Portal Absorption of Feed Oligo-peptides in Chickens

The effect of duodenal infusion with feed oligo-peptide solution on portal absorption of amino acids was investigated in poultry under unanaesthetized conditions. Four peptide solutions were used in the experiment: enzymatic hydrolysates from fish meal, soybean meal, cottonseed meal and rapeseed meal proteins with average molecular weights less than 3,000 Da and 1,000 Da, respectively. Intestinal absorptions of these oligo-peptide solutions were compared by determining the concentration of free amino acid (FAA) in portal blood after the duodenal administrations of oligo-peptide solutions. Absorptive intensity and balance were used to estimate the intestinal absorption rate of amino acids. The absorptive intensities of amino acids were highest for the fish and soybean meal oligo-peptides. The ratios of amino acids absorbed in the portal blood from fish and soybean meal oligo-peptides were more similar to the composition of the infused amino acids than that observed from the cottonseed and rapeseed meal oligo-peptides. A positive correlation was found between absorption rate and proportion of PAA in the oligo-peptides. The higher absorption rate could be contributed to the higher proportion of peptide bound amino acids (PAA). The results suggest that fish and soybean meal protein are significantly more easily hydrolyzed into oligo-peptides (p<0.05) in the gastrointestinal tracts of poultry and as such can be utilized more effectively by body tissues. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 9 : 1277-1280)


INTRODUCTION
It has been recognized in recent years that the end products of feed protein found in animal digestive tracts were mostly oligo-peptides.These play a dominant role in the absorption of amino acids (Kim, 1972;Matthews et al., 1975b).Evidence exists suggesting that these peptides are transported by systems independent of those responsible for transporting free amino acids.Peptide transports are active due to higher absorption speed, low levels of energy consumption, and less competition for carriers than those of free amino acids making peptides, especially dipeptides and tripeptides, effective donors of amino acids (Burston et al., 1972;Matthews et al., 1975b;Webb, 1990).They can be rapidly transported into intestinal mucosal cells and hydrolyzed before being absorbed as their constituent amino acids into blood flow (Webb et al., 1990;Daniel et al., 1994).
There have been many studies on the absorption of peptides, most of which indicated higher absorptive speed and intention than FAAs (Matthews et al., 1972;Hara et al., 1984;Silk et al., 1985;Rerat et al., 1988;Le et al., 1997).The peptides used in these experiments, however, were mostly standard dipeptides, tripeptides or enzymatic hydrolysates of some special proteins such as casein, albumin and lactoglobulin.These peptides have special properties and therefore have some limitation.In vivo or in vitro experiments Feng et al. (2002) and Savoie et al. (1989) reported that the proportions of PAA hydrolyzed from quality feed proteins are higher than those from poor feed proteins.This suggests that the amount of PAA in protein hydrolysate might be a reason behind the difference of feed amino acid availability.The present investigation used duodenal infusion to compare the absorption of oligopeptides hydrolyzed from common feed proteins in order to better understand the factors that affect the digestible utilization of feed proteins of different qualities.

Preparation of infused oligo-peptide solutions
The oligo-peptides utilized in this study were enzymatic hydrolysates of four common feed proteins (fish, soybean, cottonseed and rapeseed meal proteins).They were in vitro hydrolyzed using pepsin and trypsin with the following hydrolysis procedure.Eight grams of feed sample was placed in a 800 ml flask with stopper.To this was added 80 mg pepsin (Sigma Comp.USA, activity 1:10,000) dissolved in 80 ml pH 2.0 HCl.The flask was placed horizontally in an oscillator (120 r/min) and incubated at 37°C for 6h.A few drops of 50% NaOH were added to adjust pH to 7.0.Then 200 mg trypsin (BD Comp.USA, activity 1:250) was added with 400 ml pH 7.6 phosphate buffer.The incubation was continued for an additional 18 h.120 ml 5%(w/v)trichloroacetic acid (TCA) was then added and the hydrolysate centrifuged at 8,000 g, 4°C for 30 min.The supernatant was ultra filtered to discard the fractions with average molecular weights <3,000 Da and <1,000 Da. one ml of the hydrolysate fraction was analyzed for FAA and total amino acid (TAA) concentration (before and after 6 N HCl hydrolysis at 110°C for 24 h) by high performance liquid chromatography (HPLC) following derivation by FMOC.
Depending on the concentration of TAA and FAA, appropriate volumes of eight oligo-peptide fractions from four feed protein hydrolysates were measured and evaporated to dryness at 65°C, then dissolved in 5 ml of distilled water to ensure equal TAA amounts (100 umol).Eight such oligo-peptide solutions were prepared for infusion of per chicken.The molar ratio of each amino acid and PAA proportions in the eight oligo-peptide solutions are shown in Table 1.

