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

  • Park, KiYeon (Department of Animal Science and Technology, Konkuk University) ;
  • Lee, HongGu (Department of Animal Science and Technology, Konkuk University)
  • Received : 2019.11.28
  • Accepted : 2019.12.31
  • Published : 2020.01.31

Abstract

In rumen in vitro experiments, although nitrogen gas (N2) flushing has been widely used, its effects on rumen fermentation characteristics are not clearly determined. The present study is the first to evaluate the effects of N2 flushing on rumen fermentation characteristics in in vitro batch culture system by comparing with new applicable non-metabolizable gas: argon (Ar). The rumen fluid was taken from two Korean native heifers followed by incubation for 3, 9, 12, and 24 h with N2 or Ar flushing. As a result, in all incubation time, N2 flushing resulted in higher total gas production than Ar flushing (p < 0.01). Additionally, in N2 flushing group, ammonia nitrogen was increased (p < 0.01). However, volatile fatty acids profiles and pH were not affected by the flushing gases (p > 0.05). In conclusion, the present study demonstrated that N2 flushing can influence the rumen nitrogen metabolism via increased ammonia nitrogen concentration and Ar flushing can be used as a new alternative flushing gas.

Keywords

INTRODUCTION

In rumen fluid in vitro experiments, especially using batch culture, using flushing gas (headspace gas composition) in order to make an anaerobic condition in incubation bottle is a pivotal factor and thus should be chosen prudently [1]. However, the studies identifying the effects of flushing gases on rumen fermentation are limited. Nitrogen gas (N2) flushing is routinely used in rumen in vitro experiments [2–4]. It may affect rumen nitrogen metabolism as rumen microbes can utilize atmospheric N2 [5–7] and thus may confound the obtained results. But the previous studies which investigated the effects of headspace N2 with carbon dioxide (CO2) or dihydrogen on rumen fermentation characteristics in batch culture system found no changes in ammonia nitrogen (NH3-N) between the headspace gas compositions [8,9].

These results could be resulted from the gas used for the comparison. As CO2 and H2 concentration in the medium affects rumen fermentation thermodynamically [10], it is not proper to investigate the effects of N2 by comparing its effects with CO2 or H2. Conversely, if headspace N2 could influence the rumen fermentation, then investigating the effects of CO2 or H2 by comparing its effects with N2 may not be relevant. Therefore, N2 flushing effects should be demonstrated by comparing its effects with the non-metabolizable gas. In the present study, argon gas (Ar) was selected as an alternative gas because there is no possibility for Ar to influence the rumen nitrogen metabolism due to its extremely low biological availability and toxicity for microbes. Given the above, the objective of the present study was to investigate the effects of N2 flushing on rumen fermentation kinetics by comparing its effects with Ar flushing using rumen in vitro batch culture system.

Materials and Methods

Ruminal inoculum and diet

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.

Table 1. Chemical composition of the total mixed ration (TMR)

NDF, neutral detergent fiber; ADF, acid detergent fiber.

In vitro incubation procedures

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℃ 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.

Post-fermentation parameters analyses

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℃, 50℃, and 250℃, 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℃ 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℃ 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℃. 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℃, 100℃, and 250℃, 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℃. 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).

Statistical analysis

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.

Results and Discussion

Gas and methane production

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.

Table 2. Over time impacts of N2 and Ar flushing on rumen pH, TGP, NH3-N and VFA production

1)p-value for time factor were < 0.001 in all items.

N2, nitrogen gas; Ar, argon gas; TGP, total gas production; NH3-N, ammonia nitrogen; VFA, volatile fatty acids; RMSE, root of mean square error; TVFA, total volatile fatty acids; sum of acetate propionate, butyrate, iso-butyrate, valerate and iso-valerate concentration (mM); A:P ratio, acetate and propionate ratio.

Ammonia nitrogen

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 atmo-spheric 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).

VFA production and pH

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.

Conclusion

It was demonstrated that Ar flushing can be used for rumen in vitro experiments as the volatile fatty acids and methane production were not influenced by the flushing gases. The increased ammonia nitrogen in N2 flushed group demonstrated that N2 can influence the rumen nitrogen metabolism which can mislead into confounding results. Further studies should be conducted to identify how N2 flushing affect the rumen nitrogen metabolism and to reinvestigate the effects of CO2 flushing on rumen fermentation by comparing it with Ar flushing.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

This research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Agri-Bio Industry Technology Development Program (117030-03- 2-HD020) and Konkuk University Researcher Fund in 2019.

Acknowledgements

Not applicable.

Availability of data and material

Upon reasonable request, the datasets of this study can be available from the corresponding author.

