Use of nitrate-nitrogen as a sole dietary nitrogen source to inhibit ruminal methanogenesis and to improve microbial nitrogen synthesis in vitro.(Report)

INTRODUCTION

To protect the environment and improve the energetic efficiency of ruminant animals, it would be beneficial to decrease methane production in the rumen. Nitrate has been shown to suppress methane production by rumen microbes (Jones, 1972; Takahashi and Young, 1991). This is because the electrochemical reduction of 1 mol nitrate to ammonia consumes 8 mol of electrons, e.g., hydrogen (Richardson and Watmough, 1999). The dependence of methanogenesis and nitrate reduction on electron sources is described by the following two stoichiometries:

4[H.sub.2] + HC[O.sub.3.sup.-] + [H.sup.+] [right arrow] C[H.sub.4] + 3[H.sub.2]O; [DELTA][G.sub.o]' = -175 kJ/reaction (Conrad and Wetter, 1990)

N[O.sub.3.sup.-] + 2[H.sup.+] + 4[H.sub.2] [right arrow] N[H.sub.4.sup.+] + 3[H.sub.2]O; [DELTA][G.sub.o]' = -598 kJ/reaction (Allison and Reddy, 1984)

Since methane production is less when nitrate is added, it is presumed that nitrate- and nitrite-reducing microbes successfully compete with methanogenic microbes for hydrogen in the rumen (Allison and Reddy, 1984; Iwamoto et al., 1999; 2001).

Microbial growth in the rumen is a function of the amount of energy derived from ruminal fermentation processes. Energy is seldom abundant for growth of rumen microorganisms (Russell and Wallace, 1997). Reductive processes that allow for greater conservation of energy for growth should be advantageous to organisms which conduct these processes. Thermodynamics predicts that nitrate reduction should be energetically more beneficial than methanogenesis and therefore support more microbial growth. Allison and Reddy (1984) tested the effect of nitrate on cell yield by rumen microbes in continuous culture but considered their results to be inconclusive. We are not aware of other tests of this hypothesis with rumen microbes.

The objective of the present study was to investigate the effect of nitrate-nitrogen as a sole nitrogen source on ruminal fermentation characteristics and microbial nitrogen (MN) synthesis using an in vitro gas production method.

MATERIALS AND METHODS

In vitro substrates

Three substrate diets were formulated with urea (U4128, Sigma-Aldrich Chemical Co., St. Louis, MO), tryptone (Oxoid Ltd., Basingstoke, Hampshire, UK), or sodium nitrate (S5506, Sigma-Aldrich) as sole N sources, and with various amounts of soluble starch (S4251, Sigma-Aldrich) and Avicel (GH-9471, Fluka, Chemie GmbH) to balance the dietary N content (13% CP). Tryptone is a pancreatic digest of casein and a source of amino acids and peptides. The ingredient and chemical compositions are presented in Table 1.

In vitro incubations

All procedures involving animals were conducted under approval of the China Agricultural University Institutional Animal Care and Use Committee. Ruminal fluid was obtained from three cannulated Simmental x Luxi steers fed diets (8 kg DM/d), consisting of 60% roughage (corn stover cubes and alfalfa hay pellets) and 40% mixed concentrate (ground corn, soybean meal and soyhulls) twice daily. No nitrate was added to the diet of the donor steers. The chemical composition (on DM basis) of the diet was 13.2% CP, 39.6% NDF, 23.5% ADF, 1.14% Ca and 0.68% P. There was no detectable nitrate in the concentrate mix. Based on the nitrate content of alfalfa (1.11 mg/g DM) and corn stover (0.56 mg/g DM) and their diet proportions (25% and 35%, respectively), the nitrate intake of the donor steers was less than 4 g/head/d.

Rumen fluid (1 L) was collected from each steer, filtered through four layers of cheesecloth, and then pooled. The rumen fluid filtrates were poured into an anaerobic buffer solution in 8-L flasks under a constant flow of [O.sub.2]-free C[O.sub.2] and homogenized in a blender under C[O.sub.2] for 2 min. Buffer solution was prepared in the ratio of 400 ml distilled water, 0.1 ml trace element solution A, 200 ml buffer solution B, 200 ml macro-element solution C, 1 ml resazurin solution (0.1% w/v) and 40 ml reductant solution. Trace element solution A contained Ca[Cl.sub.2] x 2[H.sub.2]O, 13 x 2 g; Mn[Cl.sub.2] x 4[H.sub.2]O, 10 x 0 g; Co[Cl.sub.2] x 6[H.sub.2]O, 1.0 g and Fe[Cl.sub.3] x 6[H.sub.2]O, 8.0 g, which were dissolved in 100 ml distilled water. Buffer solution B was NaHC[O.sub.3], 35 g, dissolved in 1,000 ml distilled water. Macro-element solution C was [Na.sub.2]HP[O.sub.4] 5.7 g; K[H.sub.2]P[O.sub.4], 6.2 g and MgS[O.sub.4] x 7[H.sub.2]O, 0.6 g, dissolved in 1,000 ml distilled water. Reductant solution consisted of 1 M NaOH, 4 ml; [Na.sub.2]S x 9[H.sub.2]O, 0.625 g, and 95 ml distilled water. Forty milliliters of the mixed culture medium (ratio of ruminal fluid:buffer = 1:2) were pipetted with an automatic pump into replicate glass syringes (HFT000025, Haberle Maschinenfabrik GmbH, Germany) loaded with 0.2 g DM of substrate diets and pre-warmed to 39[degrees]C. The syringes were then incubated in a shaking water bath at 39[degrees]C. Blank syringes containing only mixed culture medium with no substrate addition were simultaneously incubated. There were nine replicate syringes for each substrate diet, three for measurement of gas production over 72 h after the procedure of Menke et al. (1979), three for measurement of 24-h gas composition and three for 24-h sampling of soluble analytes and microbial nitrogen (MN). The piston position on the syringe was noted at several incubation times for determination of the gas production …