Influence of sodium hydroxide treatment of barley grain on in vitro rumen gas production and microbial-N yield

The objective of this study was to investigate the effect of treatment of whole barley grain (WBG) with sodium hydroxide (NaOH) on in vitro rumen gas production parameters and microbial protein synthesis. Experimental treatments included untreated whole barley grain (BControl) and treated barley grain with NaOH þ water (35 g þ 220ml/kg DM WBG) for 30 days (BNaOH). In vitro gas production technique was used to determine the gas production parameters of the samples. Furthermore, gas volume, apparent substrate degradability of barley grain (ASD), true substrate degradability of barley grain (TSD) and microbial N at t1/2 for two treatments were measured. The amount of produced gas at each time of incubation in each glass serum bottle was taken as the gas production parameter. Results of the in vitro technique revealed that alkali treatment of WBG caused a significant increase in the asymptotic gas volume (b) versus BControl (113.71 and 162.86ml, respectively). Constant rate of gas production (c) significantly decreased by BNaOH compared with BControl (0.018 and 0.034ml/h, respectively). No effects of NaOH treatment were observed on ASD and TSD of whole barley grain. However, rumen microbial-N yield considerably (p , 0.05) built up (from 1.922 to 4.087mg) from the treatment of barley with NaOH. The finding of the present study leads us to conclude that treatment of WBG with NaOH improved in vitro gas production parameters and microbial protein synthesis.


INTRODUCTION
Barley grain is one of the most common starchy feed grains used in the diets of dairy and beef cattle, as it is a readily available source of dietary energy.However, the amount of starch that can be included in the diets of dairy cows is limited as it increases incidence of acidosis, laminitis and other metabolic disorders. 1In barley grain, endosperm is surrounded by the pericarp, which is overlain by a fibrous hull and is extremely resistant to microbial degradation in the rumen. 2 Therefore, barley grain needs to be processed in order to improve its utilization by beef and dairy cattle. 2 Processing makes the starch more accessible to rumen microbes and increases the digestive extent of starch degradation in the rumen. 2 Although processing is essential to maximize the utilization of barley grain by cattle, extensive grain processing increases ruminal starch degradation, which has a reductive effect on feed intake in ruminants. 3Additionally, it may alter microbial yield in the rumen.Microbial biomass is the major source of protein for the ruminant host animal and prediction of efficiency of microbial production (EMP) can be crucial in ruminant nutrition. 4,5Several ruminant feeding systems 6,7 include EMP to predict animal performance.However, lack of a practical laboratory technique to detect feed-specific differences in EMP is problematic.It has been proposed that variations in EMP can be detected by a combination of in vitro gas volume measurements 8 with a concomitant measure of true substrate degradability (TSD). 9,10The difference between the measures is assumed to be microbial biomass.The objective of this experiment was to determine the in vitro impact of barley grain treatment with NaOH on rumen fermentation, digestion response, and microbial protein synthesis using gas production technique.

MATERIALS AND METHOD 2.1. Chemical processing
In the present study an Iranian cultivar of barley grain (Dasht) containing 13.2% CP, 21.05% NDF and 6.78% ADF was used. 11The applied treatments were untreated whole barley grain (B Control ) and whole barley grain (WBG) treated with sodium hydroxide (B NaOH ).For chemical processing 35 g of NaOH was dissolved in 220 ml distilled water and was sprayed on 1 kg of WBG.Later, the WBG was packed in airless nylon bags.After 30 days, treated samples were allowed to aerate for 12 h.All samples were oven dried (658C, 48 h), then ground through a 2-mm diameter screen. 1

