Effect of form of nitrogen on populations of fi bre-associated ruminal microbes in pretreated rice straw in vitro

The study was conducted to investigate the effect of ammonium bicarbonate and casein on rumen fermentation and relative populations of fi bre-associated cellulolytic microbes in untreated and sodium hydroxide treated rice straw in vitro. Form of nitrogen had different effect on rumen fermentation and fi bre-associated microbial populations. Cumulative gas production signifi cantly increased with addition of nitrogen. Addition of casein signifi cantly improved rumen fermentation and microbial populations. Casein had higher effi ciency to supply available nitrogen to microbial growth and degradation of straw than ammonium bicarbonate. It is inferred that a supply of protein nitrogen would be needed for maximum degradation of straw.


INTRODUCTION
Straw as a low-nitrogen (N) feed resource is the largest by-products of cereal production.Treatment with ammonia or alkali compounds is now widely accepted and applied as the most successful chemical method to improve the nutritional value of straw (Liu et al., 2002).However, sodium hydroxide (NaOH) does not contribute N to straw.Since N is a limiting nutrient in low-quality forages, it is necessary to supply ammonia N for the rumen microbes in NaOH treated straw.

FORM OF NON-FIBRE ASSOCIATED RUMINAL MICROBES
Ruminal cellulolytic microbes such as Ruminococcus albus, Ruminococcus fl avefaciens, Fibrobacter succinogenes, Butyrivibrio fi brisolvens and fungi, play an important role in fi bre digestion.They require ammonia N for their optimum growth when host was fed fi bre basal diet (Ranilla et al., 2001).The form of N may affect substrate digestion (Griswold et al., 1996).Casein is readily attacked by rumen microbes (Chalmers et al., 1954), and may be used as a good source of protein N. Fujimaki et al. (1989) found no difference in digestion of cell wall carbohydrate when non-protein N replaced ammonia, while Carro and Miller (1999) assumed that N sources other than ammonia are required not only for maximal microbial growth but also substrate digestion.These contrasting results might be explained by differences in the composition of diet such as source and availability of readily fermentable carbohydrates.Quantifi cation of population sizes of ruminal microbes using real-time PCR would be appropriate method to reveal the effect of N forms on microbial growth.In this study, casein was used in comparison with ammonium bicarbonate (AB) as supplementary N source to untreated and NaOH treated rice straw to investigate their effect on fi bre degradation and populations of ruminal microbes.

Material
The straw (contained 8.8 g/kg N and 727 g/kg neutral detergent fi bre, NDF) obtained in Zhejiang Province (China) was treated with NaOH (45 g/kg straw dry matter, DM) by the method of Wang et al. (2007).The content of N and NDF of NaOH treated straw was 9.8 and 644 g/kg DM, respectively.

In vitro gas test
Incubations were carried out using the methods described by Theodorou et al. (1994).One gram of untreated or NaOH treated rice straw was incubated in triplicate with 10 ml rumen fl uid collected from three donor sheep before morning feeding and 90 ml buffer medium prepared as described by Menke and Steingass (1988), with exception that no ammonium bicarbonate (AB) was added.Casein or AB was added at a level equivalent to 140 mg/l N. Non-N treatment was used as negative control.In each incubation run, three blanks were included simultaneously to correct the gas production volume for gas release from endogenous substrates.

Sampling and analysis
At the 24 h of incubation time, the fermentation was stopped by swirling the bottle in ice water.About 30 ml of mixed fermentation medium was taken for analysis of ammonia-N, volatile fatty acids (VFAs) and microbial protein (MCP).The remaining contents were fi ltered through nylon bags (40 μm pore size) and the residues were used to extract total ruminal microbial DNA for quantifi cation of populations of R. albus, R. fl avefaciens, F. succinogenes, B. fi brisolvens and total fungi attached to straws.Species-specifi c real-time quantitative PCR was performed using the ABI 7500 real time PCR system (Applied Biosystems) with fl uorescence detection of SYBR green dye.Amplifi cation consisted of an initial denature at 95°C for 15 s followed by 40 cycles of 95°C for 5 s and 60°C for 34 s.Specifi city of amplifi ed products was confi rmed by melting temperatures and dissociation curves after each amplifi cation.Amplifi cation effi ciencies for each primer pairs were investigated by examining dilution series of total rumen microbial DNA template on the same plate in triplicate.

Calculations and statistical analysis
Relative population sizes of R. albus, R. fl avefaciens, F. succinogenes, B. fi brisolvens and total rumen fungi were expressed as a proportion of total rumen bacterial 16S rDNA.The main effects of straw, N form and their interactive effect on rumen fermentation parameters and rumen microbial populations were analysed using the general linear model (GLM) procedure of SAS.