Experimental procedure
The chickens were deprived of food for 24 h prior to infusion.A glucose vitamin added to water (50 g per chicken) was provided ad libitum during the deprivation period.The experiment was performed under unanesthetized and unrestrained conditions.A small opening was made at the right abdomen near keel follicle.The duodenum was removed and 5 ml of oligo-peptide solution was administered from the upper site of duodenum using a syringe.The duodenum was then replaced in the abdomen.The birds were allowed to move about normally.Different aminals were sampled at each period of time.

Sampling of plasma
Abdominal wall was opened rapidly and 10 ml of portal blood collected and placed in a centrifuge tube with a preadded anticoagulant of heparin sodium.The whole blood was centrifuged at 5,000 g, 4°C for 20 minutes.Plasma was added to bring the volume to 0.6 mol/L Perchloric acid (PCA) followed by another centrifuge at 8,000 g, 4°C for 15 minutes.The supernatant solutions of two chickens in one replicate were mixed at equal proportion and the final plasma sample stored at -20°C prior to analysis.
Experiment 1 : Fifty four 6 wk-old chickens of average weight (1.5±0.1 kg) were randomly divided into 9 groups with 3 replicates per group and 2 chickens for each replicate.One group was infused with 5 ml of 0.9% NaCl solution as a control.The other eight groups were all administrated 5 ml S3.Portal blood was collected at 0, 5, 10, 15, 20, 30, 40, 60 and 90 minutes, and the plasma FAA concentration was determined.
Experiment 2 : Forty two 6 wk-old chickens of average weight (1.5±0.1 kg) were randomly divided into 7 groups with 3 replicates per group and 2 chickens for each replicate.Seven groups were duodenal infused with 5ml of F3, C3, R3, F1, S1, C1 and R1, respectively.Portal blood was collected at 20 minute after infusion according to results obtained in experiment 1, and the plasma FAA concentration was determined.Absorptive intensity and balance were then calculated and analyzed in comparison with the proportion of PAA present.

Analysis of plasma FAA
A 5 ml plasma sample was evaporated to dryness at 65°C and 2 ml inner standard of bezoar acid added prior to derivation by 9-Fluorenylmethyl Chloroformate before HPLC determination of amino acids.
Met, Trp, Arg, and Cys were not measured.As such, the FAA in this study refers to 14 amino acids and TAA refers to the sum of the 14 amino acids.

Estimate of absorptive efficiency of amino acids
Two indexes of absorptive intensity and balance were used to estimate the portal absorptive efficiency of amino acids.The intensity of intestinal absorption for each amino acid was calculated from its elevated concentration in portal blood in the 20 minutes before and after infusion and expressed as slope.The intestinal absorptive balance of the amino acids absorbed was expressed as X 2 , according to the following equation: X 2 =Σ(fi-Fi) 2 /Fi, where fi is the ratio of intensity of each amino acids indicated as percent value.Fi is the molar ratio of each amino acids administrated.slope (umol/L⋅min)=(C 1 -C O )/t, where C 1 and C O indicated total FAA concentration in portal plasma in a period of time after and before infusion of oligo-peptide.
A lower X 2 value indicates that the balance of amino acids was better maintained in the absorptive ratio of each amino acid from the administrated oligo-peptide.

Statistical analysis
Statistical differences among the oligo-peptide infusion groups were determined using Duncan's multiple range test (Duncan, 1955), and the correlation analyzed by linear regression.All statistical analyses were performed using the SPSS 10.0 program.

Change of portal plasma FAA after duodenal infusion of S3 solution
Table 2 indicates the change of total FAA concentrations in portal blood after the infusion of S3.FAA concentrations immediately increased and reached a maximum 20 min after administration (p<0.05),then decreased to the control value after 60 to 90 min.

