Agricultural and Food Science in Finland, Vol. 11 (2002): 79–91 79 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 79–91. © Agricultural and Food Science in Finland Manuscript received February 2002 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 79–91. Concentration and estimated flow of soluble non-ammonia nitrogen entering the omasum of dairy cows as influenced by different protein supplements ChangWeon Choi, Aila Vanhatalo and Pekka Huhtanen MTT Agrifood Research Finland, Animal Production Research, FIN-31600 Jokioinen, Finland, e-mail: chang-weon.choi@mtt.fi Four ruminally fistulated Finnish Ayrshire cows were used to study the effects of different protein supplements on concentration and flow of soluble non-ammonia N (SNAN) into the omasum. The treatments in a 4 × 4 Latin square design were a basal diet of grass silage and barley and the basal diet supplemented with fishmeal, soybean meal and maize gluten meal. Protein supplements significantly increased concentrations of peptide N (P = 0.009) and total SNAN (P = 0.03) fractions in omasal digesta. Peptide constituted the largest proportion of SNAN flow into the omasum indicating that hydrolysis of peptides to amino acids is the most limiting step in rumen proteolysis. The microbial contribution to SNAN was on an average 0.64 indicating that a large proportion of SNAN flow leav- ing the rumen was of microbial origin. The estimated SNAN flow per kg dry matter intake from the basal diet and protein supplemented diets indicated that approximately 49, 22 and 37 g kg-1 of fish- meal, soybean meal and maize gluten meal protein, respectively, escaped from ruminal degradation as SNAN. Key words: soluble non-ammonia nitrogen, protein supplements, omasum, dairy cows, peptides Introduction Ruminal protein degradability is typically as- sessed by in situ method, which assumes that the rapidly degradable nitrogen (N) (a-fraction) is degraded at an infinite rate, and consequently that only insoluble feed N can escape ruminal degradation. However, relatively high concen- trations of soluble non-ammonia N (SNAN) con- sisting of free amino acid (AA), peptide and sol- uble protein, in rumen fluid (Chen et al. 1987a, Robinson and McQueen 1994) or omasal diges- ta (Choi et al. 2002a, b) suggest that a propor- tion of protein can escape rumen degradation in the liquid phase. Broderick (1987) reported us- ing in vitro system that the degradation rate of casein N was 0.40 – 0.60 h-1, also suggesting that a considerable portion of casein N can escape ruminal degradation. Consequently, the assump- tion of in situ method may be invalid. When skimmed milk powder was used as a protein sup- plement for dairy cows, the SNAN concentra- tion in omasal digesta was 110 mg N l-1 (Choi et mailto:chang-weon.choi@mtt.fi 80 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Choi, C.W. et al. Protein response to omasal soluble non-ammonia nitrogen al. 2002b). Assuming a rumen volume of 80 li- tres and a liquid passage rate of 0.15 h-1, approx- imately 32 g N d-1 of SNAN could potentially escape the rumen. It was calculated that 116 g kg-1 of skimmed milk powder N escaped rumi- nal degradation as SNAN in dairy cows fed a grass silage based diet (Choi et al. 2002b). How- ever, the value was not corrected for microbial contamination. Few dietary data on the concentration and the estimated flow of SNAN from the rumen and/or the omasal canal of ruminant animals have been reported. When dairy cows were giv- en grass silage based diet, inclusion of rape- seed meal increased the concentration of SNAN in the liquid phase of the omasal digesta (Choi et al. 2002a). Chen et al. (1987b) reported that high concentration of peptide N accumulated in the rumen when untreated soybean meal was given. However, when treated soybean meal was given the peptide N concentration de- creased even though the concentration was still relatively high (Chen et al. 1987b). Peptide N concentration varied when different protein supplements were given to steers, but it was poorly correlated with degradability and solu- bility of the supplements (Williams and Cock- burn 1991). More recently, our study (Choi et al. 2002b) also showed that omasal SNAN did not depend on the type of protein supplements although it was increased by all protein sup- plements. However, some of the supplements used in the experiment (skimmed milk powder and wet distiller’s solubles) are not commonly used in practice. The present experiment was designed to study effects of more widely used protein sup- plements on the concentration and the estimated flow of SNAN escaping ruminal degradation in dairy cows fed grass silage based diets. Further- more, because a proportion of SNAN in the liq- uid phase was suggested to be of microbial ori- gin (Choi et al. 2002a), we also determined the potential microbial