RESULTS
In vitro rumen fermentation variables for untreated and NaOH treated rice straw are shown in Table 1.The cumulative gas production at 24 h incubation increased when N was added compared with non-N treatment.The highest gas production (93 ml/g DM) was observed in NaOH treated straw with supply of protein and the lowest (48 ml/g DM) in untreated straw with non-N addition.Addition of AB and casein increased signifi cantly the concentration of total VFAs in NaOH treated straw.No signifi cant differences were found in total VFAs, molar proportion of acetate, propionate and butyrate between AB and non-N treatment in untreated straw.Concentration of MCP for two straws was increased signifi cantly by either AB or casein.
Population sizes of rumen microbes attached to straws relative to rumen bacterial 16S rDNA in vitro with or without N addition are shown in Table 2.In untreated straw, addition of AB and casein increased the populations of F. succinogenes, B. fi brisolvens and total fungi, while no signifi cant effect on Ruminococci was observed.In NaOH treated straw, N form had little effect on population of F. succinogenes.Addition of AB and casein signifi cantly increased the attached populations of R. albus and B. fi brisolvens, with higher populations for addition of casein than AB.Highest populations of R. fl avefaciens and fungi were found in NaOH treated straw with non-N than with addition of N.

DISCUSSION
In vitro gas production technique has been widely applied to simulate fermentation kinetics in the rumen and predict in vivo digestibility.The main fermentation end products from the in vitro gas production technique are gases (CO 2 and CH 4 ), microbial biomass and VFAs.The rumen fermentation variables in vitro refl ect digestion of substrate.The cumulative gas production after 24 h incubation increased when AB and The production of VFAs may depend on the type of fermentable organic matter, rumen microbes involved and rumen environment.In the present study, the energy and fermentation environment was constant for the same type of straw, thus fermentable organic matter, and then VFAs, varied only with addition of casein and microbes involved.From the result shown in Table 1, rumen MCP concentration was signifi cantly higher after addition of casein, compared with non-N treatment, probably resultant from the improved balance of energy and N for rumen microbial growth.Compared with untreated straw, concentration of ammonia-N in NaOH treated straw is lower.Probably more N was used in NaOH treated straw to support its faster degradation.
Recently, with the advancement of molecular enumeration methods, real-time PCR has been widely accepted to monitor microbes in the environment due to its rapid, sensitive and reproducible advantage, especially for low abundance templates.Realtime PCR using species-specifi c primers have been successfully developed to monitor bacterial populations in the rumen (Denman and McSweeney, 2006).In the present study, rumen main cellulolytic microbes had been detected using real-time PCR technique to investigate their responses to N form for treated and untreated rice straw.
Form of N and rate of fi bre degradation had effects on the rumen microbial populations attached to straw.R. albus, R. fl avefaciens and F. succinogenes are considered as the representative cellulolytic species in the rumen.Relative population of the three cellulolytic bacteria is about 5.04 to 17.1% of total rumen bacterial 16S rDNA in the present study with higher value in untreated straw.This result is consistent with other reports that higher populations of cellulolytic bacteria were observed in higher cellulose diets (Weimer et al., 1999).Rumen microbial communities varied when N was added in different straws (Table 2), resulting in improved rumen fermentation characteristics.Relative population of F. succinogenes was predominant in untreated straw compared with other microbes, which would have major contribution to rumen fermentation.Addition of AB seems to have slight effect on rumen microbial community and rumen fermentation in the untreated straw due to its lower rate of degradation, but has signifi cant effect on NaOH treated straw.There are more carbohydrate fermented in NaOH treated straw and more available N supplied by AB to meet requirement of microbial growth.The presence of N increased the population of B. fi brisolvens, suggesting B. fi brisolvens is not only cellulolytic but also proteolytic species.High N content of hay has been observed to stimulate growth of B. fi brisolvens (Koike et al., 2003).

CONCLUSIONS
The results of the present study have confi rmed the importance of N for microbial growth.Form of N could affect rumen microbial community and fermentation in vitro

Table 2 .
Population sizes of rumen microbes (% rumen bacterial 16S rDNA) attached to untreated and sodium hydroxide treated rice straw in vitro supplemented ammonium bicarbonate (AB) or casein 1 S -treatment, N -N form, S × N -interactive effect of straw and N form, **P<0.01

Table 1 .
Rumen fermentation in vitro of untreated and sodium hydroxide (NaOH) treated rice straw supplemented with ammonium bicarbonate (AB) or casein Addition of casein signifi cantly increased total VFAs concentration.