Livestock Science 126 (2009) 136–146
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Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i
Parameterization of ruminal fibre degradation in low-quality tropical forage using Michaelis–Menten kinetics Edenio Detmann a,⁎, Mário F. Paulino a, Hilário C. Mantovani b, Sebastião de C. Valadares Filho a, Cláudia B. Sampaio a, Marjorrie A. de Souza a, Ísis Lazzarini a, Kelly S.C. Detmann c a b c
Departamento de Zootecnia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36571-000, Brazil Departamento de Microbiologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36571-000, Brazil Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36571-000, Brazil
a r t i c l e
i n f o
Article history: Received 27 June 2008 Received in revised form 12 June 2009 Accepted 15 June 2009 Keywords: Degradation rate Intake Nitrogenous compounds Rumen ammonia nitrogen Signal grass Supplementation
a b s t r a c t This work aimed to parameterize the ruminal degradation of neutral detergent fibre (NDF) from low-quality tropical forage using Michaelis–Menten kinetics. The intake, rumen outflow (L), fractional degradation rate (kd), discrete lag (LAG) and effective degradability (ED) of NDF, and the microbial flow of nitrogenous compounds into the small intestine (Nmic) were assessed in two 5 × 5 Latin square experiments by using five Holstein × Zebu heifers cannulated in the rumen. The experiments were carried out sequentially and the treatments were formed by increasing the level of supplementation with nitrogenous compounds. A low-quality signal grass (Brachiaria decumbens) hay was used as roughage. The nitrogen supplement was a mixture of urea, ammonium sulfate and albumin, at the ratios of 4.5:0.5:1.0, respectively. The crude protein contents in the diets ranged from 51.9 to 136.3 g/kg of dry matter. The rumen ammonia nitrogen (RAN) concentration was used as an independent variable. The NDF intake, L and Nmic showed a quadratic pattern (P b 0.05) as a function of RAN concentration, and the critical points (maximum responses) were observed with 15.17, 16.28, and 14.52 mg of RAN/dL of rumen fluid, respectively. On the other hand, ED and LAG presented a linear-response-plateau (P b 0.05) according to the RAN concentration, with break points close to 8 mg/dL for ED (maximum estimate) and LAG (minimum estimate). The RAN concentrations to optimize NDF degradation and intake were defined as 8 and 15 mg/dL, respectively. This difference between estimates appears to be due to a better adequacy of the metabolizable protein:metabolizable energy ratio in the animal metabolism, which increases the animal intake even after the rumen NDF degradation has been optimized. This observation was supported by Nmic pattern. An adapted Michaelis–Menten model was applied to the data, where RAN was the independent variable and kd the dependent variable. The relationship between these variables was found to be significant by using the Hanes–Woolf plot (P b 0.01). Based on this model, the rate of NDF degradation as a function of RAN concentration indicates that fibre degradation in the rumen could be considered a second order process. In this context, the RAN concentration of 8 mg/dL was assumed as the limit where zero order (below limit) and first order (above limit) reactions become predominant for NDF degradation in the rumen. © 2009 Elsevier B.V. All rights reserved.
