyl rubber stopper with an aluminum cap and then incubated in a gently shaking incubator (Jeio Tech, SI-900R, Daejeon, Korea; 120×rpm) at 39°C for 72 h. The in vitro fermentation experiment was a completely randomized block design and performed in triplicate, using 60 serum bottles (4 treatments×5 incubation times×3 replicates).

Analysis of gas profiles and ruminal fermentation characteristics

Total gas production was measured according to the method by Theodorous who suggested that the head space gas pressure should be measured using a detachable pressure transducer and a digital readout voltmeter (Laurel Electronics, Inc., Costa Mesa, CA, USA) after removing serum bottles from a shaking incubator. For gas profiling during 72 h, the transducer was connected to the inlet of a disposable Luer-lock three-way stopcock and then gas pressure in the headspace above the culture medium was read from the LED display unit after insertion of a hypodermic syringe needle. Furthermore, another outlet was connected to a gas chromatograph (HP 5890, Agilent Technologies, Santa Clara, CA, USA) using a TCD detector with a Carboxen-1006 Plot capillary column (30 mm×0.53 mm, Supelco, Bellefonte, PA, USA) for detection of methane, carbon dioxide and hydrogen.

Following gas profile analysis, serum bottles were uncapped and the culture medium was subsampled for pH analysis (MP230, Mettler-Toledo, Columbus, OH, USA), ammonia and VFA concentration. Ammonia concentration was measured as optical density (OD) values using a UV/VIS spectrophotometer (Model 680, Bio-Rad laboratories, Hercules, CA, USA) at 630 nm after sample preparation. For VFA measurement, culture medium was centrifuged at 3,000 rpm for 3 min and then supernatants were filtrated using a 0.2 μM disposable syringe filter (Whatman Inc., Clifton, NJ, USA). VFA analysis was performed with high performance liquid chromatography (HPLC, Agilent-1200, Waldbronn, Germany) using a UV/VIS detector with a MetaCarb 87H column (300 mm×7.8 mm, Varian, Palo Alto, CA, USA).

In vitro DM disappearance rate was determined using the nylon bag digestion method. After incubation, the nylon bag containing the substrate was washed twice in a water-bath equipped with a Heidolphs Rotamax 120 (Heidolph Instrument, Nuremberg, Germany) at 100 rpm for 30 min and then oven dried at 60°C to a constant weight. DM disappearance was defined as weight loss before and after incubation in the serum bottle.

Microbial growth performance

At the end of each fermentation period, samples were centrifuged at 3,000 rpm for 3 min to remove feed particles and the supernatants were re-centrifuged at 14,000 rpm for 3 min to pellet. Supernatants were divided for protein and glucose analysis. The protein was evaluated as OD value at 595 nm measured by a spectrophotometer (Model 680, Bio-Rad Laboratories, USA), using the dye binding properties of Coomassie Blue G-250. For glucose measurement, 200 μL of supernatant and 600 μL of 3,5-dinitrosalicylic acid solution were mixed and incubated for 5 min in a boiling water bath. The glucose concentration was determined as OD value at 595 nm using a microplate reader (Model 680, Bio-Rad Laboratories, USA). After discarding the supernatants, pellets were washed with sodium phosphate buffer (pH 6.5) and repeated three more times. The growth rate of total microorganisms was evaluated as OD value at 550 nm as measured by spectrophotometer (Model 680, Bio-Rad Laboratories, USA).

Quantitative real-time polymerase chain reaction

Samples were placed in screw-capped tubes containing silica beads for DNA extraction with a high-speed reciprocal shaker, following a modified bead-beating protocol with a Soil kit (Macherey-nagel, Düren, Germany). Briefly, a 1.0-mL aliquot of the incubated culture solution was centrifuged at 3,000×rpm, and then placed in a NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to determine nucleic acid concentrations.

Previous reports provided primers and thermocycling protocols used for amplification of general bacteria, ciliate protozoa, methanogenic archaea, Fibrobacter succinogenes (F. succinogenes), Ruminococcus albus (R. albus), and Ruminococcus flavefaciens (R. flavefaciens) were from previously published reports [10–13].