contamination using 15N as a microbial marker. Data on nutrient flows and subsequent animal responses have been report- ed elsewhere (Korhonen et al. 2002). Material and methods Experimental procedures Four Finnish-Ayrshire dairy cows (mean live weight, 668 ± 109 kg; days in milk, 51 ± 6 days) fitted with 10-cm i.d. ruminal cannulas were used in a 4 × 4 Latin-square experiment with periods of 28 d including 4 d for omasal sampling. Dur- ing an adaptation period (14 d) dry matter (DM) intake of each cow was recorded. On day 15, DM intake was then restricted to 0.95 of the ad libi- tum intakes. The basal diet (kg kg-1 DM) con- tained grass silage (0.55) and rolled barley con- centrate (0.45) (Table 1). Part of DM intake of the basal diet (control) was isonitrogenously re- placed by one of protein supplements as follows; fishmeal (0.06) (diet FM), soybean meal (0.09) (diet SBM) or maize gluten meal (0.06) (diet MGM). Each protein supplement replaced por- tions of both silage and barley such that the ra- tio of grass silage to barley (55:45) remained constant for all treatments. Mineral and vitamin mixture was given at a rate of 300 g d-1. Grass prepared from secondary growth of swards con- taining predominately timothy (Phleum prat- ense) and meadow fescue (Festuca pratensis) and cocksfoot (Dactylis glomerata) was ensiled in a tower silo with a formic acid-based additive (AIV-2+; Kemira-Agro, Helsinki, Finland) at a rate of 5 l t-1 of the grass. Barley was coarsely milled using a roller mill prior to feeding, and all protein supplements and mineral and vitamin mixture were purchased from commercial sourc- es (Rehuraisio Ltd., Raisio; Suomen Rehu Ltd., Helsinki, Finland). The cows had a free access to water and salt block throughout the experi- ment. Feeds were offered twice daily at 0600 and 1800 and the cows were milked at 0700 and 1700. Sampling and chemical analyses Representative samples of grass silage and con- centrate were collected over the last 10 d of each 81 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 79–91. experimental period pooled over the period and stored at – 20ºC until analysis. Details of analy- sis for chemical composition of feeds have pre- viously been described by Ahvenjärvi et al. (2000). In brief, crude protein of feeds was de- termined using a Dumas-type N analyser (Leco FP-428; Leco Corporation, St Joseph, MI, USA). Soluble N content of silage was analysed using Kjeldahl method (Method No. 984.13; AOAC 1990). Neutral detergent fibre of feeds was as- sayed with α-amylase and sodium sulphite, and was expressed without residual ash (Van Soest et al. 1991). Soluble N fractions (non-protein N (NPN) and soluble true protein N) of feeds were prepared and analysed as described by Licitra et al. (1996). In the fractionation of the feed NPN, free AA and peptide N were determined using ninhydrin (Choi et al. 2002a) while ammonia N was analysed using a colorimetric method (McCullough 1967). Digesta flow into the omasum was estimated with a triple marker method (France and Siddons 1986) using indigestible neutral-detergent fibre, Yb-acetate and LiCo-EDTA as markers for large particle, small particle and liquid phase, respec- tively. Doses of LiCo-EDTA (18 g) and Yb-ace- tate (6 g) were given at 60 h before the first sam- pling time, and then the markers were continu- ously infused into the rumen (12 g d-1 of LiCo- EDTA and 4 g d-1 Yb-acetate). Microbial contri- bution to omasal SNAN was estimated using ammonium sulfate (Isotec Inc., Miamisburg, OH, USA) with 10% enrichment of 15N (250 mg 15N d-1 per cow) as a microbial marker. Infusion of the 15N-enriched ammonium sulfate was started at 48 h before the first sampling. To estimate the flow of SNAN fractions in the liquid phase of digesta, digesta entering the omasum was sampled according to the procedure described by Choi et al. (2002b). In brief, ap- proximately 30 ml of digesta was collected at 4- h intervals during a 12-h feeding cycle starting on day 25 of each period. On subsequent sam- pling days, the time of sampling was advanced by 1 h relative to the previous sampling day (i.e. totally 12 samples during 4 days). Details of the sample preparation have been described previ- ously (Choi et al. 2002a) with a modification that a portion of each supernatant of the omasal di- gesta deproteonised with trichloroacetic acid was prepared with 10 N NaOH to increase pH above 10 and incubated at 60ºC for 10 min to elimi- nate ammonia. Residual ammonia in the omasal digesta was analysed using a colorimetric meth- od (McCullough 1967). Different fractions (free AA, peptide and soluble protein) of SNAN with- in the omasal digesta were assessed using nin- hydrin (Choi et al. 2002a). In brief, each frac- Table 1. Proportion of dietary ingredients in experimental diet. Dieta Control FM SBM MGM Grass silage, kg kg-1 dry matter 0.55 0.52 0.50 0.52 Barley concentrate, kg kg-1 dry matter 0.45 0.42 0.41 0.42 Fishmeal, kg kg-1 dry matter 0.06 Soybean meal, kg kg-1 dry matter 0.09 Maize gluten meal, kg kg-1 dry matter 0.06 a Control = grass silage + barley; FM = grass silage + barley + fishmeal; SBM = grass silage + barley + soybean meal; MGM = grass silage + barley + maize gluten meal. Each diet contained 300 g d-1 of mineral and vitamin mixture (16% of Ca, 6.4% of P, 9.0% of Na, 8.0% of Mg, 150,000 IU kg-1 of vitamin A, 100,000 IU kg-1 of vitamin D, 950 mg kg-1 of vitamin E, 530 mg kg-1 of Cu, 20 mg kg-1 of Se, 4200 mg kg-1 of Zn, 20 mg kg-1 of Mo, 15 mg kg-1 of Co, 2250 mg kg-1 of Mn and 140 mg kg-1 of I). 