Abbreviations: ADFom(n), Acid detergent fibre corrected for ash and nitrogenous compounds;ADIP, Acid detergent insoluble protein;BCVFA, Branched-chain volatile fatty acids;CP, Crude protein;DM, Dry matter;ED, Effective degradability of neutral detergent fibre;EE, Ether extract;kd, Fractional degradation rate of NDF; km, The Michaelis–Menten constant;L, Time-dependent rate parameter associated with rumen flow of fibrous particles;LAG, Discrete lag for fibre degradation; Lignin (sa), Lignin determined by solubilization of cellulose with sulphuric acid;aNDFom(n), Neutral detergent fibre assayed with a heat stable amylase and corrected for ash and nitrogenous compounds;NDF, Neutral detergent fibre;pdNDF, Potentially degradable fraction of neutral detergent fibre;NFC, Non-fibrous carbohydrates;Nmic, Intestinal flow of microbial nitrogenous compounds;OM, Organic matter;RAN, Rumen ammonia nitrogen. ⁎ Corresponding author. Tel./fax: +55 31 3899 2252. E-mail address:
[email protected] (E. Detmann). 1871-1413/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2009.06.013
E. Detmann et al. / Livestock Science 126 (2009) 136–146
1. Introduction The carbohydrate components of tropical grasses insoluble fibre (cellulose and hemicellulose) are the main energy resources for cattle production in tropical regions. This can be attributed to an adequate production of digestible energy at lower costs compared to other energetic feedstuffs (Detmann et al., 2008). However, the climate characteristics of such regions, mainly rainfall and temperature, divide the year in two different seasons: rainy and dry. Although during the rainy season tropical forages under grazing have high quality, in the dry season its nutritive value is severely reduced, with increased lignin content in the cell wall and a decreased total content of nitrogenous compounds. These modifications can compromise the availability of energy from forage (Paulino et al., 2001, 2006) by reducing the neutral detergent soluble compounds and decreasing neutral detergent fibre (NDF) digestibility (Van Soest, 1994). In this context, the strategic supplementation with limiting nutrients become the best option for cattle management, specially when this supplementation is based on feeding nitrogenous compounds, which stimulates the fibrolytic activity in the rumen and increases the utilization of lowquality fibrous carbohydrates (Costa et al., 2008; Detmann et al., 2008; Paulino et al., 2006). The main challenge for nutritionists in tropical conditions is to understand the dynamics of NDF degradation for lowquality forages as a function of nitrogenous compounds supplementation, under the point of view of productive and economic optimization of interactions between basal nutrient resource (forages) and supplements. Under non-tropical conditions, some modeling approach has been developed to take into account the effect of the nitrogen deficiency on fibre utilization in the rumen for prediction of animal performance (Tedeschi et al., 2000), but similar information is not currently available in the tropics. In the ruminant nutrition literature, it is frequently assumed that NDF utilization in the rumen consists of a first order dynamic process. This argument is based on the assumption that rumen degradation parameters would be defined only by substrate characteristics (Detmann et al., 2005), without limitations in enzyme availability for NDF degradation. However, recent studies under tropical conditions indicated that enzymatic limitation could explain, at least in part, the reduction of NDF utilization in the rumen of animals fed low-quality forage, as observed for forages under grazing during the dry season (Costa et al., 2008; Lazzarini, 2007; Sampaio, 2007; Zorzi, 2008). This pattern suggests that ruminal fibre degradation is a process that follows a second order kinetics, or a Michaelis–Menten process, being defined simultaneously by enzyme availability and substrate characteristics (Detmann et al., 2005, 2008). Some experimental evidences indicated that the low nitrogen content of low-quality forages could limit the availability of microbial fibrolytic enzymes in the rumen. Thus, the main effect of the supplementation with nitrogenous compounds would be the higher supply of nitrogenous precursors for the synthesis of microbial enzymes (Costa et al., 2008; Detmann et al., 2008; Souza, 2007).
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Several parameters have been suggested to evaluate the availability of dietary nitrogenous compounds in the rumen. However, the concentration of rumen ammonia nitrogen (RAN) has been used as a qualitative reference to understand the adequacy of the rumen environment according to the microbial activity on fibrous carbohydrates (Hoover, 1986). This strategy is possibly associated with the fact that RAN is the preferred nitrogen source for the growth of fibrolytic microorganisms (Russell, 2002). Most models used to describe fibre degradation in the rumen are based on simple first order assumptions (Mertens, 2005). However, this assumption interferes with the correct interpretation of rumen degradation dynamics when the diet is based on low-quality tropical forages. In these cases, a second order model might be a better tool for interpreting NDF degradation. The Michaelis–Menten model allows the evaluation of enzyme kinetics when the ratio of enzyme:substrate is variable ((Nelson and Cox, 1999); Voet and Voet, 2006), such as the situation described above. However, it has not been verified, at least in the tropics, any research applying this model to interpret NDF degradation in the rumen. The objective of this work was to evaluate the NDF degradation dynamics in the rumen of cattle fed low-quality tropical forage as a function of rumen ammonia nitrogen concentration using the Michaelis–Menten kinetics. 2. Material and methods 2.1. Location and animals Two experiments were sequentially carried out in the Animal Laboratory at the Department of Animal Science of Federal University of Viçosa (UFV), Viçosa, Brazil, from December of 2005 to May of 2006. Five crossbred heifers (Holstein × Zebu) averaging 180 ± 21 and 209 ± 13 kg of body weight (BW), were used for the first and second experiment, respectively. The animals were surgically fitted with ruminal cannulae, approximately 60 days prior to the beginning of Experiment 1. All surgical and animal care procedures were approved by the University Animal Care Committee. Ruminal fistula and their surrounding areas were cleaned routinely during the experiments. The animals were treated for endo and ecto parasites at the beginning of the experiments and kept into individual stalls (which were cleaned daily) of approximately 10 m2. Water and mineral mixture were available to the animals at all times. 2.2. Experimental diets and feeding The forage fed to the animals consisted of low-quality signal grass (Brachiaria decumbens Stapf.) hay. The hay was produced from a dry season cutting (August 2005) of the forage available in a signal grass pasture located in the central region of Brazil (latitude 18°41′S, longitude 49°34′W, average altitude 620.2 m). The climate is classified as Aw type, hot and humid, with coldest monthly temperatures above 18 °C, annual average rainfall between 1400 and 1600 mm, rainy season from November to March and dry season from April to October.