In vitro gas production technique
In the current study, the gas production technique was conducted as described by Blu ¨mmel and Lebzien 12 and Grings and Blu ¨mmel. 13Samples (250 mg) were incubated with 20 mL incubation medium in triplicate.Substrate (250 mg) was weighed into a 120 ml glass serum bottle.Rumen content was collected from three rumen fistulated Holstein steers that were fed 5.1 kg of dry matter (DM) of alfalfa hay, 3.2 kg of DM corn silage and 2.5 kg of DM concentrate (170 g CP kg 21 of DM).Ruminal fluid and particulate matter, in the approximate proportion of 60:40, were collected before the morning feeding into a pre-warmed CO 2 -filled thermos bottle.Rumen contents were homogenized in a blender and were subsequently strained through a nylon filter (40 mm pore size), then filtered through glass wool.The filtrate was mixed with carbonate buffer [containing ammonium bicarbonate at (4 g/l) and sodium bicarbonate (35 g/l), macromineral solution (5.7 g anhydrous Na 2 HPO 4 , 6.2 g anhydrous KH 2 PO 4 and 0.6 g MgSO 4 •7H 2 O per liter), and deionized water in a ratio of 1:1:0.5:1.5].Next, 0.1 ml micromineral solution (13.2 g CaCl 2 •2H 2 O, 10.0 g MnCl 2 •4H 2 O, 1 g CoCl 2 •6H 2 O and 8.0 g FeCl 3 •6H 2 O per 100 ml) was added per litre.The medium was reduced by the addition of 41.7 ml reducing agent (40 ml deionized water, 1 ml 1N NaOH and 1 g Na 2 S•9H 2 O) per litre of medium.Twenty millilitres of medium were dispensed into a 120 ml glass serum bottle containing 250 mg of each sample and placed upright in a 398C water bath.Blank samples (i.e., medium only, no substrate) were placed throughout the water bath and used to measure gas production from the medium alone.All handling of ruminal inoculum was under a constant stream of CO 2 and all the used containers were pre-warmed and filled with CO 2 .
Cumulative gas volume measurements of treated and untreated samples were recorded manually for the incubations from 3 runs in 6 replicates each after 2, 4, 6, 8, 10, 12, 14, 16, 24, 30, 36, 48, 54, 60, 72 and 96 h of incubation.After the subtraction of gas produced from blank serum bottles, the data was fitted to an exponential model 14 as: where 'y' is the cumulative volume of gas produced at time 't' (h), 'b' is the asymptotic gas volume and 'c' is the constant rate of gas production.
Halftime of gas production (t 1/2 ) [i.e., the time (h) when half of the asymptotic gas volume (b; ml) was produced] was calculated as: In vitro digestibility and microbial-N production After the initial 96 h gas run, t 1/2 was calculated and a second incubation with the samples was conducted to obtain degradability measures at substrate-specific times (i.e., t 1/2 for each substrate).
Collection and handling of ruminal fluid was the same as that described for the 96 h incubations.
For each sample and each run (n ¼ 3), 6 serum bottles were prepared, providing 3 serum bottles for apparent and 3 for true degradability measures.The incubations were terminated at t 1/2 and the volume of gas was recorded.True substrate degradability of samples 9 at t 1/2 was measured by refluxing the incubation residue with neutral detergent (ND) solution (prepared without sodium sulphite) for 1 h with subsequent recovery of the truly undegraded substrate in sintered glass crucibles of porosity 'C' (i.e., 40-60 mm).Apparent substrate degradability was determined and calculated at t 1/2 by high-speed centrifugation of incubation residue (20000 £ g, 208C, 10 min) 12 following placement into an iced water bath (148C) to stop fermentation.Six 20 ml aliquots of media were collected, as were 4 blanks without substrate (20 ml) for each t 1/2 .All blank samples were centrifuged and the supernatant was siphoned off, frozen (2 208C) and stored.Residue was weighed and used to correct apparent substrate degradability determinations for residue from the ruminal inoculum.
Microbial N production at t 1/2 was directly estimated using the N content of the apparently degraded residue remaining after centrifugation (pellet N) and N content of truly undegraded residue (NDFN) at t 1/2 , using the equation 13 : Microbial-N production at t 1=2 ¼ pellet N at t 1=2 2 blank pellet N at 0h incubation 2 NDFN at t 1=2 :

Calculations and statistical analysis
The data was analysed using General Linear Models (GLM) procedures (SAS Inst.Inc., Ca NC).The gas production procedure, repeated in 3 runs, was conducted as a complete randomized block design with the treatment as the main effect.Statistical model was , where Yij is the observation from treatment i, m, the overall mean, Ti the mean of treatment, Bj, block and eij, the residual effect.Multiple comparisons among treatment means were performed by Duncan's New Multiple Range Test (DMRT). 15 RESULTS 3.1.In vitro gas production profiles Cumulative gas production for each of the substrate treatments were presented as gas production curves (Figure 1).Gas production profiles for two treatment incubations were adequately described by the exponential model.
Values for asymptotic gas volume (b) and rate of gas production (c) after 96 h of in vitro incubation and halftime of gas production (t 1/2 ) of B Control and B NaOH were shown in Table 1.
Asymptotic gas volume increased by B NaOH in comparison to B Control .Constant rate of gas production decreased by barley grain treatment with NaOH compared with B Control .The effect of NaOH treatment of barley grain on constant rate of gas production between the two treatments was significant ( p , 0.05).Treatment of barley grain with NaOH significantly ( p ¼ 0.007) increased the halftime of gas production (t 1/2 ) versus B Control .
3.2.Gas volumes and apparent and true substrate degradability at t 1/2 of gas production The data of gas volumes at t 1/2 and apparent (ASD) and true (TSD) degradability of B Control and B NaOH are illustrated in Table 2.
Barley grain treatment with NaOH led to an increase in the gas volume, ASD and TSD in contrast to B Control .The effect of NaOH treatment on gas volume between two treatments was significant ( p , 0.05).

In vitro rumen microbial-N yield
Values for in vitro pellet-N, nitrogen content of truly undegraded residue (NDFN) and microbial nitrogen (MN) of B Control and B NaOH were shown in Table 3.
Pellet nitrogen and MN increased with treatment of barley grain with NaOH compared with B Control , however, NDFN decreased.Differences between two treatments for pellet nitrogen and MN were significant ( p , 0.05).There was no significant difference between treatments for NDFN.