82 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Choi, C.W. et al. Protein response to omasal soluble non-ammonia nitrogen tion of SNAN in the omasal sample was esti- mated as follows: i) free AA as N from superna- tant without acid-hydrolysis, ii) peptide as N from difference between hydrolysed supernatant (6 M HCl at 110ºC for 24 h) and free AA N and iii) protein as N from the hydrolysis of trichlo- roacetic acid-precipitate. The 15N-enrichement in the liquid phase of digesta entering the omasal canal was analysed as previously described by Choi et al. (2002a) with an exception that an oven (60ºC for 48 h) instead of a freeze-drying was used. Calculation and statistical analysis Since the 15N-enrichment of ammonia was not measured, a calculation for 15N-enrichment of the liquid phase was estimated assuming 0.70 of liq- uid associated bacteria (LAB)-NAN derived from ammonia N (Firkins et al. 1987). The de- tails of calculations have been described by Choi et al. (2002a). The rumen-escape of SNAN in the liquid phase of omasal digesta (eSNAN) from each pro- tein supplement was calculated as follows: (1) eSNAN = ( S N A N s u p p – S N A N b a s a l) / (NIsupp – NIbasal) where SNANsupp and NIsupp are the flow of SNAN in the liquid phase of omasal digesta and N intake for the protein supplemented diet, re- spectively, and SNANbasal and NIbasal are the flow of SNAN in the liquid phase of omasal digesta and N intake for grass silage and barley concen- trate in the protein supplemented diet, respec- tively. These are calculated as follows: (2) SNANbasal = SNANcont × DMIbasal / DMIcont where SNANcont, DMIbasal and DMIcont are the flow of SNAN in the liquid phase of omasal di- gesta for control diet, DM intake as grass silage and barley concentrate in the protein supplement- ed diet and DM intake in control diet excluding mixture of vitamin and mineral, respectively. Finally, (3) NIbasal = NIcont × DMIbasal / DMIcont where NIcont is N intake in control diet. Data for feed N intake, liquid flow and total NAN were analysed with the GLM procedure of SAS (1996) according to the following statisti- cal model: (4) Yijk = µ + Ai + Pj + Dk + eijk where A, P and D are animal, period, diet effects, respectively. Data obtained from ammonia N and concen- tration and flow of SNAN determined at each s a m p l i n g i n t e r v a l w e r e a n a l y s e d w i t h t h e MIXED procedure of SAS (1996) for repeated measures according to the following statistical model: (5) Yijkl = µ + Ai + Pj + Dk + eijk + Tl + (A×T)il + (P×T)jl + (D×T)kl + eijkl where T is time effect, and A×T, P×T and D×T are animal by time, period by time and di- ets by time interactions, respectively. Animal ef- fect, animal by time interaction and error terms (eijk defined as between unit error and eijkl as with- in unit error) are multivariate normally distrib- uted random effects with AR (1) covariance structure. Orthogonal contrasts used in post- ANOVA comparisons were as follows; 1) effect of protein supplement (control versus protein supplements), 2) comparison between animal and plant proteins (FM versus SBM + MGM) and 3) comparison between plant proteins (SBM versus MGM). Results Feed composition, intake and digesta flow into the omasal canal The chemical composition and the soluble N fractions of experimental feeds are shown in Table 2. Fishmeal had relatively high free AA, peptide and soluble protein N but extremely low 83 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 79–91. ammonia N concentration. Peptide N in soybean meal and maize gluten meal was rather similar, but soybean meal contained more soluble pro- tein N than maize gluten meal. Proportions of free AA in soluble N (NPN + soluble true pro- tein N) of feeds were 0.47, 0.07, 0.32, 0.03 and 0.24 for grass silage, barley, fishmeal, soybean meal and maize gluten meal, respectively, where- as peptide proportions were 0.41, 0.38, 0.25, 0.17 and 0.57, respectively. Over half of the feed sol- uble N was in the form of free AA and peptide except for barley and soybean meal. Grass si- lage was restrictively fermented as indicated by relatively low concentrations