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There were five treatments in each experiment. In both cases, the treatments consisted of levels of supplementation with nitrogenous compounds set in order to raise the CP level of the diet to 0, 20, 40, 60 and 80 g/kg (Experiment 1), and to 0, 30, 50, 70 and 90 g/kg (Experiment 2), on DM basis, above the CP level of the roughage. A mixture of urea, ammonium sulfate and albumin was used as a source of nitrogenous compounds, at the ratios of 4.5:0.5:1.0, respectively. These supplements were calculated based on the DM intake computed on the previous day and placed directly into the rumen of the animals. Supplement ingredients were chosen based on the absence of carbohydrates, so supplementation effects with nitrogenous compounds could be evaluated without the interference of any additional source of fibre or energy. Albumin was included in the supplement to meet the microbial requirements for true degradable protein, allowing the supply of essential substrates, such as branched-chain volatile fatty acids (BCVFA). The forage was supplied ad libitum, allowing approximately 100 g/kg in orts. The ration was fed twice a day, in equal portions, at 8 a.m. and 4 p.m. The supplements were divided in two portions of equal weight and placed directly into the rumen of the animals when the forage was offered. The forage offered and the orts were quantified daily. Both experiments were carried out according to a 5 × 5 Latin square design, with five treatments, five experimental periods and five animals. Thus, the experiment consisted of ten 16-day experimental periods, and the first five days were allocated to the adaptation of the animals to the supplementation levels followed by 11 days of sample collection. 2.3. Handling, measurements and samples Since both experiments were conducted in a similar way, the experimental description will be performed as one experimental period. For the quantification of voluntary intake, feedstuffs supplied between the sixth and the ninth days of each experimental period were considered and the orts were measured between the seventh and tenth day. Forage and orts samples were processed in a Wiley mill (1 mm) and stored for later analysis. To evaluate the RAN concentration, samples of rumen fluid were taken on the sixth day of each experimental period at 4 a.m., 8 a.m., 12 p.m., 4 p.m., 8 p.m. and 12 a.m. The samples were collected manually from the liquid:solid interface of the rumen mat and filtered through a triple cheesecloth layer. A 40 mL aliquot was then separated, fixed with 1 mL H2SO4 (1:1) and frozen (−20 °C) for later analysis. Rumen microorganisms were isolated on the 10th day of each experimental period. Samples of rumen contents were taken immediately before and 6 h after morning feeding according to Cecava et al. (1990). On the 12th, 14th and 16th days of each experimental period urine samples were obtained, approximately 4 h after morning feeding. The samples were filtered through a cheesecloth and a 10 mL aliquot was separated and diluted with 40 mL H2SO4 (0.036 N) (Valadares et al., 1999). The evaluation of transit kinetics of fibrous particles was done between 11th and 16th days of each experimental period through a pulse dose of fibre mordant chromium