Quantitative real-time polymerase chain reaction (PCR) assays (CFX96 Real-Time system; Bio-rad, USA) were performed with the SYBR Green Supermix (QPK-201, Toyobo Co., LTD., Tokyo, Japan), following Denman and McSweeney [14] and Denman et al [11]. Abundance of these microbes was determined by the following equation:

Relative change in gene expression

= 2^{−ΔCt(Target)−ΔCt(Control)}

Where Ct represents threshold cycle. All quantitative (q) PCR reaction mixtures (final volume of 20 μL) contained forward and reverse primers, the SYBR Green Supermix and DNA template. Table 1 shown the PCR amplification of the target DNA, included the annealing and the extension temperature, was conducted following the same conditions described in the PCR primer references [10–13].

Table 1

Polymerase chain reaction primer sets for real-time polymerase chain reaction assays

Statistical analysis

All experimental data were analyzed using the general linear model procedure of SAS [15] as a completely randomized block design. The effects of supplementation of G. amansii extract on pH, total gas production, DM disappearance, gas profiles, VFA profiles and microbial growth were compared to CON and significant differences were conducted using polynomial regression to measure the linear and quadratic effects of increasing concentrations of G. amansii extract. Variability in the data was expressed as standard error of the mean and p<0.05 was considered to be statistically significant, whereas p<0.10 was considered a tendency.

In vitro fermentation characteristics (cumulative pH, gas production, dry matter disappearance rate)

The effect of dose response of G. amansii extract on in vitro fermentation parameters is shown in Table 2. G. amansii extracts increased pH correlating positively with G. amansii extract concentration. With regards to cumulative gas production by mixed ruminal microorganisms, G. amansii extracts reduced cumulative gas production at 3 h, but increased it at 12, 24 and 72 h as compared with CON, respectively. Lastly, DM disappearance with supplementation of G. amansii extracts was lower than that of CON during the whole experimental period.

Table 2

Effect of Gelidium amansii extract on in vitro cumulative pH, gas emission, dry matter disappearance by mixed rumen microbial fermentation

Volatile fatty acid profile and acetic acid/propionic acid ratio

The effect of dose response of G. amansii extract on VFA profiles of acetate, propionate, butyrate, and acetic acid/propionic acid (A/P) ratio are shown in Table 3. The supplementation of G. amansii extract increased total VFA concentration, acetate concentration, propionate concentration, butyrate concentration and A/P ratio as compared with CON, respectively.

Table 3

Effect of Gelidium amansii extract on total volatile fatty acids (VFA), acetic acid, propionic acid, butyric acid and acetic acid/propionic acid (A/P) ratio by mixed rumen microbial fermentation

Emission gas profile (methane, carbon dioxide, and ammonia)

The effect of dose response of G. amansii extract on the gas profiles of methane, carbon dioxide and ammonia are shown in Table 4. G. amansii extract reduced methane emissions relative to CON at 3 h, carbon dioxide emission was increased relative to CON at 12 h. Futhermore, G. amansii extract incerased hydrogen, ammonia emission as compared with CON.

Table 4

Effect of Gelidium amansii extract on in vitro methane, carbon dioxide, hydrogen and ammonia emission by mixed rumen microbial fermentation

In vitro ruminal change in microbial diversity

The effect of dose response of G. amansii extract on microbial growth rate, protein and glucose concentration is shown in Table 5. The supplementation of G. amansii extract increased microbial growth rate and glucose concentration, while reducing protein concentration as compared with CON, respectively.

Table 5

Effect of Gelidium amansii on rumen microbial growth rate, protein and glucose concentration

Changes in ruminal microbial diversity supplemented with G. amansii extracts are shown in Figure 1a (after 12 h incubation) and Figure 1b (after 24 h incubation), respectively. Supplementation with G. amansii extracts lead to an increase in the ciliate-associated methanogen population at 12 h, while reducing the methanogenic archaea population at 24 h as compared with CON, respectively. With regards to major fibrolytic microorganisms, G. amansii extracts reduced R. albus at 12 h, 24 h and F. succinogenes at 12 h, while increasing R. flavefaciens at 24 h as compared with CON, respectively.

Figure 1

Relative quantification analysis of rumen microorganism populations under in vitro ruminal fermentation by the addition of marine alga Gelidium amansii after 12 h (a) and 24 h (b) incubation. Microorganism populations examined include ciliate-associated methanogens, methanogenic archaea, and cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens). Dietary treatments were as follows: CON, basal diet (without G. amansii); TRT 1, CON+1% G. amansii; TRT 2, CON+3% G. amansii; TRT 3, CON+5% G. amansii as basis of substrate (timothy). ^ab Means with different superscripts in the same row differ significantly (p<0.05).