of total acids and a low proportion of ammonia N in total N. Table 3 shows the DM intake of experimen- tal feeds and liquid and total NAN flows into the omasal canal. The liquid flow into the oma- sal canal was not affected by dietary treatment (mean 200 l d-1). However, the liquid flow tend- ed to be higher for MGM than that for SBM (P = 0.08). Protein supplements increased total NAN flow entering the omasal canal (P = 0.05) mainly as a result of increased total dietary NAN flow (P < 0.001). Total NAN (P = 0.04) and total dietary NAN flow (P < 0.001) were higher in cows fed diet MGM than diet SBM. Soluble N entering the omasal canal Concentration Protein supplements significantly increased the concentration of ammonia N in omasal digesta (P = 0.006) (Table 4). The ammonia N concen- tration peaked at 2 h post-feeding (data not shown). Protein supplementation increased the ammonia N concentration compared to the con- trol diet throughout the feeding cycle. Protein supplements significantly increased the concen- trations of peptide (P = 0.009) and total SNAN (P = 0.03) fractions in omasal digesta. However, Table 2. Chemical composition of experimental feeds. Grass silage Barley Fishmeal Soybean meal Maize gluten meal Component, g kg-1 dry matter (DM) DM, g kg-1 222 888 909 912 917 Organic matter 914 973 881 940 969 Nitrogen 21 22 122 75 108 Neutral detergent fibre 506 189 134 112 60 Acid detergent fibre 257 39 0 59 7 Feed soluble N fractionsa, g kg-1 total N NPN 453 126 107 42 61 Ammonia 40.0 1.2 1.0 0.6 3.7 Free amino acid 219 20 60 6 17 Peptide 194 105 47 35 40 Soluble true protein N 15 151 80 163 9 Silage fermentation quality pH 4.00 Lactic acid, g kg-1 DM 12.2 Acetic acid, g kg-1 DM 9.77 Water soluble carbohydrate, g kg-1 DM 146 Soluble N, g kg-1 total N 458 a Non-protein N (NPN) and soluble true protein N of feeds were determined according to Licitra et al. (1996), and each fraction of NPN was analysed using ninhydrin assay (Choi et al. 2002a). 84 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Choi, C.W. et al. Protein response to omasal soluble non-ammonia nitrogen Table 3. Intake of dietary ingredients and effect of protein supplements on flow measurements into the omasal canal. Dieta Orthogonal contrastsc Control FM SBM MGM SEMb C1 C2 C3 Dry matter intake, kg d-1 Grass silage 10.9 10.7 10.2 10.5 Barley concentrate 8.2 7.3 7.6 7.5 Fishmeal 1.0 Soybean meal 1.8 Maize gluten 1.1 Mineral and vitamin mixture 0.3 0.3 0.3 0.3 Total 19.4 19.4 19.9 19.5 0.23 Nitrogen, g d-1 403 501 511 496 6.9 <0.001 0.69 0.17 Omasal canal flow Liquid, l d-1 210 199 182 209 9.1 0.25 0.75 0.08 Total NANd, g N d-1 358 396 368 413 11.2 0.05 0.95 0.04 Microbial NAN 246 232 226 221 9.5 0.12 0.46 0.75 Dietary NAN 112 164 142 192 4.6 <0.001 0.55 <0.001 a Control = grass silage + barley; FM = grass silage + barley + fishmeal; SBM = grass silage + barley + soybean meal; MGM = grass silage + barley + maize gluten meal. b SEM = standard error of the mean. c C1 = control vs. other diets; C2 = FM vs. SBM + MGM; C3 = SBM vs. MGM. d Total NAN = total non-ammonia nitrogen. Table 4. Effect of protein supplements on concentration (mg N l-1) of ammonia N, soluble non-ammonia N (SNAN), soluble microbial non-ammonia N (SMNAN) and soluble dietary non-ammonia N (SDNAN) in the liquid phase of digesta entering the omasal canal. Dieta Orthogonal contrastsc Control FM SBM MGM SEMb C1 C2 C3 Ammonia N 61.1 91.4 92.5 84.2 10.58 0.006 0.69 0.35 SNAN Free amino acids 13.5 16.9 16.8 18.9 3.73 0.37 0.84 0.69 Peptide 56.0 81.0 78.4 72.8 9.74 0.009 0.40 0.44 Protein 0.21 0.87 0.32 0.28 0.298 0.44 0.15 0.93 Total 69.8 98.8 95.5 92.0 11.30 0.03 0.61 0.76 SMNAN Total 50.0 55.1 56.4 52.2 8.64 0.55 0.92 0.65 SDNAN Total 19.8 43.7 39.1 39.7 5.94 0.02 0.56 0.94 a Control = grass silage + barley; FM = grass silage + barley + fishmeal; SBM = grass silage + barley + soybean meal; MGM = grass silage + barley + maize gluten meal. b SEM = standard error of the mean. c C1 = control vs. other diets; C2 = FM vs. SBM + MGM; C3 = SBM vs. MGM. 85 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 79–91. there were not significant differences in SNAN fractions entering the omasal canal between the protein supplements. Approximately 0.64 of SNAN in the liquid phase of digesta was of mi- crobial origin. Protein supplements did not af- fect microbial SNAN concentration whereas di- etary SNAN concentration was significantly in- creased by the supplements (P = 0.02). Flow Protein supplements tended to increase total SNAN (P = 0.12) and peptide N flow (P = 0.06) (Table 5). Approximately 0.80 of SNAN flow into the omasum was in the form of peptides, while free AA and protein N accounted for pro- portionately of 0.19 and 0.02 SNAN, respective- ly. The contribution of dietary SNAN to the to- tal SNAN was 0.28, 0.46, 0.43 and 0.44 for