Forage; Microbial contamination; Nylon bag; Protein degradability; Rumen
Abstract :
[en] The use of stable15N as a marker to determine microbial contamination in nylon bag incubation residues to estimate protein degradability was investigated. Three methods using15N were compared:15N-labeled forage (dilution method, LF),15N enrichment of rumen solids-associated bacteria (SAB), and 15N enrichment of rumen liquid-associated bacteria (LAB). Herbage from forages differing in protein and fiber contents (early-cut Italian ryegrass, late-cut Italian ryegrass, and red clover) were freeze-dried and ground and then incubated in situ in the rumen of 3 steers for 3, 6, 12, 24, and 48 h using the nylon bag technique. The15N-labeled forages were obtained by fertilizing the plots where herbage was grown with15NH4 15NO3. Unlabeled forages (obtained from plots fertilized with NH4NO3) were incubated at the same time that (15NH4)2SO4 was continuously infused into the rumen of the steers, and then pellets of labeled SAB and LAB were isolated by differential centrifugation of samples of ruminal contents. The proportion of bacterial N in the incubation residues increased from 0.09 and 0.45 g bacterial N/g total N at 3 h of incubation to 0.37 and 0.85 g bacterial N/g total N at 48 h of incubation for early-cut and late-cut ryegrass, respectively. There were differences (P < 0.001) between uncorrected N degradability values and those corrected for microbial contamination with all of the methods. Apparent N degradability of the low-N, high-fiber forage (latecut ryegrass) was 0.51, whereas the corrected values were 0.85, 0.84, and 0.77 for the LF, SAB, and LAB methods, respectively. With early-cut ryegrass and red clover, the differences between uncorrected and corrected values ranged between 6% and 13%, with small differences among the labeling methods. Generally, methods using labeled forage or labeled SAB and LAB provided similar corrected degradability values. The accuracy in estimating the extent of degradation of protein in the rumen from in situ disappearance curves is improved when values are corrected for microbial contamination of the bag residue.
Disciplines :
Animal production & animal husbandry
Author, co-author :
Kamoun, Mohamed; Département Sciences Agronomiques, Animal Science Unit, Gembloux Agro Bio-Tech, Université de Liège, Passage Deportes 2Gembloux, Belgium, Ecole Nationale de Médecine Vétérinaire, Département des Productions AnimalesSidi Thabet, Tunis, Tunisia
Ammar, H.; Ecole Supérieure d’Agriculture de MograneMograne-Zaghouan, Tunisia
Thewis, André ; Université de Liège - ULiège > Sciences agronomiques > Zootechnie
Alexandrov, A. N. 1998. Effect of ruminal exposure and subsequent microbial contamination on dry matter and protein degradability of various feedstuffs. Anim. Feed Sci. Technol. 71:99–107.
AOAC. 1999. Official methods of analysis. 16th ed. Assoc. Off. Anal. Chem, Gaithersburg, MD.
Beckers, Y., A. Thewis, B. Maudoux, and E. François. 1995. Studies on the in situ nitrogen degradability corrected for bacterial contamination of concentrate feeds in steers. J. Anim. Sci. 73:220–227.
Belanche, A., G. de la Fuente, E. Pinloche, C. J. Newbold, and J. Balcells. 2012. Effect of diet and absence of protozoa on the rumen microbial community and on the representativeness of bacterial fractions used in the determination of microbial protein synthesis. J. Anim. Sci. 90:3924–3936.
Bremner, J. 1965. Isotope ratio analysis of nitrogen-15 tracer investigations. In: C. Black, editor, Methods of soil analysis, part 2. Agronomy No. 9. Am. Soc. Agron., Madison, WI. p. 1256–1286.
Broderick, G. A., and N. R. Merchen. 1992. Markers for quantifying microbial protein synthesis in the rumen. J. Dairy Sci. 75:2618–2632.
Carro, M. D., and E. L. Miller. 2002. Comparison of microbial markers (15N and purine bases) and bacterial isolates for the estimation of rumen microbial protein synthesis. Anim. Sci. 75:315–321.
Cecava, M. J., N. R. Merchen, L. C. Gay, and L. L. Berger. 1990. Composition of ruminal bacteria harvested from steers as influenced by dietary energy level, feeding frequency, and isolation techniques. J. Dairy Sci. 73:2480–2488.
Colombini, S., and G. A. Broderick. 2008. In vitro ruminal protein degradation and microbial protein formation of seed legumes. J. Dairy Sci. 91(Suppl. 1):43.
Craig, W. M., D. R. Brown, G. A. Broderick, and D. B. Ricker. 1987. Post-prandial compositional changes of fluid and particle-associated ruminal microorganisms. J. Anim. Sci. 65:1042–1048.
Dixon, R. M., and S. Chanchai. 2000. Colonization and source of N substrates used by microorganisms digesting forages incubated in synthetic fiber bags in the rumen. Anim. Feed Sci. Technol. 83:261–272.