(Udén et al., 1980). Samples of the signal grass hay were used for mordant production. On the 11th day of each experimental period, approximately 100 g of the fibre mordant was placed into the rumen of the animals at 8 a.m. Feacal samples were taken from the rectum of the animals at 0, 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, 60, 72, 84, 96, 120 and 144 h after marker administration. The samples were oven dried (60 °C/72h) and processed in a Wiley mill (1 mm). Simultaneously to the transit evaluation, it was carried out through in situ incubation to estimate the rumen degradation parameters of NDF. Samples of hay were processed in a Wiley mill (2 mm) and were placed in non-woven textile (100 g/ m2) bags (Casali et al., 2008) in a ratio of 20 mg DM/cm2 of bag surface (Nocek, 1988). The bags, in duplicate for each incubation time, were placed in the rumen of the animals. The following incubation times were used: 0, 3, 6, 9, 12, 18, 24, 36, 48, 72, 96 and 120 h. After incubation, the bags were cleaned with tap water and oven dried (60 °C). 2.4. Laboratory analysis Samples of hay were analyzed regarding DM (index no. 934.01), OM (index no. 942.05), CP (index no. 954.01), EE (index no. 920.39), and ADF (index no. 973.18) contents according to the methods of the Association of Official Analytical Chemistry — AOAC (1990). In the NDF analysis, the samples were treated with a heat stable alpha amylase, without using sodium sulphite, and corrected for residual ash (Mertens, 2002). The corrections of NDF and ADF regarding nitrogenous compounds and ash were done according to Licitra et al. (1996) and Mertens (2002), respectively. The lignin content was obtained by solubilization of cellulose by sulphuric acid (Van Soest and Robertson, 1985). The orts samples were analyzed for DM and CP while supplements were analyzed for DM, OM and CP as described above (Table 1). The RAN content in the rumen fluid was determined in the supernatant of the samples (1000 ×g, for 15 min) by the micro-Kjeldahl system, without acid digestion and after distillation with potassium hydroxide (2 N). The concentrations obtained at the different sampling times were combined by animal and period in order to obtain a single value that represented the average daily RAN concentration.
Table 1 Chemical composition of hay and nitrogen supplement according to the experiments. Nutrient
DM a OM b CP b EE b aNDF b aFDNom(n) b NFC b c ADFom(n) b Lignin (sa) b ADIP d a b c d
Experiment 1
Experiment 2
Hay
Supplement
Hay
Supplement
899.6 942.6 48.6 15.3 830.4 769.8 108.9 531.1 79.6 324.5
896.4 989.1 2512.0 – – – – – – –
877.4 943.5 50.8 6.2 835.5 797.3 89.2 464.3 75.1 244.6
967.0 987.5 2353.4 – – – – – – –
g/kg as fed. g/kg DM. CNF = OM − [CP + EE + aNDFom(n)]. g/kg CP.
E. Detmann et al. / Livestock Science 126 (2009) 136–146
The rumen microorganism samples were assessed for DM, CP (Association of Official Analytical Chemistry — AOAC, 1990), and purine bases (Ushida et al., 1985) contents. The urine samples, after thawing, were composed per animal and experimental period. The creatinine concentrations in the urine were obtained by a modified Jaffé method (Bioclin® K016-1). The contents of allantoin and uric acid in the urine were estimated using colorimetric methods, as reported by Chen and Gomes (1992). The total urinary volume was estimated by the ratio of creatinine concentration in the urine on its excretion per unit of live weight, which was estimated as described by Chizzotti (2004): CE = 32:27−0:01093 × LW;
ð1Þ
where CE is the daily creatinine excretion (mg/kg of LW), and LW is the live weight (kg). Purine derivatives excretion was calculated by the sum of the quantities of allantoin and uric acid excreted in the urine. The absorbed purines were calculated from purine derivatives excretion by the equation (Verbic et al., 1990): AP =
PD−0:385 × LW 0:85
0:75
;
ð2Þ
where AP is the absorbed purines (mmol/d), PD is the purine derivatives excretion (mmol/d), 0.85 is the recovery of absorbed purines as purine derivatives in the urine (mmol/ mmol), and 0.385 is the endogenous purine derivatives excretion in the urine per unit of metabolic size (mmol). Microbial synthesis of nitrogenous compounds in the rumen was estimated as function of the absorbed purines and the NRNA: NTOTAL ratio in the microorganisms (Chen and Gomes, 1992): Nmic =