con- trol, FM, SBM and MGM diets, respectively. The dietary SNAN flow significantly increased when protein supplements were given (P = 0.05). Proportion of soluble N in total NAN flow Examination of individual nitrogenous fractions in SNAN indicated that the proportion in the form of peptide present in total NAN was mark- edly higher than the other two fractions (Table 6). Mean proportions of free AA, peptide, pro- tein and total SNAN were 8.6, 37.5, 0.23 and 46.3 g kg-1 total NAN, respectively. Based on 15N enrichments, the proportions of microbial SNAN and dietary SNAN were on average 28.0 and 18.4 g kg-1 total NAN, respec- tively. The proportion of microbial SNAN in to- tal microbial NAN was on an average 46.4 g kg- 1, whereas the proportion of dietary SNAN in total dietary NAN was 46.8 g kg-1. Protein sup- plements increased the proportion of dietary SNAN in total NAN flow (P = 0.03). Diurnal variation in SNAN Diurnal variations in SNAN and dietary SNAN only tended to be influenced by diet × time in- teraction (P < 0.10). Mean diurnal pattern of SNAN fractions for the experimental diets are shown in Fig. 1. Mean peptide N peaked at 1 – 3 h post-feeding and declined thereafter, while the peak of mean free AA was shown only at 1 h post-feeding. Soluble protein N concentration was very low and remained relatively constant throughout the feeding cycle. Diurnal pattern of free AA (P = 0.02) and peptide (P = 0.005) frac- Table 5. Effect of protein supplements on flow (g N d-1) of soluble non-ammonia N (SNAN), soluble microbial non- ammonia N (SMNAN) and soluble dietary non-ammonia N (SDNAN) in the liquid phase of diegeta entering the omasal canal. Dieta Orthogonal contrastsc Control FM SBM MGM SEMb C1 C2 C3 SNAN Free amino acids 2.93 3.59 3.12 3.73 0.976 0.64 0.90 0.67 Peptide 11.8 16.1 13.5 14.3 1.17 0.06 0.15 0.62 Protein 0.05 0.15 0.06 0.06 0.048 0.45 0.15 0.96 Total 14.7 19.8 16.7 18.1 1.73 0.12 0.29 0.59 SMNAN Total 10.7 10.6 9.6 10.1 1.12 0.69 0.58 0.74 SDNAN Total 4.08 9.16 7.11 7.96 1.403 0.05 0.37 0.67 a Control = grass silage + barley; FM = grass silage + barley + fishmeal; SBM = grass silage + barley + soybean meal; MGM = grass silage + barley + maize gluten meal. b SEM = standard error of the mean. c C1 = control vs. other diets; C2 = FM vs. SBM + MGM; C3 = SBM vs. MGM. 86 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Choi, C.W. et al. Protein response to omasal soluble non-ammonia nitrogen tions of SNAN were affected by the diet as indi- cated by significant diet × time interaction. At 1 h post-feeding diet MGM had the highest (74.8 mg N l-1) concentration of free AA followed by diet SBM (Fig. 2). Free AA concentration for control and FM diets were higher at 0 h than in samples taken at 1 h post-feeding, and those for the other diets were also relatively high. Pep- tide N concentration in the liquid phase of di- gesta entering the omasum remained to be high- er for protein supplemented-diets than that for control diet during the feeding cycle (Fig. 3). Table 6. Effect of protein supplements on the proportion (g kg-1) of soluble non-ammonia N (SNAN), soluble microbial non-ammonia N (SMNAN) and soluble dietary non-ammonia N (SDNAN) in the liquid phase of omasal digesta in total non-ammonia N (NAN). Dieta Orthogonal contrastsc Control FM SBM MGM SEMb C1 C2 C3 SNAN in total NAN Free amino acids 7.9 8.6 8.6 9.3 1.76 0.66 0.89 0.77 Peptide 33.8 40.7 39.6 35.8 5.30 0.15 0.37 0.33 Protein 0.1 0.5 0.2 0.1 0.16 0.52 0.16 0.93 Total 41.9 49.8 48.4 45.2 5.97 0.22 0.54 0.58 SMNANd Proportion in total NAN 30.0 27.4 28.8 25.6 4.64 0.48 0.97 0.50 Proportion in TMNAN 43.5 46.8 46.3 48.8 7.64 0.55 0.90 0.74 SDNANd Proportion in total NAN 11.9 22.4 19.6 19.6 2.89 0.03 0.41 1.00 Proportion in TDNAN 39.0 54.3 52.1 41.8 7.72 0.21 0.39 0.30 a Control = grass silage + barley; FM = grass silage + barley + fishmeal; SBM = grass silage + barley + soybean meal; MGM = grass silage + barley + maize gluten meal. b SEM = standard error of the mean. c C1 = control vs. other diets; C2 = FM vs. SBM + MGM; C3 = SBM vs. MGM. d Proportions of SMNAN and SDNAN expressed as g kg-1 total microbial NAN (TMNAN) and g kg-1 total dietary NAN (TDNAN), respectively. Fig. 1. Diurnal variations of ni- trogenous fractions of soluble non- ammonia N (SNAN) in the liquid phase of digesta entering the omasum during a 12 h feeding cy- cle (� = free amino acid; � = pep- tide; � = protein; × = total SNAN; standard error of the mean for the free amino acid, peptide, protein and total SNAN were 4.87, 9.36, 0.28 and 11.25, respectively). 