In vitro investigation of the ruminal digestion kinetics of different nitrogen fractions of 15N-labelled timothy forage.

An in vitro method based on 15N-labelled forage nitrogen (N) was developed to study ruminal N metabolism of soluble N (SN), insoluble N (ISN) and neutral detergent insoluble N (NDIN) fractions of timothy forage. Timothy grass was grown on replicated experimental plots with one plot receiving 15N-labelled and the other unlabelled N fertilizer. Harvested grass was preserved as dried grass or as formic acid treated or untreated silage. The intact forages and their corresponding N fractions were incubated in buffered rumen fluid in vitro to determine degradation parameters based on the 15N fluxes between labelled feed N and ammonia N pools. A high percentage (25-38%) of 15N-labelled ammonia disappeared from ammonia N pool during the first 15 min of incubation. Microbial uptake of dried grass SN fraction was higher than of silage SN fractions. Fractional degradation rates of SN from formic acid treated silage, untreated silage and dried grass during the first 6 hours of incubation were 0.145, 0.125 and 0.115 /h, respectively. By the end of the incubation period (28 h), 69, 66 and 43%, of the SN fraction of formic acid treated silage, untreated silage and dried grass, respectively were recovered as ammonia. The percentage of ISN fractions degraded to ammonia N were 9, 34 and 27%, respectively. Based on the changes in 15N-labelled ammonia N pool in blank incubation and appearance of 15N to ammonia N pool from 15N-labelled NDIN fractions, it was estimated that a significant portion of microbial lysis occurred when incubations were carried out for longer than 20 hours. With dried grass the contribution of ammonia N for microbial N synthesis was greater than with silages. Use of 15N-labelled forages together with this in vitro method is a promising technique for determining soluble N degradation parameters, but it requires further development to be used for determining degradation parameters of insoluble N fractions and work with whole feeds.

Ruminal metabolism of grass silage soluble nitrogen fractions.

The present study was conducted to investigate ruminal N metabolism in dairy cows using 15N-labeled N sources and dynamic models. The data summarized in this study were obtained from 2 of 4 treatments whose effects were determined in a 4 × 4 Latin square design. Soluble N (SN) isolated from timothy grass silage labeled with 15N and ammonia N (AN) labeled with 15N were administered into the rumen contents of 4 ruminally cannulated dairy cows. Ruminal N pool sizes were determined by manual evacuation of rumen contents. The excess 15N-atom% was determined in N-fractions of rumen digesta grab samples that were collected frequently between 0 to 72 h and used to determine 15N metabolism in the rumen. Calculations of area under the curve ratios of 15N were used to estimate proportions of N fractions originating from precursor N pools. A model including soluble nonammonia N (SNAN), AN, bacterial N, and protozoal N pools was developed to predict observed values of 15N atomic excess pool sizes. The model described the pool sizes accurately based on small residuals between observed and predicted values. An immediate increase in 15N enrichment of protozoal N suggests physical attachment of bacteria pool to protozoa pool. The mean proportions of bacterial N, protozoal N, and feed N in rumen solid phase were 0.59, 0.20, and 0.21, respectively. These observations suggest that protozoal N accounted for 0.25 of rumen microbial N. About 0.90 of the initial dose of AN was absorbed or taken up by microbes within 2 h. Faster 15N enrichment of bacterial N with SN than with AN treatment indicates a rapid adsorption of SNAN to microbial cells. Additionally, the recovery of 15N as microbial and feed N flow from the rumen was approximately 0.36 greater for SN than for the AN treatment, indicating that SNAN was more efficiently used for microbial growth than AN. The present study indicated that about 0.15 of microbial N flowing to the duodenum was of protozoal origin and that 0.95 of the protozoal N originated from engulfed bacterial N. The kinetic variables indicated that 0.125 of SNAN escaped ruminal degradation, which calls into question the use of in situ estimations of protein degradation to predict the flow of rumen undegradable protein.

