Intestinal digestibility of protein and amino acid of ruminant feeds with the mobile nylon bag and in vitro digestion technique

The study was to provide the Chinese Feedbank with intestinal digestibility (Idg) of crude protein (CP) and AA for feedstuffs commonly used in China. The various AA of feedstuffs weren’t synchronously digested, while a larger variation was found in the same AA between feedstuffs. The CP Idg estimated by mobile nylon bag technique in soyabean meal, cottonseed meal, peanut cake meal, rapeseed meal, rice bran, maize germ meal, wheat bran and lucerne hay were 0.964, 0.813, 0.950, 0.826, 0.586, 0.793, 0.679 and 0.400, respectively. It has a strong correlation with the value obtained by three-step in vitro procedure.


INTRODUCTION
According to the new protein evaluation systems, the protein value of a feed for ruminants is now commonly expressed as amino acids truly absorbed from the small intestine (Hvelplund and Weisbjerg, 2000).Currently, as the lack of intestinal digestibility (Idg) parameters of individual feedstuffs commonly used in China, Feeding Standard of Beef Cattle (Feng et al., 2005) assumed a constant value of 0.65 of rumen undergradable protein for concentrate, and 0.60 for roughage etc., which obviously sound much more recognized.The objective of this study was fi rstly, the choice of residuals of soyabean meal, cottonseed meal, peanut cake meal, rapeseed meal, rice bran, maize germ meal, wheat bran and lucerne hay after in situ nylon bag incubation was discussed for further Idg determination with the mobile nylon bag technique (MNBT).Secondly, apparent Idg of amino acids or CP was determined using both MNBT and the three-step in vitro procedure.Correlation of Idg between the two methods was further discussed for their reliability and applicability.

MATERIAL AND METHODS
Two steers (Simmental × Chinese Yellow cattle crossbreed) were fi tted with a permanent fl exibal ruminal cannula and a T-type proximal duodenal cannula.They were housed in individual stall and fed with the same amount of a standardized diet containing 4.2 kg Chinese wild rye day -1 and 4.8 kg concentrate mixture day - 1 which was equally offered at 08.00 and 20.00.
The tested feeds included soyabean meal (SBM), cottonseed (CSM), peanut cake meal (PCM), rapeseed meal (RSM), rice bran (RB), maize germ meal (MGM), wheat bran (WB) and lucerne hay (LH).Dry matter and CP were determined according to AOAC (1999).The AA profi les were estimated by a HPLC system (Waters 26954, Waters Corporation, US).The in situ procedures applied to measure rumen disappearance and intestinal digestion were mainly as described by Hvelplund and Weisbjerg (2000).The three-step in vitro procedure was performed according to Calsamiglia and Stern (1995).
Ruminal degradation characteristics of CP and AA were calculated according to Ørskov and McDonald (1979) using PROC NLIN (SAS, 1999).The effective degradability (ED) was calculated using a rumen fractional outfl ow rate (k) of 0.05 h -1 .PROC GLM was used to analyse the differences between the disappearance rate (DR) at the rumen incubation time of 12, 16 and 24 h and ED of CP or AA within feedstuff, with Duncan's multiple range test used for the comparison of means (SAS, 1999).The same procedure was used to analyse the differences within feed residue remaining after rumen incubation in Idg of CP and AA.The relationship between MNBT and three-step procedure digestibility was measured for linear regression using the PROC REG (SAS, 1999).