70 × AP ; 0:83 × R × 1000
ð3Þ
where Nmic is the microbial nitrogenous compounds flow in the small intestine (g N/d), R is the NRNA:NTOTAL ratio in the microorganisms (mg/mg), 70 is the nitrogen content in purines (mg/mol), and 0.83 is the intestinal digestibility of the microbial purines (mg/mg). The faeces samples to evaluate transit kinetics were analyzed regarding DM (Association of Official Analytical Chemistry — AOAC, 1990) and chromium (Willians et al., 1962). The degradation residues were analyzed regarding NDF contents using a heat stable alpha amylase, without using sodium sulphite (Mertens, 2002). In this case, there were no corrections for residual ash or nitrogenous compounds. 2.5. Rumen dynamics modeling The parameters of transit kinetics were estimated through adjustment of a gamma-2 time-dependent model to chromium excretion profile (Ellis et al., 1994): Ct = Z × ðt−τÞ × L × exp½−L × ðt−τÞ;
ð4Þ
where Ct is the faecal concentration of chromium at time t (ppm), t is the time after marker administration (h), L is the time-dependent rate-parameter related to rumen flow of
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fibrous particles (h− 1), Z is a parameter without biological meaning (ppm × h), and τ is the time of intestinal transit (h). The degradation profiles of NDF were interpreted according to a logistic model described by Van Milgen et al. (1991): Rt = B × ð1 + λ × tÞ × expð−λ × tÞ + I;
ð5Þ
where Rt is the non-degraded residue of NDF at time t (g/g), B is the potentially degradable fraction of NDF (pdNDF), and λ is the common rate of lag and degradation (h− 1) of pdNDF. The fractional degradation rate of NDF was estimated from gamma-2 distribution properties (Ellis et al., 1994): kd = 0:59635 × λ;
ð6Þ
where kd is the fractional degradation rate of pdNDF (h− 1). The estimates of discrete lag were obtained according to Vieira et al. (1997): LAG =
Rð0Þ−Rðti Þ + ti ; R′ ðti Þ
ð7Þ
where LAG is the discrete lag (h), R(0) is the non-degraded NDF residue at t = 0 (g/100 g), R(ti) is the non-degraded NDF residue at the inflexion point of the adjusted profile (g/100 g), R′(ti) is the mathematical derivative at the inflexion point (maximum degradation rate) (h− 1), and ti is the time at inflexion point (h). The ti values were obtained according to Van Milgen et al. (1991): ti =
1 : λ
ð8Þ
The effective degradability of NDF was estimated by adapting the procedures of Ørskov and McDonald (1979), according to the equation: t
ED = limt→∞ ∫0
f ðtÞ ×
−
dRt dt
dt;
ð9Þ
where ED is the effective degradability of NDF (g/g), and ƒ(t) is the mathematical function that describes the particles flow in the rumen. The function ƒ(t) was obtained by re-parameterization of Eq. (4), where it was assumed a rumen resident particles profile (Ellis et al., 1994): f ðtÞ = ð1 + L × tÞ × expð−L × tÞ:
ð10Þ
The non-linear adjustments of models described in Eqs. (4) and (5) followed the iterative algorithm of Gauss–Newton, which is implemented in the NLIN procedure of SAS (Statistical Analysis System). 2.6. Statistical analysis and Michaelis–Menten modeling The average CP levels in the diets, according to the supplementation levels described above, were 51.9, 71.1, 86.0, 116.7 and 130.2 g/kg of DM (Experiment 1), and 52.8, 80.8, 98.2, 118.7 and 136.3 g/kg of DM (Experiment 2). Those values were produced through ratio of actual CP intake on actual DM intake and used for the initial statistical evaluation.
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Michaelis–Menten model (Nelson and Cox, 1999; Plowman, 1972; Voet and Voet, 2006): V0 =
Fig. 1. Relationship between rumen ammonia nitrogen (RAN) concentration and diet content of crude protein (CP) (Ŷ = 4.012 + 0.06142X, ∀ X ≤ 109.038; Ŷ = − 29,829 + 0.37178X, ∀ X N 109.038; R2 = 0.7520) (n = 50; + = least square means of treatments).
