The estimation of feed protein degradability in the rumen using nylon bag, microbial labelling (S35) and plant labelling (N15) techniques.

Abstract

Animal Production in Australia Vol. 15 THE ESTIMATION OF FEED PROTEIN DEGRADABILITY IN THE RUMEN USING NYLON BAG, MICROBIAL LABELLING (S35) AND PLANT LABELLING (N15) TECHNIQUES P.G. CHAPMAN* and B.W. NORTON* SUMMARY Three techniques for estimating rumen protein degradability were used together with in vivo apparent nitrogen digestibility estimates from sheep fed dried chopped Pangola grass or Siratro. The techniques used were 1) nitrogen disappearance from nylon bags suspended in the rumen, 2) S labelling of rumen microbial protein with plant nitrogen degradability calculated as the difference between microbial and total non-ammonia nitrogen flows, using a correction for endogenous nitrogen, and 3) N labelled plant protein incorporating an endogenous nitrogen determination. Significant differences (P < 0.05) between the two feeds for rumen protein degradability estimates were shown for all three techniques. The nylon bag technique gave significantly higher estimates of protein degradability for Siratro (79.6%) compared with Pangola grass (68.3%), but for the two isotope techniques, Panola grass was of higher degradability than Siratro. Degradability estimated from 3 5S labelling gave significantly lower estimates (91.5% Pangola, 79.6 Siratro) than those determined by the 15N plant labelled technique (98.6%) Pangola, 95.5% Siratro). INTRODUCTION The Agricultural Research Council proposals for assessing protein requirements of ruminants (ARC 1980) requires an accurate estimate of the extent to which different dietary protein sources are degraded in the rumen. However, a suitable technique for the estimation of rumen protein degradability which will be easily adopted and standardised between laboratories and cover all feed types, has yet to be selected. Problems associated with interpretation of feed degradability in still exist primarily due to the current lack of standardisation of the with sample preparation, bag pore size, incubation time and basal diet It is still not known whether standardised degradability estimates will accurately with in vivo protein degradability in feeding ruminants. nylon bags technique variations. compare Isotope labelling techniques have proved useful for quantitatively determining the rate of microbial synthesis and the combined flows of undegraded and endogenous protein nitrogen (N) to the intestines. There is presently no established technique which permits the separation of endogenous and plant protein at the intestines, and consequently estimates of protein degradability must assume values for endogenous secretions. The following experiment used 15N labelled plant proteins to estimate the true degradability of two plant species, and these values were compared with the more conventional estimates of degradability, determined in the same experiments. MATERIALS AND METHODS Animals and Diets Three mature Border Leicester x Merino wethers fitted with rumen and abomasal T-piece cannulae were held in metabolism cages in a constant environment room, and were fed two diets in two separate experiments. The diets used were: 1) Pangola grass (Digitaria decumbens, Stent) harvested after five weeks' regrowth *Department of Agriculture, University of Queensland, St. Lucia, Qld 4067. 286 Animal Production in Australia Vol. 15 Table 1 Mean values for the intake, digestion and balance of nitrogen in sheep given Pangola grass and Siratro hay *Means differ significantly (P < 0.05) (16.6% crude protein), dried (60�C), and chopped (2-2.5 cm), and 2) Siratro (Macroptilium atropurpureum) was harvested at 11 weeks (15.1% crude protein) and processed in a similar way to the Pangola grass. The diets were offered ad lib from hourly feeders for five weeks during which an in vivo digestibility trial and isotope experiments were carried out. Experimental Methods Microbial protein synthesis and flow rate of di esta was determined by the continuous infusion of Na 35S04 (8.9 p Ci/lOO mls), 2 'Cr EDTA (4.6 1-1 Ci/lOO mls) and lo3Ru Phenanthroline if 1.9 p Ci/lOO mls) into the rumen through an indwelling probe. Abomasal samples were collected at six hourly intervals over the last two days of the infusion. Whole abomasal and an isolated abomasal microbial fraction (centrifugation) were stored at 20�C for the S analysis. The 15N labelled pangola grass and siratro were grown in hydroponic solutions (Hoaglands and Arnon 1950) in a controlled environment glasshouse. The 15N in the form of (15NH4)2 SO4 (96.6% enriched, Isocommerz, German Democratic Republic) was added to the culture solutions (1.17 moles 15N/1 of culture solution) to give an enrichment of 13.3% of the total culture N available to the plants. A total of 7.5 g of the (15NH4) SO4 was given to both plant species in order to obtain a calculated 10% enric 2ment in the harvested material. This plant material was harvested at a stage of growth corresponding to the field grown feed at harvest. Three sheep were given, in two separate periods, labelled feed replacing one of the hourly unlabelled feeds (30 g), and rumen fluid was sampled at frequent intervals over the following two days. Table 2 Mean values of the distribution of plant nitrogen between the ammonia pool and microbial protein synthesis * Means differ significantly (P < 0.05). 