L. E. Phillip, M.V. Simpson, E. S. Idziak H and S.F. Kubow*

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1 Ruminal and metabolic effects of pure lignin in sheep fed low and high fibre diets. L. E. Phillip, M.V. Simpson, E. S. Idziak H and S.F. Kubow* Introduction Previous studies with cattle indicated that addition of pure lignin to a grain diet improved growth rate and altered faecal excretion of ammonia and total nitrogen. is thought to have the capability of binding ammonia in the gut and may therefore, affect ammonia kinetics in ruminants. Any impact of A-lignin on ammonia metabolism would have important implications for the efficiency of protein utilisation by ruminants.to ascertain whether lignin exerted an effect merely as dietary fibre, we studied the nutritional significance of pure lignin when it was incorporated into pelleted concentrate diets with low and high NDF content. is a component of plant cell wall fibre but the specific effects of pure, isolated lignin (eg. ALCELL7 lignin, REPAP-ALCELL Technologies Inc.) on rumen fermentation and metabolism are largely unknown. Objectives The aims of the research were: 1) to evaluate effects of Alcell lignin (Alignin) on rumen fermentation, liver function and excretion of nitrogenous constituents in urine; 2) to determine whether the fibre (NDF) content of the diet alters ruminal and metabolic responses to A-lignin. We hypothesized that if A-lignin acts merely as a component of dietary fibre, the magnitude of responses to A-lignin would be less at the high than the low level of fibre intake. Experimental Protocol Page 96 Animals and Diets Four adult sheep (initial weight 60 kg) were fitted with rumen cannulae and maintained in individual floor pens. For six weeks prior to the initiation of the study, the animals were offered, twice daily (0900 and 1700), a diet of hay (ad libitum) plus a restricted amount of a commercial grain supplement. During the last two weeks of this pre-trial period, the sheep were introduced gradually to their respective experimental diets. The diets consisted of pelleted concentrate feeds, formulated to be isocaloric and to contain 13 % protein and either 10% or 24 % NDF (Table 1). Soyhulls was used to alter the NDF content of the diets. Pure lignin was either incorporated (1.25% of the pelleted feed ) or not incorporated into the low (LF) and high fibre (HF) diets. During the entire experiment, the sheep were offered their diets at a restricted level of feeding (85% of ad libitum intake). We found it necessary to offer some hay to ensure proper rumen function, so all animals were fed 150g of chopped hay and had free access to water. Experimental Design and Measurements Ammonia in rumen fluid and urine was determined using the indole- The experiment was conducted as a 4 x 4 Latin square with four 12 d periods; the treatments were arranged as a 2 x 2 factorial. During the first 7d (diet adaption phase) the sheep were maintained in the floor pens; they were then transferred to stainless steel metabolic cages for the phenol colorimetric method; urine was diluted 1 in 100 before being remaining 5d (collection phase). Samples of blood, urine and rumen fluid were collected on the last day. The 24-h output of urine was collected in 50 ml of 10 N H 2 S0 4 and the volume was recorded. Representative samples of urine was frozen at -20 C for subsequent analysis. The samples of rumen fluid and blood were collected 20 min before feeding and at 2h and 4h thereafter.the rumen fluid was strained through four layers of cheese cloth and ph was determined immediately. The samples were then acidified with 5 drops of 10 N H 2 S0 4 and placed in an ice bath before being centrifuged at 3000 x g for 15 min; portions of individual samples were stored at - 20 C for subsequent analyses of ammonia and VFA. Blood samples were collected via jugular puncture into EDTA-coated evacuated tubes. The samples were placed in an ice bath then centrifuged at 3000 x g for 15 min; plasma was stored at - 20 C for subsequent analysis of ammonia, urea, creatinine and determination of enzyme activity. Samples of the experimental diets and of chopped hay were collected daily during the 5d collection phase. The samples were composited and ground for subsequent chemical analysis. The sheep were weighed at the beginning and at the end of each 12 d experimental period. assayed. Urea in plasma and urine was determined with the SIGMA kit