Edmunds, B., K.-H. Sudekum, H. Spiekers, and F. Schwarz. 2012. Estimating ruminal crude protein degradation of forages using in situ and in vitro techniques. Anim. Feed Sci. Technol. 175:95–105.
Elliott, R., B. W. Norton, and C. W. Ford. 1985. In vivo colonization of grass cell walls by rumen microorganisms. J. Agric. Sci. 105:279–283.
Firkins, J. L., S. M. Lewis, L. Montgomery, L. L. Berger, N. R. Merchen, and J. R. Fahey. 1987. Effects of feed intake and dietary urea concentration on ruminal dilution rate and efficiency of bacterial growth in steers. J. Dairy Sci. 70:2312–2321.
France, J., J. H. M. Thornley, S. López, R. C. Siddons, M. S. Dhanoa, P. J. Van Soest, and M. Gill. 1990. On the two-compartment model for estimating the rate and extent of feed degradation in the rumen. J. Theor. Biol. 146:269–287.
González, J., J. M. Arroyo, M. Ouarti, J. Guevara-González, C. A. Rodríguez, M. R. Alvir, V. J. Moya, and O. Piquer. 2012. Composition of free and adherent ruminal bacteria: Inaccuracy of the microbial nutrient supply estimates obtained using free bacteria as reference samples and 15N as the marker. Animal 6:468–475.
González, J., C. Centeno, N. Morujo, J. Faria-Marmol, and A. Martínez. 2009. In situ ruminal amino acid degradability of green and ensiled Italian rye-grass according to particle transit model and microbial contamination correction. Livest. Sci. 123:209–214.
González, J., J. Farià-Màrmol, C. A. Rodríguez, and A. Martínez. 2007. Effects of ensiling on ruminal degradability and intestinal digestibility of Italian rye-grass. Anim. Feed Sci. Technol. 136:38–50.
González, J., M. Ouarti, C. A. Rodríguez, and M. R. Alvir. 2006. Effects of considering the rate of comminution of particles and microbial contamination on accuracy of in situ studies of feed protein degradability in ruminants. Anim. Feed Sci. Technol. 125:89–98.
Ipharraguerre, I. R., S. M. Reynal, M. Lineiro, G. A. Broderick, and J. H. Clark. 2007. A comparison of sampling sites, digesta and microbial markers, and microbial references for assessing the postruminal supply of nutrients in dairy cows. J. Dairy Sci. 90:1904–1919.
Jarrige, R. 1987. Ingestion et digestion des aliments. In: R. Jarrige, editor, Alimentation des bovins, ovins et caprins. INRA Publ., Paris. p. 29–56.
Kamoun, M., S. López, Y. Beckers, P. Lecomte, E. Francois, and A. Théwis. 2007. Effect of feed 15N incorporation into solid-associated bacteria on the in situ nitrogen degradability of 15N labelled Italian ryegrass. Anim. Feed Sci. Technol. 135:353–361.
Kamoun, M., and A. Thewis. 1990. Influence du mode de conditionnement d’un fourrage vert sur sa composition chimique, la digestibilité in vitro de la matière organique et dégradabilité in sacco de l’azote dans le rumen. Reprod. Nutr. Dev. 30 (Suppl. 2):159s–160s.
Kamoun, M., A. Théwis, L. Couvreur, and E. François. 1993. Etude de la contamination microbienne et de dégradabilité ruminale in sacco des matières azotées de 2 variétés de ray-grass italien marquées au moyen de 15N et récoltées à 2 stades végétatifs. Ann. Zootech. 42:122–123.
Klopfenstein, T. J., R. A. Mass, K. W. Creighton, and H. H. Patterson. 2001. Estimating forage protein degradation in the rumen. J. Anim. Sci. 79(Suppl.):E208–E217.
Krawielitzki, K., T. Schmidt, J. Voigt, J. Kowalczyk, and M. Gabel. 2006. Dynamics of microbial contamination of protein during ruminal in situ incubation of feedstuffs. J. Anim. Feed Sci. 15:313–328.
Legay-Carmier, F., and D. Bauchart. 1989. Distribution of bacteria in the rumen contents of dairy cows given a diet supplemented with soya-bean oil. Br. J. Nutr. 61:725–740.
Lindberg, J. E. 1985. Estimation of rumen degradability of feed proteins with in sacco technique and various in vitro methods. Acta Agric. Scand. 25(Suppl.):64–69.
Lindberg, J. E., A. Kaspersson, and P. Ciszuk. 1984. Studies on pH, number of protozoa and microbial ATP concentrations in rumen incubated nylon bags with different pore size. J. Agric. Sci. 102:501–504.
López, S. 2005. In vitro and in situ techniques for estimating digestibility. In: J. Dijkstra, J. M. Forbes, and J. France, editors, Quantitative aspects of ruminant digestion and metabolism. 2nd ed. CAB Int., Wallingford, UK. p. 87–121.
Makkar, H. P. S., B. Singh, and S. S. Negi. 1989. Relationship of rumen degradability with microbial colonization, cell wall constituents and tannin levels in some tree leaves. Anim. Prod. 49:299–303.
