Effects of nitrogen gas flushing in comparison with argon on rumen fermentation characteristics in in vitro studies

Two fistulated Korean native heifers fed a total mixed ration (TMR; Table 1) were used to obtain rumen fluid samples. The chemical analyses for the TMR were conducted in accordance with the AOAC [11], but the content of NDF was analyzed with a neutral detergent solution [12] containing sodium sulfite and a heat stable amylase. The rumen fluid was collected from ventral and dorsal sac of the heifers at 2 h after morning feeding and filtered through 4 layers of cheese cloth and then mixed in the same ratio. Afterwards, the mixed rumen fluid was transported to laboratory using preheated thermos bottles.

The TMR fed to the heifers was milled through a 1-mm screen and used as a substrate for the incubation. McDougall’s buffer [13] which was heated at 39°C and purged continuously with CO₂ was mixed with the rumen fluid in a 3:1 (vol:vol) ratio. Next, 40 mL of the buffered rumen fluid was dispensed into 120 mL serum bottle filled with 0.4 ± 0.002 g of substrates. The ultra-high purity (99.999%) N₂ and Ar was flushed into headspace of the serum bottles, respectively. For each treatment, N₂ or Ar flushing, four replications were incubated at 3, 9, 12, and 24 h.

Total gas production (TGP) was calculated from headspace gas pressure measured by a pressure transducer (Sun Bee Instrument Inc., Seoul, Korea) [14]. To measure the methane concentration, 0.3 mL of head space gas was collected by gas tight syringe (Gastight#1001; Hamilton Co., Reno, NV, USA) and then injected manually to a gas chromatograph (HP 6890 series GC system; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a thermal conductivity detector and a capillary column (HP-PLOT/Q; Agilent Technologies Inc., Santa Clara, CA, USA). The temperatures of the inlet, oven and detector were 50°C, 50°C, and 250°C, respectively. The helium gas was used as carrier gas. The standard gas with known composition: H₂ 1.0%, CH₄ 10.1%, CO₂ 20.1% and N₂ 19.9% in He (MS Dong Min Specialty Gases, Inc., Pyeongtaek, Korea) was used to quantify CH₄ concentration.

After measuring the pH values using a digital pH meter (S20 SevenEasy pH; Mettler Toledo Co. Ltd., Greifensee, Switzerland), residual rumen fluid samples were stored at −20°C immediately for volatile fatty acids (VFA) and NH₃-N analysis. After being thawed, 10 mL of sample was mixed with 1 mL of HgCl₂ 2% (wt/vol) solution and briefly centrifuged at 2,000 ×g for 10 min at 4°C in order to remove feed particles. The supernatants were used for VFA and NH₃-N analysis.

To prepare samples for VFA analysis, 1.4 mL of the supernatants were mixed with 0.28 mL of 25% (wt/vol) meta-phosphoric acid and then centrifuged again at 20,000 ×g for 20 min at 4°C. Next, a 1 mL of the supernatant was mixed with 50 μL of 2% (wt/vol) pivalic acid as an internal standard. The gas chromatograph (HP 6890 series GC system; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a flame ionization detector and a capillary column (DB-FFAP; Agilent Technologies Inc., Santa Clara, CA, USA) was used for measuring VFA profile. The temperatures of the inlet, oven and detector were set at 220°C, 100°C, and 250°C, respectively. Each sample for VFA analysis was duplicated.

For the NH₃-N analysis, the previously centrifuged samples were centrifuged again at 20,000×g for 20 min at 4°C. The supernatants were used to determine the NH₃-N concentration by catalyzed indophenol reaction [15] using spectrophotometry (Synergy2; Biotek Instruments, Inc., Winooski, VT, USA). Each sample for the NH₃-N analysis was triplicated and measured 3 times (3 × 3).

Data were analyzed as a two-way ANOVA with source of flushing gases and time as separate factors using general linear model procedure of SAS software (SAS Institute, Cary, NC, USA). The significant differences were accepted if p < 0.05.

Nitrogen flushing resulted in higher TGP than Ar flushing (p < 0.01; Table 2). This result could be attributed to the lower Henry’s law constant of N₂ than Ar [16]. Due to the lower Henry’s law constant, N₂ is less soluble in water than Ar, which resulted in higher TGP. As time does not affect the Henry’s law constant, TGP did not show time gas interaction. Additionally, as VFA and methane production were not influenced by the flushing gases, the only assumption for the lower TGP in Ar flushed bottles could be the solubility differences between flushing gases.

Ammonia nitrogen was higher in N₂ flushed group rather than Ar flushed group (p < 0.01). If Ar flushing lowered NH₃-N concentration (e.g., by inhibiting amino acids biosynthesis), it should be accompanied with the different concentration of the branched chain fatty acids including iso-butyrate and iso-valerate which are used to synthesis valine and leucine [17,18]. However, these parameters were not influenced by the flushing gases which demonstrate that Ar flushing did not inhibit the rumen fermentation and thus can be used as an alternative flushing gas. On the other hand, N₂ flushing enhanced NH₃-N which was partially due to the N₂ fixation. Although N₂ fixation in rumen is not quantitively significant [5–7], the N₂ fixation may be facilitated by flushed N₂ in headspace which could result in the higher NH₃-N concentration. On the contrary, Patra and Yu [8] showed that the different headspace gas composition including CO₂ and N₂ did not affect the NH₃-N concentration in rumen in vitro fermentation. These contrasting results might arise from different experimental methods including diets, donor animals, rumen fluid sampling time, buffer [1,19,20] and, especially, the gas used for the comparison. In the study of Patra and Yu [8], the CO₂ in headspace was used to compare its effects with the effects of N₂ in headspace on rumen fermentation characteristics. As noted, if the flushed CO₂ dissolved into rumen fluid and influenced NH₃-N concentration, then the different NH₃-N between CO₂ and N₂ flushing group could not be detected. For example, as Patra and Yu noted the increased methane production by CO₂ flushing [8], CO₂ flushing could promote the activity of Methanosarcina barkeri and Methanobacterium bryantii which also can fix the atmospheric nitrogen [21]. However, it cannot be easily deemed that the nitrogen fixation was promoted simply due to the higher concentration of N₂ in headspace because the nitrogen fixation requires 16 ATPs [22] and only a few methanogens can fix the atmospheric nitrogen among the rumen microbes [21,23]. Nevertheless, increased NH₃-N still indicates that N₂ flushing influences rumen N metabolism which should be considered when choosing a flushing gas. Therefore, further studies should demonstrate how rumen N metabolism was affected by N₂ flushing (e.g., acetylene reduction assay [24], detecting the nitrogenase activity and its gene expression).

In the present study, VFA production and pH were not affected by the flushing gases. These results imply that the impacts of N₂ flushing on nitrogen metabolism did not dramatically change the overall rumen fermentation characteristics. Conversely, as VFA, methane and pH were not affected, this is the demonstration that Ar can be used as an alternative flushing gas to N₂.