2 (Procedure No. 67-UV); creatinine in plasma and urine was measured using the SIGMA kit (procedure No. 555) and plasma enzymes were analysed using Abbott biochemical kits (Abbott Laboratories).Volatile fatty acids in rumen fluid were determined by gas liquid chromatography. Diet samples were analysed for protein, NDF, ADF, ash and minerals. All data were statistically analysed using the GLM procedure of SAS. During the fourth period of the study one animal became unthrifty and lost appetite; it was removed from the experiment and the data were analysed with missing observations. Results and Discussion The composition of the diets is shown in Table 1. The HF diets contained approximately twice the level of NDF as the LF diets. The protein content of the diets was higher than expected (13%) but the values were similar among diets. The major role of the hay was to stabilize rumen function and maintain the animals= appetite. The hay undoubtedly contributed to the total intake of fibre. However, because all animals received the same amount of hay this would not have been a confounding factor in the experiment. Furthermore, the fibre intake from hay accounted for only 22 % of total dietary NDF intake indicating that the experimental diets made that principal contribution to fibre inake. All animals consumed similar amounts of food (Table 4). At an average intake of 1578 g/d from the pelleted experimental diets, those sheep in the HF group would have consumed approximately 358g of NDF from concentrate (93g from hay); corresponding values for the LF group would be 213g (and 47 g). Thus, the HF group consumed 68% more dietary fibre (as NDF) than the LF group. At the specified level of lignin incorporation into the diet (1.25%) the "lignin group" of animals would have consumed approximately 20g/d of A-lignin ( 0.3g/kg BW/d). We hypothesized that if A-lignin were acting merely as a component of dietary fibre (measured as NDF) the responses to A-lignin would be less with animals in the HF group than in the LF group. Hence we would observe a significant interaction of "fibre x lignin". The results of all ruminal and metabolic measurements revealed that, with the exception of LDH activity and urinary excretion of ammonia, no such interaction existed. This suggests that in cases where there were significant effects of lignin, they may be unique to A- lignin. Table 2 reveals that lignin had no significant effect on any measure of rumen fermentation either before (0 h) or after feeding. There was a trend towards lower concentrations of ammonia in ruminal fluid of animals fed the HF diet.there were no significant effects of dietary treatment on plasma concentrations of creatinine or urea (Table 3). Activity of liver enzymes in plasma (Table 3) are typical of what would be expected for healthy animals. In contrast, the estimates for gamma glutamyl transferase (GGT) in all treatment groups exceeded the normal range of values (10-40 U/L). Plasma activity of lactate dehydrogenase (LDH) in animals from control LF group also exceeded the limit value (530 U/L) expected for healthy animals.of the liver enzymes analysed, only alanine amino transferase (ALT) and LDH were affected by dietary treatment. addition led to significant (P<0.05) reduction in plasma activity of ALT at 4h after feeding but there was no significant interaction with fibre intake. With the addition of lignin to the LF diet, plasma activity of LDH declined. However, lignin addition to the HF diet resulted in an elevation in LDH activity resulting in an interaction between (P<0.10) of fibre intake and lignin addition. An elevation in plasma activity of liver enzymes, particularly GGT, is an indicator of liver dysfunction. Given the lack of any adverse effect of lignin on most of the measures of liver function, it seems unlikely that feeding A-lignin to ruminants would lead liver dysfunction.the dietary level of lignin used in this study was equivalent to that which stimulated growth rate of calves in our previous study. Estimates of urinary excretion of urea and ammonia, and of plasma concentrations of creatinine are presented in Table 4. Urine volume was reduced (P<0.05) with diets containing lignin. When expressed relative to creatinine, urinary excretion of ammonia increased with the LF diet and decreased with the HF diet; this led to a significant interaction of fibre intake and lignin, and implies that lignin may have had more impact on "ammonia clearance" in the low fibre diet than in the high fibre diet. The data also indicate that part of the effect of A-lignin on ammonia kinetics may be a "fibre effect". This phenomenon requires further study. Given that plasma creatinine concentrations were all within normal limits and that the plasma levels tended to be even lower (p.<10) in lignin-fed animals, it appears that the addition of pure lignin to the diet had no adverse effects on creatinine clearance, hence on kidney function. Impact The results of this study revealed that incorporation of pure lignin at 1.25% into pelleted concentrate diets had no Page 97

3 detrimental effects of measures of liver and kidney function. decreased plasma activity of the liver enzyme, ALT, but the nutritional significance of this finding is uncertain. The current finding that lignin increased urinary excretion of ammonia in sheep fed a low fibre diet but decreased ammonia excretion in those fed a high fibre diet should stimulate more research into the specific influence of pure lignin on gut ecology. H Department of Natural Resource Sciences * School of Dietetics and Human Nutrition. Table 1.Ingredient and chemical composition of control and lignin containing pelleted diets Low fibre High fibre Low fibre High fibre Ingredient composition, %: Corn grain Barley Canola meal Soybean meal (48%) Soy hulls ALCELL lignin Molasses Dicalcium phosphate Limestone Salt (NaCl) Magnesium oxide Vitamin-trace mineral mix Chemical composition (as is) 1 : DM, % Protein, % NDF, % ADF, % Ash, % Ca, % P, % Mg, % Values are means of 4 samples Page 98

4 Table 2.Measures of rumen fermentation in sheep fed low (LF) and high fibre (HF) with added lignin. Time after feeding Rumen fluid LF HF LF HF SE 0h ph Ammonia, mg/l Total VFA, mm h ph Ammonia, mg/l Total VFA, mm h ph Ammonia, mg/l Total VFA mm Table 3. Plasma concentrations of creatinine and urea, activity of liver enzyumes in plasma and urinary constituents in sheep fed low (LF) and high (HF) fibre diets with added lignin. LF HF LF HF SE Creatinine, FM Urea, mm GOT, U/L GGT, U/L ALT, U/L LDH, U/L Page 99

5 Table 4.Food consumption and 24 h urinary excretion of urea and ammonia in sheep fed low (LF) and high (HF) fibre diets with added lignin. LF HF LF HF SE Food consumption, g/w kg /d Urine volume, ml/d Urinary urea, mmol/mol creatinine Urinary ammonia mmol/mol of creatinine Page 100

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