87 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 79–91. Peptide N concentration in omasal digesta peak- ed at 1 h (diet SBM), 2 h (control and FM diets) and 3 h (diet MGM) post-feeding and declined to the pre-feeding level. Discussion Contribution of microbial N to total SNAN Previous studies have neglected the potential microbial contamination of SNAN (Chen et al. 1987a, b, Broderick and Wallace 1988). How- ever, extracellular AAs are probably excreted by rumen bacteria and protozoa cells or released during the cell lysis (Nolan 1993). Our recent study using 15N as a microbial marker suggested that a high proportion of SNAN in the liquid phase of digesta entering the omasal canal was of microbial origin (Choi et al. 2002a). It may also be partly explained by high concentration of glutamic acid, as an intermediate of bacterial AA metabolism, in free AA fraction of SNAN in omasal digesta (Choi et al. 2002a). In the present study, 15N was also used to es- timate microbial contamination of the liquid phase of digesta. The NA15N was assumed to be entirely derived from liquid associated bacteria because engulfed LAB-NAN is spilled outside of protozoa cells (Jouany et al. 1988). The Fig. 3. Influence of dietary treat- ment on the extent of diurnal var- iation in peptide N of the liquid phase of digesta entering the omasum during a 12 h feeding cycle (� = grass silage + barley; � = grass silage + barley + fish- meal; � = grass silage + barley + soybean meal; × = grass silage + barley + maize gluten meal; stand- ard error of the mean for peptide N was 11.7). Fig. 2. Influence of dietary treat- ment on the extent of diurnal var- iation in free amino acid (AA) N of the liquid phase of digesta en- tering the omasum during a 12 h feeding cycle (� = grass silage + barley; � = grass silage + barley + fishmeal; � = grass silage + barley + soybean meal; × = grass silage + barley + maize gluten meal; standard error of the mean for free AA N was 7.98). 88 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Choi, C.W. et al. Protein response to omasal soluble non-ammonia nitrogen present microbial contribution to SNAN in the liquid phase of digesta (mean 0.64) is consistent with that of 0.63 – 0.86 (Hristov and Broderick 1 9 9 6 ) a n d o f 0 . 6 1 ( C h o i e t a l . 2 0 0 2 a ) . The proportion of microbial contribution to the SNAN could, however, be overestimated as microbial lysis could have occurred during acid treatment of digesta and freezing after sam- pling. Here, the microbial contribution to SNAN was similar to the microbial contribution to to- tal NAN flow (mean 0.61). Besides, neither mi- crobial NAN nor microbial SNAN were signifi- cantly affected by the supplements, supporting the previous result that rapeseed meal supple- mentation had no influence on microbial N flow in dairy cows (Ahvenjärvi et al. 1999). An in- crease of dietary SNAN by protein supplements may indicate that release of dietary N in the form of peptide and/or protein exceeds the proteoly- sis in the rumen. Consequently the peptide N could have an opportunity to escape the rumen and be absorbed in the small intestine as AA source by the host animal. Soluble NAN Protein supplements Robinson et al. (1998) using dairy cows fed tim- othy silage, whole-crop barley silage and a mixed concentrate showed that protein supplements gave no significant increase in peptide N con- centration in the liquid phase of ruminal digesta (mean 83.6 and 83.0 mg N l-1 for without and with protein supplements, respectively). In the present study, peptide N concentration was, how- ever, increased by protein supplements (mean 56.0 and 77.4 mg N l-1). This is consistent with a considerable increase in peptide N flow from the rumen when the cows were fed grass silage based diets received protein supplements (Choi et al. 2002a, b). Soybean meal has produced a higher SNAN concentration than other protein supplements e.g. maize gluten meal and/or blood meal (Robinson 1997) and fishmeal (Chen et al. 1987b) but not always (Williams and Cockburn 1991). Peptide N concentration in ruminal digesta at 1 h post- feeding was much higher for maize gluten meal than for fishmeal supplement (Williams and Cockburn 1991). However, the present concen- tration of peptide N and/or total SNAN was rath- er similar between protein supplemented-diets, even though the soluble N composition differed in protein feeds. Different peptide N concentra- tions on similar protein feeds between the stud- ies could be influenced by variation in the qual- ity of protein feeds, e.g. it is well known that the quality and composition of fishmeal can be different depending on a fish-drying and adding antioxidants (Mehrez et al. 1980). In addition, a rate of slowly degradable N fraction (b-fraction) is very different for experimental supplements, e.g. a rate (% h-1) of b-fraction of soybean meal (9.4) is the fastest rate and that of maize gluten meal (2.3) is the slowest (NRC 2001). The sim- ilar peptide N concentration for the protein sup- plemented-diets supports the claim that peptide N concentration is poorly correlated with degrad- ability and