The influence of diet and intake level on hepatic ammonia metabolism and ureagenesis by the ovine liver : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand

Abstract

The New Zealand agricultural industry is based on the efficient utilisation of fresh forages, a characteristic of which is a high soluble protein content. A large proportion of the ingested protein is highly soluble in the rumen. A significant proportion of the ingested N is removed from the rumen as ammonia with the bulk of this ammonia being removed from the venous blood by the liver for detoxification to urea. Hepatic urea-N production, or ureagenesis, typically exceeds the rate of hepatic ammonia-N extraction, consequently it has been suggested that the shortfall in N required for ureagenesis is contributed by amino acid-N (Parker et al. 1995; Lobley et al., 1995). This study tested the hypothesis that elevated hepatic ammonia extraction would require a concomitant increase in hepatic amino acid catabolism to supply the additional N required for ureagenesis. In order to evaluate the level of rumen ammonia production and consequently the rates of hepatic ammonia extraction, ureagenesis and amino acid catabolism. the following feeding regimens were tested in sheep held indoors in metabolism crates in three separate experiments; Firstly, lucerne pellets (Medicago sativa) were compared with fresh white clover (Trifolium repens), secondly fresh white clover was offered at either a low or high intake and finally the daily allowance of fresh white clover was fed in two 2 hour periods per day. In each experiment, silicone based catheters were surgically inserted into the posterior aorta and the mesenteric (2), portal and hepatic veins. Following a ten day dietary adjustment period and a ten day nitrogen balance, the sheep were infused with para-aminohippurate (pAH) and 15NH4Cl via the mesenteric vein. The pAH was infused to allow the blood flow across the splanchnic tissues to be estimated, whilst the 15NH4Cl was infused to trace hepatic ammonia metabolism to urea. Blood samples were collected to determine the ammonia, urea, oxygen and amino acid concentrations in the mesenteric, portal and hepatic veins, as well as the posterior aorta. Despite similar DM intakes, the nitrogen intake of the sheep fed fresh white clover was 60% higher (P < 0.001) than that of the same animals fed lucerne pellets. The difference in rumen protein fermentation in these two contrasting diets resulted in higher (P < 0.001) rumen ammonia production in the animals offered fresh white clover. There was, however, only a trend (P = 0.072) toward elevated hepatic ammonia extraction in these animals and urea production was not significantly different to the animals fed lucerne pellets. Hepatic amino catabolism was not elevated in the sheep fed fresh white clover, nor was there a significant difference in the proportion of ME intake that was utilised for ureagenesis between the two groups. In the second experiment the DM intakes of the two groups were different (P < 0 001), with the sheep offered the low intake of fresh white clover consuming 807 g DM/d whilst the high intake group consumed 1118 g DM/d. Even with these differences in intake, portal vein ammonia and urea concentrations were similar. Therefore the rate of hepatic ammonia extraction and urea production were also similar between the two intake groups. However, hepatic extraction of 15N-ammonia was higher (P = 0.033) in the high intake group compared to the low intake group. There was no evidence to suggest that the level of hepatic amino acid catabolism increased with intake level, consequently the proportion of ME intake attributed to urea synthesis was similar for the two intake groups When the experimental animals were restricted to two 2 hour feeding periods per day the DM and N intake decreased by 31% from that of the low intake group in the second experiment. There was no significant effect of time after the onset of feeding on portal ammonia or urea concentrations, hepatic ammonia extraction or hepatic urea production. However portal ammonia concentration and consequently hepatic ammonia extraction and urea production tended to be higher 4-6 hours after ingestion of fresh white clover. However this trend was not observed when the 15N tracer data was used to calculate the hepatic ammonia transfer rate. The ammonia, urea and amino acid hepatic transfer values in this experiment were largely comparable to those recorded for the low and high intake treatments in the second experiment. In these studies, there was no evidence of elevated hepatic amino acid catabolism occurring in response to elevated rates of hepatic ammonia extraction and hence ureagenesis. Additionally there was no suggestion that ammonia provided both of the N atoms of the urea molecule. It is concluded that the liver adapted to the changes in dietary nitrogen supply without incurring significant increases in the metabolic cost of ammonia detoxification to urea. However the nutritional challenges presented to the liver may not have been severe enough to induce measurable changes in hepatic ammonia metabolism. A possible mechanism to account for these observations may be that the liver adapted to the changes in nitrogen supply by altering the activity of the primary regulator of the rate of ureagenesis, carbamoyl phosphate synthetase (CPSI).