Mass, R. A., G. P. Lardy, R. J. Grant, and T. J. Klopfenstein. 1999. In situ neutral detergent insoluble nitrogen as a method for measuring forage protein degradability. J. Anim. Sci. 77:1565–1571.
Mathers, J. C., and E. M. Aitchison. 1981. Direct estimation of the extent of contamination of food residues by microbial matter after incubation within synthetic fiber bags in rumen. J. Agric. Sci. 96:691–693.
Mathis, C. P., R. C. Cochran, E. S. Vanzant, I. E. O. Abdelgadir, J. S. Heldt, K. C. Olson, D. E. Johnson, J. Caton, D. B. Faulkner, G. Horn, S. Paisley, R. Mass, K. Moore, and J. Halgerson. 2001. A collaborative study comparing an in situ protocol with single time-point enzyme assays for estimating ruminal protein degradability of different forages. Anim. Feed Sci. Technol. 93:31–42.
Olubobokum, J. A., and W. M. Craig. 1990. Quantity and characteristics of micro-organisms associated with ruminal fluid or particles. J. Anim. Sci. 68:3360–3370.
Olubobokum, J. A., W. M. Craig, and W. A. Nipper. 1988. Characteristics of protozoal and bacterial fractions from microorganisms associated with ruminal fluid or particles. J. Anim. Sci. 66:2701–2710.
Ørskov, E. R., and I. McDonald. 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92:499–503.
Pérez, J. F., C. A. Rodríguez, J. González, J. Balcells, and J. A. Guada. 1996. Contribution of dietary purine bases to duodenal digesta in sheep. In situ studies of purine degradability corrected for microbial contamination. Anim. Feed Sci. Technol. 62:251–262.
Ranilla, M. J., and M. D. Carro. 2003. Diet and procedures used to detach particle-associated microbes from ruminal digesta influence chemical composition of microbes and estimation of microbial growth in Rusitec fermenters. J. Anim. Sci. 81:537–544.
Reynal, S. M., G. A. Broderick, and C. Bearzi. 2005. Comparison of four markers for quantifying microbial protein flow from the rumen of lactating dairy cows. J. Dairy Sci. 88:4065–4082.
Rodríguez, C. A., and J. González. 2006. In situ study of the relevance of bacterial adherence to feed particles for the contamination and accuracy of rumen degradability estimates for feeds vegetable origin. Br. J. Nutr. 96:316–325.
Rodríguez, C. A., J. González, M. R. Alvir, and R. Caballero. 2008. Effects of feed intake on in situ rumen microbial contamination and degradation of feeds. Livest. Sci. 116:108–117.
Sadik, M. S., J. T. Huber, K. King, R. Wanderley, D. De Young, A. Al Dehneh, and C. Dudas. 1990. Comparison of nitrogen-15 and diaminopimelic acid for estimating bacterial protein synthesis of lactating cows fed diets of varying protein degradability. J. Dairy Sci. 73:694–702.
Setala, J. 1983. The nylon bag technique in the determination of ruminal feed protein degradation. J. Sci. Agric. Soc. Finl. 55:1–78.
Théwis, A., D. Lemal, F. Rodríguez, E. François, N. Bartiaux-Till, E. Baudart, and G. Dardenne. 1987. Influence de la complémentation de l’herbe de pâturage par de l’orge et de la pulpe de betterave sur la digestion et la synthèse microbienne dans le rumen. Reprod. Nutr. Dev. 27:269–270.
Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fibre, neutral detergent fibre and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597.
Varvikko, T., and J. E. Lindberg. 1985. Estimation of microbial in nylon bag residues by feed 15N dilution. Br. J. Nutr. 54:473–481.
Vérité, R., and C. Démarquilly. 1978. Qualité des matières azotées des aliments pour ruminants. In: M. Journet and A. Hoden, editors, Vache laitière: Aspects génétiques, alimentaires et pathologiques. INRA, Paris. p. 143–157.
Wanderley, R. C., G. A. Alhadhrami, M. Pessarakli, J. L. Aquino- Ramos, and J. T. Huber. 1999. An assessment of the microbial colonization of forage in the rumen of dairy cows and camels. Anim. Feed Sci. Technol. 76:207–218.
Wanderley, R. C., J. T. Huber, Z. Wu, M. Pessarakli, and C. Fontes. 1993. Influence of microbial colonization of feed particles on determination of nitrogen degradability by in situ incubation. J. Anim. Sci. 71:3073–3077.
Weakley, D. C., M. D. Stern, and L. D. Satter. 1983. Factors affecting disappearance of feedstuffs from bags suspended in the rumen. J. Anim. Sci. 56:493–507.
Yang, W. Z., K. A. Beauchemin, and L. M. Rode. 2001. Effect of dietary factors on distribution and chemical composition of liquidor solid-associated bacterial populations in the rumen of dairy cows. J. Anim. Sci. 79:2736–2746.
Zinn, R. A., and F. N. Owens. 1986. A rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis. Can. J. Anim. Sci. 66:157–166.