DISCUSSION
The present study was conducted to evaluate the influences of NaOH treatment of WBG on degradability and digestibility characteristics and microbial nitrogen yield using gas production technique.The agreement of volume of gas with the in situ parameters indicates that in vitro gas production accurately reflects substrate fermentation. 16The objective of grain processing is to obtain a balance between maximizing the extent of ruminal starch fermentability and controlling the rate of starch fermentation in order to avoid digestive and metabolic disturbances. 17esults of gas production measurement revealed that WBG treated with NaOH resulted in a decrease in constant rate of gas production compared with B Control .The reason is that slower digestion of treated barley results in a decrease in fluctuations in ruminal pH, and lower metabolic disorders of rumen such as acidosis.Ørskov and Greenhalgh 18 reported that the rate of digestion of NaOH-treated grain increased along with an increase in applying NaOH.Finally, Barnes and Ørskov 19 concluded that   7 20.03 a 37.92 b 1.064 0.007 a,b Means within a row with different letters differ ( p , 0.05).
1 Untreated barley grain, 2 Treated WBG with NaOH (35 g þ 220 ml/kg DM), 3 Standard error of mean, 4 Probability, 5 Asymptotic gas volumes, 6 Constant rate of gas production, 7 Halftime of gas production [the time (h) when half of the asymptotic gas volume (b; ml) was produced] was calculated as: treating barley with 40 g NaOH/kg results in a slightly lower starch digestibility than if the grain is rolled.The major site of cereal grain starch digestion is usually the rumen.Processing increases microbial degradation of starch in the rumen and decreases amounts of starch digested post-ruminally. 20he rate of gas production directly affects t 1/2 .The effect of NaOH treatment on rate of gas production between the two treatments was significant ( p , 0.05) and thus time to half maximal gas production (t 1/2 ) of incubated barley grains significantly increased from 20.03 to 37.92 h in B Control and B NaOH incubations, respectively.In this study, treatment of barley grain with NaOH caused an increase in the gas volume in contrast to B Control .This is because barley treatment resulted in an increase in potential gas production and a consequent rise in gas volume.Apparent substrate degradability of barley grain (ASD) and true substrate degradability of barley grain (TSD) increased with NaOH-treated barley versus B Control .It may originate from reducing the resistance of seed coat by treatment.Dehghan-banadaky et al. 1 reported that NaOH treatment of whole barley disrupts the seed coat by partial hydrolysis of hemicellulose and lignin and causes a swelling of the outer starch granules, so the ruminal bacteria and enzymes gradually gain access to the starchy endosperm.This confirmed the findings of Ørskov et al., 21 who reported that for whole barley, the digestibility of all fractions improved by treatment with NaOH.In addition, Barnes and Ørskov 20 indicated that the digestibility of starch increased linearly with NaOH application.The present experiment showed a significant ( p , 0.05) difference between the two treatments for estimating the microbial-N yield by applying in vitro technique.In comparison with B Control , NaOH-treated barley significantly increased MN and because of MN's key role in protein synthesis, the microbial protein synthesis improved.

CONCLUSION
The findings of the present study lead us to conclude that treatment of WBG with sodium hydroxide causes a decrease in rate of gas production, and an increase in the half time of maximal gas production (t 1/2 ).This might demonstrate a phenomenon under which a reduction in rumen starch digestion takes place.NaOH-treatment of WBG enhanced ASD and TSD; therefore, it indicates an improvement in rumen barley availability.To sum up, treatment of WBG with NaOH causes an improvement in rumen microbial-N production and therefore, it may increase nutritive value of the grain in ruminants.Pellet N (mg) 5 3.239 a 5.219 b 0.403 0.025 NDFN (mg) 6 1.316 1.131 0.240 0.617 MN (mg) 7 1.922 a 4.087 b 0.266 0.004 a,b Means within a row with different letters differ ( p , 0.05).
1 Untreated barley grain, 2 Treated WBG with NaOH (35 g þ 220 ml/kg DM), 3 Standard error of mean, 4 Pprobability, 5 N content of the apparently degraded residue remaining after centrifugation, 6 N content of truly undegraded residue, 7 Microbial N production at t 1/2 , MN ¼ pellet N at t 1/2 -blank pellet N at 0 h incubation 2 NDFN at t 1/2 .The data relating to the incubation of 250 mg of dry substrate obtained by terminating the incubation at t 1/2 .a,b Means within a row with different letters differ ( p , 0.05).

Figure 1 .
Figure 1.In vitro gas production profiles of untreated (B Control ) and treated WBG with NaOH (B NaOH ), p , 0.05.

Table 1 .
Asymptotic gas volume (b), constant rate of gas production (c) and halftime of gas production (t 1/2 ) of untreated and treated WBG with NaOH.

Table 3 .
Nitrogen of pellet, N content of truly undegraded residue (NDFN), microbial nitrogen (MN) of untreated and treated WBG with NaOH at t 1/2 .