solubility of protein feeds (Williams and Cockburn 1991). Many studies have also shown that there was no difference in peptide N and SNAN concentration between diets contain- ing different type of protein supplements (Chen et al. 1987b, Robinson and McQueen 1994, Choi et al. 2002b). Here, protein supplements were replaced not by barley concentrate but by a part of control diet in order to keep grass silage to barley ratio (55:45) constant. However, the actual ratio of DM intake failed to keep the ratio planned (see Table 3), but it was still similar between the di- ets (57:43). The purpose of this was to allow to estimate SNAN flow in the liquid phase of oma- sal digesta per kg DM intake from the basal diet, and then calculate eSNAN from each protein supplement. Based on the equations 1–3, eSNAN was 49, 22 and 37 g kg-1 of fishmeal, soybean meal and maize gluten meal, respectively. Daily intake of soluble NAN in feed N of each protein supplement, calculated as soluble NAN in total N of protein feed × daily N intake of protein feed, appeared to be approx. 23, 28 89 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 11 (2002): 79–91. and 8 g N d-1 for fishmeal, soybean meal and maize gluten meal, respectively. Overall, the eSNAN increased by each pro- tein feed does not seem to be subjected to the intake of soluble NAN in protein feeds. The present results support our previous observation that the eSNAN is not related to a-fraction of in situ determination, and effective protein degrad- ability can not necessarily be estimated as a sum of the escape calculated from a- and b-fractions of in situ determination (Choi et al. 2002b). Metabolism of SNAN Wallace and McKain (1990) reported that a col- ourimetric method using ninhydrin does not give estimate of peptide concentration reliably be- cause of extremely high ammonia concentration in acid-hydrolysates after alkaline-heating. We have also observed markedly high ammonia in samples after the acid-hydrolysis (Choi et al. 2002a). However, our preliminary analysis showed that proportionately 0.99 of ammonia in the pre-hydrolysis samples was eliminated by the alkaline-heating method. Therefore, in the present study, SNAN obtained in the acid-hy- drolysates was corrected for ammonia N concen- tration determined in the pre-hydrolysis. In the present study, markedly higher pep- tide N in total SNAN than the other two frac- tions is consistent with the previous observation of high peptide N in ruminal fluid (Chen et al. 1987a, Choi et al. 2002b) and in omasal fluid (Choi et al. 2002a, b). Free AA concentration in ruminal digesta is relatively low (Williams and Cockburn 1991) even during the period imme- diately post-feeding (see Nolan 1993). Howev- er, in the present study, free AA concentration in omasal digesta was relatively high immedi- ately post-feeding. On an average, the present free AA concentration is consistent with our pre- vious results (mean 15.3 mg N-1) (Choi et al. 2002a). In the diurnal pattern of free AA in the present study, the reason for the lacks of the peaks for control and FM diets was unclear (Fig. 2). However, although many studies reported clear peaks in free AA concentration (Broderick and Wallace 1988, Choi et al. 2002b), some pro- tein supplements produced the constant diurnal pattern without peaks in free AA concentration (Choi et al. 2002a). Extremely low soluble pro- tein fraction of SNAN in the present study is consistent with other studies, in which different protein supplements were given (Williams and Cockburn 1991, Choi et al. 2002b). The low sol- uble protein N could be explicated by that solu- ble protein in feed N is rapidly degraded to non- precipitable peptides (Choi et al. 2002b). Parti- tioning of SNAN fractions may be less impor- tant as regards to determination of the supply of AA from soluble N fraction, since all N frac- tions are assumed to be completely digested in the small intestine. Concentrations of peptide N varied between 82 and 111 mg N l-1 (Chen et al. 1987a, b, Rob- inson and McQueen 1994, Robinson et al. 1998). The present peptide N concentration in omasal fluid (mean 72 mg N l-1) was marginal- ly lower than the values reported previously. However, the previous peptide concentrations included free AA (Robinson and McQueen 1994, Robinson et al. 1998) since free AA frac- tion was not determined before hydrolysis. Tak- ing this into account, the sum of peptide and free AA concentration for control, FM, SBM and MGM was 70, 98, 95 and 92 mg N l-1, re- spectively, estimates that are in good agreement with the reported values. Our most recent study showed that peptide N concentration excluding free AA in omasal digesta was 54 – 64 mg N l- 1 when protein supplements were given to cows (Choi et al. 2002b). Diurnal pattern in ruminal or omasal peptide concentration (and/or total SNAN) that peaked immediately post-feeding and declined thereafter has been observed in many studies (Chen et al. 1987a, Williams and Cockburn 1991, Robinson et al. 1998, Choi et al. 2002a, b). When ryegrass hay and maize based concentrate were fed with urea or oval- bumin diurnal pattern in peptide concentration in the rumen was rather constant (Broderick and Wallace 1988). In their study, however, rumi- nal peptide N reached a maximum immediate- ly post-feeding, when the diet was supplement- ed with casein. 