The dataset were initially evaluated through analysis of variance, according to the model: Yijkl = μ + Ei + LðiÞj + AðiÞk + PðiÞl + εijkl ;
ð11Þ
Vmax × ½S kd × ½RAN ⇔kd = max km + ½S km + ½RAN;
ð12Þ
where V0 is the velocity of the enzyme activity on substrate (mass of product/time), Vmax is the maximum velocity of the enzyme activity (mass of product/time), [S] is the substrate concentration, kd is the fractional degradation rate of pdNDF (h− 1), kdmax is the maximum fractional degradation rate of pdNDF (h− 1), [RAN] is the RAN concentration in the rumen fluid (mg/dL), and km is the Michaelis–Menten constant, which corresponds to [S] when V0 = ½ Vmax (classic model) or [RAN] when kd = ½ kdmax (adapted model). The Hanes–Woolf plot was used to adjust the adapted model described in Eq. (12) (Bracht et al., 2003): ½RAN km + = kd kdmax
1 kdmax
ˆ × X: ˆ +β × ½RAN⇔ Yˆ = β 0 1 ð13Þ
All statistical procedures were performed using SAS (Statistical Analysis System) (PROC GLM and PROC MIXED) and SAEG (Sistema de Análises Estatísticas) (α = 0.05).
where μ is the general constant, Ei is the effect of ith experiment, L(i)j is the effect of jth CP level in the diet nested to the ith experiment, A(i)k is the effect of kth animal nested to the ith experiment, P(i)l is the effect of lth experimental period nested to the ith experiment, and εijkl is the random error, which was presupposed of normal distribution with average 0 and variance σ2. The estimable linear functions produced from the model described in Eq. (11) were used for the adjustment of experimental observations regarding the effects of experiment, animal and experimental period. This procedure was adopted to clarify the functional relationships between variables, once the adjusted observations became composed only by general constant, the effect of CP level in the diet and experimental error. After that, the relationships between variables were analyzed according to linear regression techniques (Myers, 1990). In a particular way, the relationship between kd and RAN concentration was interpreted by analogy with the classic
When CP levels in the diet ranged from 50 to approximately 140 g/kg DM, the concentration of rumen ammonia nitrogen (RAN) increased in a linear relationship (P b 0.05). However, the positive association between these variables changed in each curve section, and the increase in RAN concentration was more pronounced at CP levels greater than 109.0 g/kg DM (Fig. 1). The NDF intake (g/kg of LW) showed a quadratic profile as function of RAN concentration (P b 0.05), and the critical point for maximum response was obtained at 15.17 mg RAN/dL (Fig. 2). A similar pattern was observed for the passage rate of fibrous particles (P b 0.05), which reached the critical point (maximum response) at 16.28 mg RAN/dL (Fig. 3). The ED of NDF had a linear-response-plateau pattern (P b 0.01) as a function of RAN concentration (Fig. 4). The ED of NDF increased until the RAN concentration reached
Fig. 2. Relationship between neutral detergent fibre intake (NDFI) and rumen ammonia nitrogen (RAN) concentration (Ŷ = 8.6387 +0.8353X − 0.027525X2; R2 = 0.5087) (n = 50; + = least square means of treatments).
Fig. 3. Relationship between estimates of time-dependent rate-parameter related of rumen flow of fibrous particles (L) and rumen ammonia nitrogen (RAN) concentration (Ŷ = 0.0083 + 0.000775 − 0.00002379X2; R2 = 0.5576) (n = 50; + = least square means of treatments).
3. Results
E. Detmann et al. / Livestock Science 126 (2009) 136–146
Fig. 4. Relationship between effective degradability (ED) of neutral detergent fibre and rumen ammonia nitrogen (RAN) concentration (Ŷ=−0.35439+0.1032X, ∀ X≤8.0048; Ŷ=0.4719, ∀ XN 8.0048; R2 =0.9124) (n=50; + = least square means of treatments).
8.00 mg/dL. At higher RAN concentrations ED estimates became unchangeable. A linear-response-plateau relationship was also observed between LAG and RAN concentration (P b 0.05). However, in this latter case, the LAG estimates decreased as the RAN concentration increased up to 8.17 mg/dL of rumen fluid (Fig. 5). At higher RAN values, no change in LAG estimates was detected. A quadratic pattern was verified between Nmic and RAN concentration (P b 0.05), and the critical point for maximum response was reached at a RAN concentration of 14.52 mg/dL (Fig. 6). The Hanes–Wolf plot indicated a linear relationship between kd and RAN concentration in the rumen fluid (Fig. 7). This relationship was significant (P b 0.01) and showed a high coefficient of determination (R2 = 0.9218). Based on these results, it seems reasonable to describe the rumen degradation NDF through a Michaelis–Menten kinetics model. 4. Discussion Diet supplementation with nitrogenous compounds caused a positive effect on RAN concentration in the rumen fluid. However, the observed pattern indicates that a greater increase in RAN concentration at CP levels higher than 109.0 g/kg DM might be associated with a decreased assimilation of nitrogenous compounds by rumen microorganisms (Fig. 1).