287 Animal Production in Australia Vol. I5 Table 3 .Mean values of rumen protein degradability estimates, using nylon bag, 35S and l5 N labelling techniques * Means differ significantly (P < 0.05) If: Assumed 2 g/d endogenous NAN to small intestine assumption in calculation (see Mather and Miller 1980). The ammonia pool size, recycling and disappearance from the rumen were measured by a single injection of (15NH4)2 S04, (100 mg in 20 mls/sheep, 96.6% enriched) into the rumen, three days before the 15N labelled plant material was fed. Analvtical Methods Microbial protein synthesis in the rumen was estimated from the 35 S labelling in freeze dried abomasal digesta and abomasal microbial fraction (Mathers and Miller 1980). Water and dry matter flows from the rumen were determined on the cabosil suspended whole abomasal digesta following counting of the 51Cr and lo3Ru (Packard Auto Scintillation Spectrometer). 15 N labelled rumen ammonia samples were prepared by steam distillation The with magnesium oxide and the l5N enrichment (atoms % excess) was measured on a mass spectrometer (MS20 AEI). Both the labelled ammonia and labelled feed injection dilution curves were analysed by the methods of Nolan and Leng (1974). The plant N at the abomasum was estimated by: Total abomasal 15N(mM/d) - Microbial flow 15N(mM/d) x 14 Plant N(mg/d) = 15N abundance in plant N (Atoms %) The endogenous N at the abomasum was calculated as the difference in abomasal nonammonia-nitrogen (NAN) not accounted for as plant or microbial N. The 15N labelled plant N rumen protein degradability estimate was calculated by: (Plant N intake - Plant N at abomasum) g/d x 1Oo Plant N intake (g/d) Degradability % = The nylon bag rumen protein degradability estimates were not corrected for rumen digesta dilution rates. These values are for masticated feeds and have been previously published (Chapman and Norton 1982). RESULTS The l5 N labelled Pangola grass and Siratro feeds were 11.20 and 2.62% 15N enriched, respectively. Table 1 shows mean values for N intake and balance for microbial protein synthesis and the distribution of abomasal NAN between endogenous and plant N. There was no significant difference between the two feeds in N intake or apparent N digestibility. Sheep given Pangola grass had a significantly higher total NAN flow (P < 0.05) at the abomasum than did the sheep given Siratro. The proportion of microbial NAN in the abomasal digesta NAN was significantly higher (P < 0.05) for the Pangola diet than the Siratro diet. Conversely, the amount of plant N at the abomasum was significantly higher for the Siratro diet than the Pangola diet. Endogenous N at the abomasum was similar for both plant species. The l5 N determinations of the rumen ammonia (NH3) pool recycling and irreversible loss (IRL) estimates are shown in Table 2. The recycling within the 288 Animal Production in Australia Vol. I5 However, the rumen NH 3 pool IRL was significantly higher (P < 0.05) for the Pangola grass- The Pangola grass values for the proportion of plant N passing through the rumen NH3 pool were not different from the Siratro. The proportion of rumen degraded plant N not appearing in the rumen ammonia pool (assumed to be directly incorporated as NAN into the microbial protein) was not significantly different for the Siratro, and the actual flows were not significantly different for the two feeds. A summary of the comparison between the two feeds using the three techniques is presented in Table 3. All three techniques showed a significant difference in rumen protein degradability estimates between the two feeds, however the nylon bag technique showed the Siratro to be of higher degradability than Pangola grass, while the two isotope techniques showed that Pangola grass was of higher degradability than Siratro. The 15N labelled plant N gave a comparatively high estimate of rumen protein degradability. DISCUSSION The estimates of endogenous protein N secretion found in our experiment (4.2 and 5.2 g N/kg dry matter intake (DMI)) are similar to those (5 g N/kg DMI) calculated by Kennedy and Milligan (1980). The values for the proportion of dietary N entering the rumen NH3 pool of 46 and 39% for the Pangola and Siratro are higher than the values of Nolan (1975) of 30% for dried diets. The rumen NH3 pool recycling rate was found to be 24% and 28% of the ammonia flux for the sheep given the Pangola and Siratro diets, this recycling rate represents a daily turnover of the microbial population of 26.9% in sheep given the Pangola grass diet and 48.7% for sheep given the Siratro diet. The forages used were at an early stage of growth and were considered to be of high quality. The estimation of degradability using either 35S or 15N gave much higher degradability estimates than with the nylon bag technique. The higher degradability estimates are consistent with the net loss of N across the rumen, high apparent N digestibilities and the high rumen NH3 concentrations. The nylon bag estimates were not only lower than that estimated by the isotope techniques but in the reverse order. It would seem that more studies using 15N labelled plant material need to be conducted to determine whether or not the nylon bag technique can rank feeds, let alone give quantitively accurate estimates of the true degradability of plant or feed proteins in the rumen. REFERENCES ARC (1980). 'Nutrient Requirements of Ruminant Livestock'. Comm. Ag. Bureaux Farnham Royal. CHAPMAN, P.G. and NORTON, B.W. (1982). Proc. Aust. Soc. Anim. Prod. 14: 580. HOAGLAND, D.R. and ARNON, D-1, (1950). California Agric. Exp. Station= Circular 347. KENNEDY, P.M. and MILLIGAN, L.P. (1980). Can. J. Anim. Sci. 60: 1029. MATHERS, J.C. and MILLER, E.L. (1980). Br. J. Nutr. 43: 503.= NOLAN, J.V. (1975). In 'Digestion and Metabolism in ge Ruminant' p. 416 editors 1-W. McDonald and A.C.1, Warner (Uni. of New England, Armidale). NOLAN, J.V. and LENG, R.A. (1974). Proc, Nutr. Soc. 33: 1. rumen ammonia pool did not differ for the two feeds. 289