90 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Choi, C.W. et al. Protein response to omasal soluble non-ammonia nitrogen Conclusions Present data confirm the previous observation (Choi et al. 2002a) that a substantial proportion of SNAN in the liquid phase of omasal digesta can escape ruminal degradation. Protein supple- ments increased peptide N and total SNAN frac- tions in the liquid phase of omasal digesta, whereas there were not differences in the SNAN concentration between different protein supple- ments. The omasal SNAN provided by protein feeds was not equated with soluble N the pro- tein feeds. Quantitatively peptides rather than free AA or soluble protein were the most impor- tant N fraction of SNAN in the liquid phase of digesta. The potential microbial contribution to SNAN in omasal digesta using 15N as a microbi- al marker suggested that the SNAN was substan- tially contaminated by microbes. Acknowledgements. The authors thank Mrs A. Matilainen and her staff for care of experimental animals and Mr V. Toivonen and his laboratory staff for chemical analyses. C.W. Choi appreciates the financial support of the Agricul- tural Research Foundation of August Johannes and Aino Tiura. References Ahvenjärvi, S., Vanhatalo, A., Huhtanen, P. & Varvikko, T. 1999. Effects of supplementation of a grass silage and barley diet with urea, rapeseed meal and heat- moisture-treated rapeseed cake on omasal digesta flow and milk production in lactating dairy cows. Acta Agriculturæ Scandinavica Section A, Animal Science 49: 179–189. Ahvenjärvi, S., Vanhatalo, A., Huhtanen, P. & Varvikko, T. 2000. Determination of reticulo-rumen and whole- stomach digestion in lactating cows by omasal canal or duodenal sampling. British Journal of Nutrition 83: 67–77. AOAC 1990. Official methods of Analysis. 15th ed. As- sociation of Official Analytical Chemists, AOAC, Ar- lington, VA. USA. Broderick, G.A. 1987. Determination of protein degrada- tion rates using a rumen in vitro system containing inhibitors of microbial nitrogen metabolism. British Journal of Nutrition 58: 463–475. Broderick, G.A. & Wallace, R.J. 1988. Effects of dietary nitrogen source on concentrations of ammonia, free amino acids and fluorescamine-reactive peptides in the sheep rumen. Journal of Animal Science 66: 2233–2238. Chen, G., Russell, J.B. & Sniffen, C.J. 1987a. A proce- dure for measuring peptides in rumen fluid and evi- dence that peptide uptake can be a rate-limiting step in ruminal protein degradation. Journal of Dairy Sci- ence 70: 1211–1219. 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SELOSTUS Valkuaistäydennyksen vaikutus lypsylehmän pötsistä virtaavan liukoisen rehuperäisen typen pitoisuuteen ja määrään säilörehuruokinnalla Chang Weon Choi, Aila Vanhatalo ja Pekka Huhtanen MTT (Maa- ja elintarviketalouden tutkimuskeskus) Valkuaistäydennyksen vaikutusta lypsylehmän pötsis- tä virtaavan liukoisen rehuperäisen typen (N) pitoi- suuteen ja määrään tutkittiin 4 × 4 latinalaisen neli- ön koemallin mukaisessa kokeessa. Koe-eläiminä oli neljä pötsifistelöityä lypsylehmää, jotka saivat kont- rolliruokinnalla nurmisäilörehua ja ohraa siten, että syönti oli 95 % vapaasta syönnistä ja säilörehun ja ohran suhde oli 55:45. Muilla ruokinnoilla perusre- huja korvattiin kalajauholla (6 %), soijarouheella (9 %) ja maissigluteenilla (6 %) siten, että ohran ja säilörehun suhteelliset osuudet pysyivät vakioina ja valkuaistäydennyksenä tulevan typen määrä oli sama kaikilla valkuaisrehuilla. Valkuaistäydennys lisäsi pötsistä virtaavan pep- tidi-N:n ja liukoisen rehuperäisen N:n pitoisuuksia satakerran ruokasulassa, mutta erot valkuaisrehujen välillä olivat pieniä. Peptidi-N muodosti suurimman osan liukoisesta rehuperäisestä N:stä osoittaen, että peptidien hydrolyysi aminohapoiksi on rajoittavin vaihe pötsin proteolyysissä. Mikrobi-N:n osuus liu- koisesta rehuperäisestä N:stä oli 0,64 osoittaen, että suuri osa satakertaan virtaavasta rehuperäisestä liu- koisesta N:stä oli mikrobista alkuperää. Laskelma liu- koisen rehuperäisen N:n virtauksesta syötyä kuiva- ainekiloa kohti osoitti, että kalajauhosta n. 49 g kg-1, soijarouheesta n. 22 g kg -1 ja maissigluteenista n. 37 g kg-1 virtasi ulos pötsistä hajoamatta liukoi- sessa muodossa. Title Introduction Material and methods Results Discussion Conclusions References SELOSTUS