Componente Formal

The results of the study reported in the previous Chapter of this thesis demonstrated that endogenous ileal nitrogen and amino acid flows determined in digesta collected from pigs 30 hours after the pigs had received the last protein-containing meal did not significantly differ from those determined after the pigs had received a protein­ free diet for eight days. Given that endogenous ileal nitrogen and amino acid flows determined in digesta collected from animals that have received a protein-free diet for seven to ten days are thought to be underestimated, this suggests that the protein-free method is not a suitable method for the determination of endogenous ileal nitrogen and amino acid flows. Other methods that may be used to determine endogenous ileal protein flow include the enzyme hydrolysed protein, isotope dilution and guanidination methods. These latter methods allow determination of endogenous ileal protein flows in animals that are receiving a diet that contains protein or peptides. No direct comparison has been made previously among the latter three methods to determine whether they give similar estimates of endogenous flows. Therefore, the aim of this study was to compare endogenous ileal nitrogen and lysine flows determined using the isotope dilution and guanidination methods, respectively, with those determined using the enzyme hydrolysed protein method in the growing pig and rat.

Direct Comparison of the Enzyme Hydrolysed Protein, Guanidination and Isotope Dilution Methods for Determining

Endogenous Ileal Protein Flow in the Growing Rat and Pig

Suzanne M. Hodgkinson 1 , Wolfgang B. Souffranf and Paul J . Moughan1

1 Institute of Food , Nutrition and Human Health , Massey University, Palmerston North , New Zealand.

20epartment of Nutritional Physiology "Oskar Kellner" . Research Institute for the Biology of Farm Animals, Rostock, Germany.

ABSTRACT

The aim of this study was to determine whether the isotope dilution and guanidination methods give similar estimates of endogenous ileal nitrogen and lysine flows, respectively, as the enzyme hydrolysed protein method in the growing rat and pig. The test diet contained casein labelled with 15N, guanidinated and enzymatically hydrolysed (MW < 5,000 Oa). Male rats (n=30; mean bodyweight 1 78 g) and entire male pigs (n=6; mean bodyweight 1 9.2 kg) received a preliminary enzyme hydrolysed casein based diet for 7 days. The rats had free access to the diet for 1 0 minutes each hour for 8 hours per day; the pigs were given the diet at a rate of 1 0% of their metabolic body weight per day divided into 8 hourly feeds. The test diet was given to the rats and pigs on the following day in the same manner as the preliminary diet. Oigesta were sampled at the terminal ileum after the animals had been anaesthetised. The guanidination method gave significantly (P<0.05 and P<0.01 for the rat and pig, respectively) lower endogenous lysine flows than the enzyme hydrolysed protein method (means ·of 298 vs 382 and 2 1 4 vs 287 /1g/g dry matter intake, OMI, in the rat and pig, respectively). Endogenous nitrogen flows determined using the enzyme hydrolysed protein method were significantly (P<0.001 for the rat and P<0.05 for the pig) greater than those determined using the isotope dilution method (means of 1 942 vs 1 034 and 1 543 vs 1 01 1 /1g1g dry matter intake, OMI, in the rat and pig, respectively). The guanidination method may underestimate endogenous ileal lysine flow in both the rat and pig. The isotope dilution method (labelled diet) may also underestimate endogenous ileal nitrogen flow in the rat and pig.

INTRODUCTION

When calculating amino acid requirements for growing animals or true ileal digestibility coefficients it is important to be able to determine with accuracy the amount of protein of endogenous origin that is present in ileal digesta (Tamminga et al., 1 995; Boisen and Moughan, 1 996a; Nyochoti et al., 1 997a). Several methods have been developed to this end. Three of these approaches, the enzyme hydrolysed protein, isotope dilution and guanidination methods, allow the determination of endogenous protein flow through the ileum when dietary peptides are present in the gut lumen.

The enzyme hydrolysed protein method, proposed by Moughan et al. (1 990), involves feeding the test animal a semi-synthetic diet containing enzymatically hydrolysed casein (molecular weight <5,000 Da) as the sole source of nitrogen. After the digesta are collected, the high molecular weight fraction (>1 0,000 Da), which contains the endogenous protein, is separated by centrifugation and ultrafiltration and is analysed for total nitrogen and amino acids.

The isotope dilution method involves the use of the stable isotope 15N and can be applied by feeding diets labelled with 15N to test animals (Leterme et al. , 1 993; Leterme et al., 1 994; Roos et al., 1 994). Endogenous nitrogen'in the digesta can then be distinguished from 15N-labelled exogenous (dietary) nitrogen.

The guanidination method involves partially transforming dietary lysine to homoarginine. The latter amino acid (an analogue of lysine) is absorbed but not used for body protein synthesis. It is not present in endogenous gut protein and thus acts as a marker for lysine uptake. By determining both the homoarginine and lysine contents of ileal digesta collected after feeding an animal a guanidinated diet, the endogenous lysine flow can be calculated ( Hagemeister and Erbersdobler, 1 985;

Rutherfurd and Moughan, 1 990).

The enzyme hydrolysed protein method allows valid measurement of endogenous ileal nitrogen and amino acid flow, by definition, in the specific case pertaining to the administration of the hydrolysed protein itself. The determined endogenous ileal amino acid flow will be a valid estimate for the particular hydrolysate being fed to the animals. It should be noted, however, that there may be a slight underestimation of flow due to the loss of endogenous amino acids discarded with the ultrafiltrate but this is a defined systematic error. In the present study the endogenous total nitrogen and lysine flows determined using the enzyme hydrolysed protein method

were accepted as a suitable baseline and compared to those determined by the