Ethics approval and consent to participate

All animal procedures were performed in accordance with the Institution of Animal Care and Use Committee (IACUC) at Konkuk University, Seoul (Approval no. KU19075)

References

  1. Yanez-Ruiz DR, Bannink A, Dijkstra J, Kebreab E, Morgavi DP, O'Kiely P, et al. Design, implementation and interpretation of in vitro batch culture experiments to assess enteric methane mitigation in ruminants: a review. Anim Feed Sci Technol. 2016;216:1-18. https://doi.org/10.1016/j.anifeedsci.2016.03.016
  2. Machado L, Magnusson M, Paul NA, de Nys R, Tomkins N. Effects of marine and freshwater macroalgae on in vitro total gas and methane production. PLoS ONE. 2014;9:e85289. https://doi.org/10.1371/journal.pone.0085289
  3. Roque BM, Brooke CG, Ladau J, Polley T, Marsh LJ, Najafi N, et al. Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage. Anim Microbiome. 2019;1:3. https://doi.org/10.1186/s42523-019-0004-4
  4. Choi YY, Lee SJ, Lee YJ, Kim HS, Eom JS, Jo SU, et al. In vitro and in situ evaluation of Undaria pinnatifida as a feed ingredient for ruminants. J Appl Phycol. 2019;2019:1-11.
  5. Granhall U, Ciszuk P. Nitrogen fixation in rumen contents indicated by the acetylene reduction test. J Gen Microbiol. 1971;65:91-3. https://doi.org/10.1099/00221287-65-1-91
  6. Hobson PN, Summers R, Postgate JR, Ware DA. Nitrogen fixation in the rumen of a living sheep. J Gen Microbiol. 1973;77:225-6. https://doi.org/10.1099/00221287-77-1-225
  7. Li Pun HH, Satter LD. Nitrogen fixation in ruminants. J Anim Sci. 1975;41:1161-3. https://doi.org/10.2527/jas1975.4141161x
  8. Patra AK, Yu Z. Effects of gas composition in headspace and bicarbonate concentrations in media on gas and methane production, degradability, and rumen fermentation using in vitro gas production techniques. J Dairy Sci. 2013;96:4592-600. https://doi.org/10.3168/jds.2013-6606
  9. Qiao JY, Tan ZL, Guan LL, Tang SX, Zhou CS, Han XF, et al. Effects of hydrogen in headspace and bicarbonate in media on rumen fermentation, methane production and methanogenic population using in vitro gas production techniques. Anim Feed Sci Technol. 2015;206:19-28. https://doi.org/10.1016/j.anifeedsci.2015.05.004
  10. Janssen PH. Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics. Anim Feed Sci Technol. 2010;160:1-22. https://doi.org/10.1016/j.anifeedsci.2010.07.002
  11. AOAC [Association of Official Analytical Chemists] International. Official methods of analysis of AOAC International. 13th ed. Arlington, VA: AOAC International; 1990.
  12. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. 1991;74:3583-97. https://doi.org/10.3168/jds.s0022-0302(91)78551-2
  13. McDougall EI. Studies on ruminant saliva. 1. The composition and output of sheep's saliva. Biochem J. 1948;43:99-109. https://doi.org/10.1042/bj0430099
  14. Theodorou MK, Williams BA, Dhanoa MS, McAllan AB, France J. A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Anim Feed Sci Technol. 1994;48:185-97. https://doi.org/10.1016/0377-8401(94)90171-6
  15. Chaney AL, Marbach EP. Modified reagents for determination of urea and ammonia. Clin Chem. 1962;8:130-2. https://doi.org/10.1093/clinchem/8.2.130
  16. Sander R. Compilation of Henry's law constants (version 4.0) for water as solvent. Atmos Chem Phys. 2015;15:4399-981. https://doi.org/10.5194/acp-15-4399-2015
  17. Allison MJ, Bryant MP. Biosynthesis of branched-chain amino acids from branched-chain fatty acids by rumen bacteria. Arch Biochem Biophys. 1963;101:269-77. https://doi.org/10.1016/S0003-9861(63)80012-0
  18. Kand D, Raharjo IB, Castro-Montoya J, Dickhoefer U. The effects of rumen nitrogen balance on in vitro rumen fermentation and microbial protein synthesis vary with dietary carbohydrate and nitrogen sources. Anim Feed Sci Technol. 2018;241:184-97. https://doi.org/10.1016/j.anifeedsci.2018.05.005
  19. Leedle JA, Bryant MP, Hespell RB. Diurnal variations in bacterial numbers and fluid parameters in ruminal contents of animals fed low- or high-forage diets. Appl Environ Microbiol. 1982;44:402-12. https://doi.org/10.1128/aem.44.2.402-412.1982
  20. Muetzel S, Hunt C, Tavendale MH. A fully automated incubation system for the measurement of gas production and gas composition. Anim Feed Sci Technol. 2014;196:1-11. https://doi.org/10.1016/j.anifeedsci.2014.05.016
  21. Leigh JA. Nitrogen fixation in methanogens: the archaeal perspective. Curr Issues Mol Biol. 2000;2:125-31.
  22. Hoffman BM, Lukoyanov D, Dean DR, Seefeldt LC. Nitrogenase: a draft mechanism. Acc Chem Res. 2013;46:587-95. https://doi.org/10.1021/ar300267m
  23. Kim M, Morrison M, Yu Z. Status of the phylogenetic diversity census of ruminal microbiomes. FEMS Microbiol Ecol. 2011;76:49-63. https://doi.org/10.1111/j.1574-6941.2010.01029.x
  24. Dilworth MJ. Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochim Biophys Acta. 1966;127:285-94. https://doi.org/10.1016/0304-4165(66)90383-7

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