Fig. 5. Relationship between discrete lag (LAG) and rumen ammonia nitrogen (RAN) concentration (Ŷ = 2.6660 + 0.2210X, ∀ X ≤ 8.1672; Ŷ = 0.8601, ∀ X N 8.1672; R2 = 0.7642) (n = 50; + = least square means of treatments).
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Fig. 6. Relationship between intestinal flow of microbial nitrogen (Nmic) and rumen ammonia nitrogen (RAN) concentration (Ŷ = 3.1825 + 6.0213X − 0.207380X2; R2 =0.5485) (n=50; + = least square means of treatments).
Some experimental evidences suggest that diets containing high quantities of amino acids stimulate the growth of hyper-ammonia producing bacteria, and this growth often results in excess ammonia production (Russell, 2002). However, this effect is not expected to occur in this work, as the true protein content in the supplement was relatively low. On the other hand, it must be emphasized that both forage and supplement presented low contents of non-fibrous carbohydrates (Table 1). Therefore, it should be expected a wide predominance of fibrolytic species in the rumen environment compared to species that degrade non-fibrous carbohydrates. Thus, the point where the relationship between rumen ammonia nitrogen (RAN) concentration and crude protein content changes (as described in Fig. 1) seems to indicate the break point where the microbial assimilation of nitrogenous compounds using the energy extracted from NDF become severely inefficient. This fact could implicate in an increment on nitrogenous compounds losses in the rumen. The increase of NDF intake until the RAN concentration was near to 15 mg/dL supports the idea of a higher productive efficiency for cattle fed low-quality tropical forage when supplemented with nitrogenous compounds (Leng, 1990; Paulino et al., 2006). In this case, the increases on low-quality forage intake result from the greater digestibility of fibrous compounds, which have high rumen fill effect (Lazzarini, 2007; Sampaio, 2007). This behavior is frequently associated with increases in forage
Fig. 7. Relationship between fractional degradation rate of NDF (kd − h− 1) and rumen ammonia nitrogen (RAN — mg/dL) concentration expressed through Hanes–Woolf plot (Ŷ = 0.02892 +0.000926X; R2 = 0.9218) (n = 50; + = least square means of treatments).
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degradation and passage rates as a result of nitrogenous compounds supplementation (Hannah et al., 1991). The supplementation causes higher removal of undegradable residues and increases the turnover of fibre in the rumen (Allen, 1996), as observed in this work (Figs. 3 and 4). Low-quality tropical forages are deficient not only in nutrients for animal performance, but also in substrates for microbial metabolism (in this case, mostly nitrogenous compounds). This situation causes the inclusion of nitrogen supplements to be helpful to the rumen environment (Dixon and Stockdale,1999), and increases microbial growth on fibrous carbohydrates (Costa et al., 2008). On the other hand, the BCVFA have been pointed out as a limiting factor for microbial growth in diets with low levels of plant protein (McCollum et al., 1987) and the benefits of its supplementation on fibre degradation have been demonstrated by in vitro studies (Yang, 2002). This fact justified the inclusion of a true protein source in the nitrogen supplement. However, there was no supplement in the lower diet levels of CP in both experiments, as previously described. So, in this case a BCVFA deficiency could be added to the nitrogen deficiency, confounding the results. Nevertheless, despite of the in vitro results (Yang, 2002), several results from the in vivo trials indicate that BCVFA supplementation did not increase the number of cellulolytic microorganisms in the rumen (Oltjen et al., 1971) or improve fibre intake and digestibility of low-quality forages (Gunter et al., 1990; Hefner et al., 1985; McCollum et al., 1987). Thus, under these circumstances, a BCVFA deficiency should not be the first-limiting factor affecting animal performance (Hefner et al., 1985), once low-quality diets could provide adequate quantities of BCVFA for microbial growth (McCollum et al., 1987), and its supplementation should not be justified (Gunter et al., 1990). Under this point of view, the nitrogen deficiency must be assumed as the main nutritional constraint for fibre utilization in the rumen. NDF intake decreased when RAN concentration was higher than 15 mg/dL (Fig. 2). Considering that microbial nitrogen assimilation reduced (Fig. 1), it