Digestion and metabolism of sulphur containing amino acids in sheep fed fresh forage diets : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand

Sulphur amino acids (SAA) are important in the sheep industry because they appear in many instances, to limit wool protein synthesis. Experiments were carried out to study two methods with the potential to increase the absorption of SAA from the small intestine of sheep fed fresh forage diets. The first experiment evaluated the effects of condensed tannins (CT) in Lotus pedunculatus upon the following; (1) Rumen-S metabolism, and (2) the digestion of methionine and cystine. (3) The metabolism of plasma SAA and inorganic sulphate, and (4) The solubility and (5) degradation of protein in the rumen. The second experiment identified proteins that contained a high proportion of SAA, and assessed their potential for expression in the leaves of forage legumes using genetic engineering techniques. Two aspects were measured; (6) Rates of rumen degradation of ovalbumin and sunflower albumin 8 (SF8) proteins in vitro, and (7) The expression of SF8 protein from sunflower seeds in the leaves of transgenic tobacco. The nutritional consequences of CT in Lotus pedunculatus were assessed by infusing polyethylene glycol (PEG), into the rumen of one group of sheep (PEG sheep; CT not operating), whilst a separate group of sheep received an infusion of water (CONTROL sheep; CT operating). PEG selectively binds CT, preventing CT from binding plant proteins in the rumen, so that CT effects on digestion could be evaluated. Polyethylene glycol was also added to in vitro incubations as required. The principal results were; (1) Condensed tannins had a major effect on rumen sulphur (S) metabolism. The irreversible loss rate (IRL) of reducible-S (total non-protein S in rumen fluid, measured as sulphate after performic acid oxidation of rumen fluid) from the rumen was lower (P0.05) for control (1.40g/d) and PEG (l.34g/d) sheep. The apparent digestibility of methionine in the small intestine was similar (0.78) for both groups, whilst for cystine, it was lower (P<0.01) in control (0.42) than PEG (0.53) sheep. The increased absorption of methionine from the small intestine with CT was due to an increased flux from the rumen, whereas the digestibility of cystine in the small intestine may have been lower as a consequence of binding to CT complexes. (3) Condensed tannin had a major effect on plasma SAA fluxes, especially cystine. Plasma cystine concentration and IRL were higher (P<0.001) in control (41.7μmol/l and 39.8:μmol/min) than PEG (27.5μmol/l and 22.4μ.mol/min) sheep. Condensed tannins resulted in a 79% increase in the transulphuration of methionine to cystine (11.7 and 6.5μ:mol/min; P<0.05) and a decrease (P<0.05) in the oxidation of cystine (3.33 and 5.2μ.mol/min) and methionine (0.2μ.mol/min and 1.2μmol/min) to sulphate in control compared to PEG sheep. The net effect of CT was to increase (P<0.05) the flux of plasma cystine to productive processes and maintenance by 110% in control (36.5μmol/min) compared to PEG (17.4μmol/min) sheep. This represented 91% of total plasma cystine flux in control sheep, compared to only 74% of total cystine flux in PEG sheep (P<0.05). Since wool growth is generally accepted as the major productive process utilising plasma cystine, these results indicate that a major effect of CT in the diet would probably be to increase wool growth. The IRL of plasma methionine was similar in control (20.5μmol/min) and PEG (19.9μmol/min) sheep, whilst the IRL of plasma sulphate was lower (P