The effect of duodenal infusion of different oligo-peptide solutions on the intestinal absorptive efficiency of amino acids
Table 3 shows the FAA concentrations in experiment 2. Compared with the control, the portal plasma FAA concentrations increased significantly after infusion of eight oligo-peptide solutions (data of S3 are from Experiment 1).The F3 and S3 groups were significantly higher than other groups, with C3 higher than C1 and R1 (p<0.05).There was no difference between C3 and R3, F1 or S1, and R1 and C1.
Table 3 also indicates that the absorptive efficiencies were significantly different among the eight groups.The absorptive intensities of amino acids from F3 and S3 are markedly higher than the other groups (p<0.05).

Regression analysis of PAA proportion in the administrated oligo-peptides and intestinal absorptive efficiency of amino acids
Two linear regression processes were carried out on the PAA/TAA using the oligo-peptide solution as the independent variable and absorptive intensity and balance as dependent variables.The coefficients between the proportion of PAA in the oligo-peptide solution and absorptive intensity and balance are 0.92 and 0.93.Regression equations were Y=4.23X-234.09and Y= -2.70X+280.24.

DISCUSSION
The intestinal absorption rates of peptides and corresponding amino acid mixtures were compared in many experiments.Silk et al. (1985) used a method of intestinal perfusion with a double balloon catheter.Hara et al. (1984) installed cannulation of the portal vein and placed the tip of the catheter behind the neck of rat skin.Studies of Matthews et al. (1972), Guowei Le et al. (1997) used the similar method of duodenal infusion as the present investigation except that theirs were performed under anesthesia disturbing the movement of intestines or blood flow.This experiment aimed to a rapid and sensitive determination and to minimize the individual differences in animals and consequently to allow a good comparison among infusion groups of different oligo-peptide solutions.
The peptides used in reported experiments were mostly standard dipeptides, tripeptides, or enzymatic hydrolysates of some special proteins such as casein, albumin and lactoglobulin.While the oligo-peptide solutions used in this study were prepared from enzymatic hydrolysis of fish meal, soybean meal, cottonseed meal and rapeseed meal to simulate the gastrointestinal conditions of poultry.Accordingly, the results may better illuminate the intestinal absorption of digestive products of common feed proteins.
To estimate the intestinal absorption rate of amino acids, an index of absorptive intensity and balance was used according to that of Hara et al. (1984).Both slope and X 2 were calculated from FAA concentrations taken 20 minutes after infusion.This may have had a slight effect on the recycled amino acids, although the groups were still comparable.In order to discuss the factors affecting the rate of absorption, correlations were assessed between PAA content in the oligo-peptide solution and absorptive intensity and balance.The rate of intestinal absorption was positively correlated with the proportion of PAA in the oligo-peptide solution.Thus, the higher rate of absorption of amino acids from F3 and S3 oligo-peptide solutions could have contributed to their higher PAA proportions.
The ratio of absorptive intensity of each amino acid from those oligo-peptide solutions containing more PAA was in better balance.The loss of absorptive balance among amino acids in groups having less PAA may be due to the competition from amino acids on the brush border membrane.Based on these results, the proteins from fish meal and soybean meal were easily hydrolyzed to oligopeptides by enzymes in animal gastrointestinal tracts.These oligo-peptides provided more rapidly absorbed sources of amino acids from intestine and maintained a better balance after absorption compared to hydrolysates from cottonseed meal and rapeseed meal protein.This may be the reason why fish meal and soybean meal protein supply better availability of amino acids and are more efficiently utilized by body tissues in monogastinal animals than cottonseed meal and rapeseed meal protein.

Table 1 .
Molar percent of each amino acids and PAA proportion in oligo-peptide solutions C3 and R3 represent hydrolysate fraction of <3,000 Da of fish meal, soybean meal, cottonseed meal and rapeseed meal, respectively.F1, S1, C1 and R1 represent hydrolysate fraction of <1,000 Da of the same ingredients.

Table 2 .
Changes in plasma total FAA concentration after infusion of S3 (n=3)

Table 3 .
Comparison of absorptive intensity and balance of AA from different peptides (n=3)