Comparison of two buffers on in vitro gas production , pH and NH 3N concentration of feedstuffs for ruminants

The objective of the study was to compare Menke et al.’s buffer (1979) with Zhao and Lebzien’s buffer (2000) on gas production, pH and NH3-N of 16 feedstuffs for ruminants in in vitro incubation. The gas production was recorded after incubation at 12 and 24 h. The pH and NH3-N concentration of incubation residues were determined after incubation for 24 h. The results showed that there was no signifi cant difference in in vitro gas production, pH and NH3-N concentration between the two buffers (P>0.05). It was found that in vitro gas production, pH or NH3-N concentration of the two buffers were signifi cantly correlated (P<0.05). It was concluded that the two buffers had similar results on in vitro gas production, pH and NH3-N concentration in in vitro incubation. It is suggested to compare these two buffers on volatile fatty acids (VFA) concentration and rumen microorganism activities in the future.


INTRODUCTION
has been widely used in the determination of in vitro gas production.Zhao and Lebzien's buffer (2000) has been used for the measurement of utilizable crude protein, utilizable amino acids (Zhao and Lebzien, 2002) and utilizable true protein (Li and Zhao, 2007) of feedstuffs for ruminants.Some components of the two buffers are the same whereas others are different.When in vitro incubation technique is used in ruminant nutrition research, it is interesting to know whether Menke et al.'s buffer (1979) and Zhao and Lebzien's buffer (2000) have similar experimental results and could replace each other.Gas production, pH, NH 3 -N concentration, VFA concentration and rumen microorganism activities are important indices of in vitro incubation.The aim of the study is to compare Menke et al.'s buffer (1979) and Zhao and Lebzien's buffer (2000) on gas production, pH and NH 3 -N concentration in in vitro incubation.If no signifi cant difference in these indices between the two buffers was found, then the further step of the study is to compare the two buffers on VFA concentration, rumen microorganism activities and other indices for complete comparison.

Animal and feedstuffs
An adult Yellow cattle (550 kg BW), fi tted with a rumen fi stula, was used as the donor of rumen fl uid.The animal was fed with lucerne hay twice daily and fresh water was freely available.Sixteen air-dried feedstuffs for ruminants, grounded through 3 mm sieve, were used for incubation.The air-dried feedstuffs were lucerne hay, brewer's dried grain, Chinese rye-grass, maize grain, cottonseed meal, peanut meal, rapeseed meal, sorghum grain, soyabean meal, straw, maize stover, soyabean stalk, wheat fl our middling, wheat bran and wheat grain.Menke et al. (1979): Buffer A:13.2 g CaCl 2 •2H 2 O, 10.0 g MnCl 2 •4H 2 O, 1.0 g CoCl 2 •6H 2 O and 8.0 g FeCl 3 •6H 2 O were dissolved in 100 ml distilled water.Buffer B:39.0 g NaHCO 3 and 2.0 g NH 4 HCO 3 were dissolved in 1000 ml distilled water.Buffer C: 5.7 g Na 2 HPO 4 and 0.6 g MgSO 4 •7H 2 O were dissolved in 1000 ml distilled water.Deoxidize fl uid: 4 ml 1 N NaOH and 0.625 g Na 2 S•9H 2 O were dissolved in 95 ml distilled water.0.1 ml buffer A, 200 ml buffer B, 200 ml buffer C, 40 ml deoxidize fl uid and 400 ml distilled water were mixed.Then 600 ml mixed buffer were mixed with 300 ml rumen fl uid.Zhao and Lebzien (2000): Buffer A: 23.5 g Na 2 HPO 4 •12H 2 O, 12.5 g NaHCO 3 and 11.5 g NH 4 HCO 3 were dissolved in 400 ml distilled water.Buffer B: 23.5 g NaCl, 28.5 g KCl, 6.0 g MgCl 2 •6H 2 O and 2.63 g CaCl 2 •2H 2 O were dissolved in 1000 ml distilled water.50 ml buffer B was mixed with 400 ml buffer A. An adequate amount of distilled water was added to the mixed buffer to yield a fi nal volume of 500 ml.Then 250 ml of the mixed buffer was diluted with 1000 ml distilled water and pre-warmed to 38°C.Then 312.5 ml rumen fl uid were added and continuously gassed with CO 2 .

Experimental design
Syringes with volume of 100 ml were used as incubation vessels.About 0.20 g of feed sample was weighed into each syringe.Each feed sample was incubated in two duplicates.The rumen fl uid was taken 2 h after morning feeding.Thirty ml of buffer-rumen fl uid mixture was transferred into each syringe.Three blank syringes that only contained buffer-rumen fl uid mixture were used in incubation.The syringes were kept at 38°C in a water bath and shaken occasionally to mix the liquids with the solids.The incubation time was set to 24 h.The 12 and 24 h gas production were recorded.The 24 h pH was measured immediately after incubation.Then the incubation residues were fi ltered through four layers of surgical cloth and then distilled for determination of NH 3 -N.

Statistical analysis
The SAS 9.0 software (SAS, 2005) was used for regression analysis of gas production, pH and NH 3 -N between the two buffers.

RESULTS
The obtained results indicated (Table 1) that there was no signifi cant difference in 12 and 24 h in vitro gas production between the two buffers.It was found that there was a signifi cant linear regression relationship between 12 h in vitro gas production of Menke et al.'s buffer (1979) (x, ml/0.2 g air-dried feed) and that of Zhao and Lebzien's (2000) (y, ml/0.2 g air-dried feed): y=0.76x +3.33, r 2 =0.82, n=16; P<0.05.A similar relationship was also found between 24 h in vitro gas production of Menke et al.'s buffer (1979) (x, ml/0.2 g air-dried feed) and that of Zhao and Lebzien's buffer (2000) (y, ml/0.2 g air-dried feed): y= 0.73x +8.55, r 2 =0.89, n=16; P<0.05.

DISCUSSION
One reason for the results that in vitro gas production and pH had no signifi cant difference between the two buffers could be that both buffers, Menke et al. (1979) or Zhao and Lebzien (2000) contained NaHCO 3 and Na 2 HPO 4 as the main buffer salts.Another reason could be that the rumen fl uid used in incubations contained some of minerals and trace elements which were important for incubation.Therefore, although the components of the two buffers were not the same, the difference in in vitro gas production, pH and NH 3 -N between the two buffers would not reach statistically signifi cant level.

CONCLUSIONS
There was no signifi cant difference in in vitro gas production, pH and NH 3 -N concentration between Menke et al. buffer (1979) and Zhao and Lebzien's buffer (2000).It is suggested to compare VFA concentration and rumen microbial activities in in vitro incubation between the two buffers in the future.

Table 1 .
Gas production, pH and NH