Predicting omasal flow of nonammonia N and milk protein yield from in vitro-determined utilizable crude protein at the duodenum.

This study evaluated the relationship between utilizable crude protein (uCP) at the duodenum estimated in vitro and omasal flow of crude protein (CP; omasal flow of nonammonia N × 6.25) measured in lactating dairy cows. In vivo data were obtained from previous studies estimating omasal digesta flow using a triple-marker method and 15N as microbial marker. A total of 34 different diets based on grass and red clover silages were incubated with buffered rumen fluid previously preincubated with carbohydrates for 3 h. The buffer solution was modified to contain 38 g of NaHCO3 and 1 g of (NH4)HCO3 in 1,000 mL of distilled water. Continuous sampling of the liquid phase for determination of ammonia-N was performed at 0.5, 4, 8, 12, 24, and 30 h after the start of incubation. The ammonia N concentrations after incubation were used to calculate uCP. The natural logarithm of uCP [g/kg of dry matter (DM)] at time points 0.5, 4, 8, 12, 24, and 30 h of incubation was plotted against time to estimate the concentration of uCP (g/kg of DM) at time points 16, 20, and 24 h using an exponential function. Fixed model regression analysis and mixed model regression analysis with random study effect were used to evaluate the relationships between predicted uCP (supply and concentration) and observed omasal CP flow and milk protein yield. Residual analysis was also conducted to evaluate whether any dietary factors influenced the relationships. The in vitro uCP method ranked the diets accurately in terms of total omasal CP flow (kg/d) or omasal CP flow per kilogram of DM intake. We also noted a close relationship between estimated uCP supply and adjusted omasal CP flow, as demonstrated by a coefficient of determination of 0.87, although the slope of 0.77 indicated that estimated uCP supply (kg/d) was greater than the value determined in vivo. The linear bias with mixed model analysis indicated that uCP supply overestimated the difference in omasal CP flow between the diets within a study, an error most likely related to study differences in feed intake, animals, and methodology. Predicting milk protein yield from uCP supply showed a positive relationship using a mixed model (coefficient of determination = 0.79), and we observed no difference in model fit between the time points of incubation (16, 20, or 24 h). The results of this study indicate that the in vitro method can be a useful tool in evaluating protein value of ruminant diets.

Effects of heat treatment on protein feeds evaluated in vitro by the method of estimating utilisable crude protein at the duodenum.

The effect of heat treatment on the protein value of field beans, lupins and peas was studied using an in vitro method. Protein feeds were subjected to heat treatment for 30, 60 and 90 min in forced air oven at 120, 140 and 160 °C and in autoclave at 105, 120 and 135 °C. The heat-treated protein feeds were incubated in buffered rumen fluid together with grass silage and barley in complete isonitrogenous diets. The gas production (GP) was recorded continuously, and ammonia-N (NH3 -N) concentrations were determined during the in vitro incubation at 8, 24 and 48 h and used to determine the utilisable crude protein (uCP) at the duodenum at 16 h of incubation (uCP16 ). Heat treatments decreased the concentration of soluble crude protein and increased neutral detergent insoluble CP (NDICP) in all protein feeds compared to untreated. Inclusion of protein feeds to basal diet showed no increase in the uCP16 in untreated field bean diet and only a small numerical increase in the uCP16 concentrations from 160 g/kg dry matter (DM) to 166 and 172 g/kg DM in untreated lupine and pea diets, respectively, indicating high degradability of untreated feeds. Increasing the time and temperature of the heat treatment linearly increased the uCP16 concentrations in field bean and pea diets, but not in lupin diets. Autoclave treatment was more effective in decreasing uCP16 than oven treatment despite the lower temperatures used. However, the combination of highest temperatures and treatment time in autoclave increased acid detergent insoluble CP (ADICP) concentrations in protein feeds, indicating protein damage and decreased intestinal digestibility. Determining in vitro uCP and ADICP shows to be a promising method for evaluating protein value in heat-treated animal feeds.