RESULTS AND DISCUSSION
The variation of CP was pronounced from 121.4 g kg -1 DM in LH to 497.5 g kg 1 DM in SBM.SBM and RB had a relatively high content of essential amino acids (EAA), and WB had a relatively low content.The proportion of Lys varied from 2.69 g in WB to 5.78 g 100 g -1 CP in SBM.The content of total amino acids (TAA) and EAA in SBM, CSM, WB and LH was higher than found by Taghizadeh et al. (2005) but EAA in LH.The individual EAA in SBM was lower than literature data presented by Harstad and Prestløkken (2000) except Ile.These differences might be due to different plant varieties, producing areas, processing, chemical analyse methods, etc.
The disappearance rate after rumen incubation for 12, 16 and 24 h and effective degradability for CP, TAA, EAA, and non-essential amino acids (NEAA) were obtained, and the values of SBM were presented in Figure 1.It's obvious from our results, the DR after 24 h of rumen fermentation was much Figure 1.The in situ disappearance rate in rumen (DR) at the time of 12, 16 and 24 h and effective degradability (ED) of crude protein (CP), total amino acids (TAA), essential amino acids (EAA), non-essential amino acids (NEAA) using a rumen fractional outfl ow rate (k) of 0.05 h -1 for soyabean meal (SBM).The fi gures in parentheses represent standard error of means within a same class, and means in the same column within a class followed by different letter differ (P<0.05)higher than the values after 12 or 16 h of rumen fermentation and those values of ED, and the difference between the ED and DR after 12 or 16 h of rumen incubation was not signifi cant except WB.Hence, for most feedstuffs the CP and AA profi le in the residue of 12 or 16 h incubation time can be used to refl ect the rumen undegraded CP and AA profi le reaching the small intestine.Furthermore, Hvelplund and Weisbjerg (2000) pointed that incubation for 16 h could be used for all feeds at present, as this incubation time was believed to refl ect the infl uence of rumen metabolism on the feed before passage to the intestine.For the standardization of the mobile nylon bag technique adopted in China, the choice of residuals of tested feedstuffs after in situ nylon bag incubation for 16 h was strongly recommended for further Idg determination of CP and AA.
The digestion of CP and AA in small intestine was calculated (Table 1) basing on the residue preincubated 16 h in rumen.The intestinal digestibility among feedstuffs varied considerably.RB, WB and LH, showing higher disappearance of CP and AA in the rumen, presented lower digestion in small intestine.Weisbjerg et al. (1996), who studied 15 different concentrates, indicated that the Idg of TAA was similar to that of CP in the residues.But it only agreed with SBM, PCM, RSM, RB and WB in our study.Therefore, using the Idg of CP to replace the values of TAA is problematic.Moreover, the assumed constant value of 0.65 of RUP for concentration, and 0.60 for roughage etc., adopted in Feeding Standard of Beef Cattle (Feng et al., 2005) cannot account for real supply of available amino acids in the intestinal tract for host animal.As the limiting amino acid, Lys in SBM, WB and LH had a higher Idg than TAA as well as most other individual AA, but in the other 5 feedstuffs it had a lower Idg.In the study by Taghizadeh et al. (2005), which included 10 different feedstuffs, it was shown that Lys Idg was higher than TAA Idg except for maize grain, fi sh meal and barley grain.The lowest Idg of Lys was observed in RB, a feedstuff that has some trypsin-inhibitor (TI).The TI can inhibit the action of trypsin which is an endopeptidase to hydrolyse only Lys or Arg bonds.
In general, the digestibility obtained from three-step procedure was lower than corresponding data obtained via MNBT.That might be relative to protein digestion that occurs in the large intestine.A much higher RSM digestibility examined with MNBT might attributive to the antinutritional factors in RSM.Furthermore, Stern et al. (1997) measured results of three-step procedure with in vivo intestinal protein digestion and found a high correlation (R 2 =0.91).Therefore, the three-step technique might be a rapid, reliable procedure for evaluating intestinal digestion of proteins in ruminants.

CONCLUSIONS
For the standardization of the mobile nylon bag technique adopted in China, the choice of residuals of tested feedstuffs after in situ nylon bag incubation for 16 h was strongly recommended for further intestinal CP and AA digestibility determination.Using an assumed constant or the digestibility of CP to replace those of TAA or individual AA is problematic for individual feeds in feed evaluation for new protein system.The three-step in vitro technique might be a rapid, reliable procedure for evaluating intestinal digestion of proteins in ruminants.

Figure 2 .
Figure 2. Relationship and linear regression equations between CP digestions for the residuals of soyabean meal (SBM), cottonseed meal (CSM), peanut cake meal (PCM), rapeseed meal (RSM), rice bran (RB), maize germ meal (CGM), wheat bran (WB) and lucerne hay (LH) after preincubated in the rumen for 16 h measured by the mobile nylon bag technique (MNBT) and the three-step in vitro procedure

Table 1 .
The apparent intestinal digestibility of crude protein (CP), total amino acids (TAA) and individual amino acids for the residue of feedstuffs after preincubated 16 h in the rumen estimated by the mobile nylon bag technique CSM -cottonseed meal, PCM -peanut cake meal, RSM -rapeseed meal, RB -rice bran, MGM -maize germ meal, WB -wheat bran, LH -lucerne hay; 2 SEM -standard error of the mean; a-k means within a column with different subscripts differ (P<0.05)