appears that nitrogen losses increased in the rumen. Under this condition nitrogenous compounds in animal metabolism would be in excess, which could have several negative effects on voluntary intake, such as: ATP deficiency in liver metabolism due to excessive utilization of the urea cycle (Visek, 1984), increased body heat production (Poppi and McLennan, 1995), and animal indisposition due to excess ammonia in the blood (Detmann et al., 2007). Decreases in voluntary intake associated with excess use of nitrogenous compounds supplements were also observed by other authors when they fed cattle with low-quality forage (DelCurto et al., 1990a; DelCurto et al., 1990b; Lazzarini, 2007; Sampaio, 2007). Most assumptions applied to rumen NDF degradation dynamics consider that fibre degradation can be described by first order models. In this case, the biological events depend on a single pool, assuming substrate as the limiting variable. Therefore, intrinsic characteristics of the substrate would be the single restrictive factor for rumen degradation, as a large and non-limiting enzymatic pool would exist in the rumen (Detmann et al., 2008). However, the relationship between ED and rumen ammonia nitrogen (Fig. 4) indicates that two different phases of rumen
degradation must be analyzed. When RAN concentrations were higher than 8 mg/dL, ED stabilized on a maximum plateau. This result indicated that enzyme activity was not limited in the rumen, and suggested a typical first order reaction. However, the lower ED estimates obtained when RAN concentrations were below 8 mg/dL indicate that degradation was also limited by enzymes activity, characterizing a zero order reaction. The relationship based on RAN concentration characterizes the NDF degradation dynamics in the rumen as a second order process (or Michaelis–Menten kinetics) (Mertens, 2005; Detmann et al., 2005, 2008). In other words, at RAN concentration below 8 mg RAN/dL the availability of nitrogenous compounds for microbial enzymes synthesis is low, which implies a zero order reaction. When the nitrogen availability in the medium is increased, NDF degradation follows first order kinetics. In this way, nitrogen supplementation elevates the availability of microbial enzymes for fibre degradation in the rumen. A second order interpretation for NDF degradation was also presented by Detmann et al. (2005). These authors carried out a cumulative gas production assay and verified that the degradation rate of NDF in tropical forages was higher when supplements and forage were incubated together as compared to forage and supplements incubated separately. The difference between ED estimates on RAN concentrations lower than 8 mg/dL and plateau values constitutes the latent energy of low-quality tropical forages. This concept was defined by Paulino et al. (2001) and represents a possible source of lowcost energy for animal production, which is unavailable due to restriction of nitrogenous precursors for synthesis of microbial enzymes. This argument emphasizes that nitrogen supplements play a major role in cattle fed low-quality tropical forages. The first phase of the Michaelis–Menten kinetics, from the perspective of time, is called transitory phase. In this phase, there is no enzymatic activity while homogenization between substrate and enzyme is occurring (Voet and Voet, 2006). Under controlled laboratory conditions, the kinetics interpretation do not take into account the transitory phase due to its very short time (fractions of second). Because the NDF is a substrate that shows high physical and chemical heterogeneity, the contact between enzyme and substrate can be complex. Therefore, from the perspective of time the transitory phase should not be considered irrelevant. Under the context of fibre degradation in the rumen, the discrete lag represents an estimate of the transitory phase in the Michaelis–Menten kinetics. This parameter estimates the time dispended with preparatory events for fibre degradation, such as physical (e.g. particle hydration, mastication, etc.) and microbiological aspects (e.g. adhesion, enzymes synthesis, etc.). In this context, if physical aspects of transitory phase could be assumed constant under different RAN concentrations (e.g. rate of particles hydration), any alteration on its estimates would be due only to microbiological aspects. The pattern of LAG estimates (Fig. 5) brings in evidence that nitrogen deficiency at RAN concentrations below 8 mg/dL would cause a microbial deficiency in the synthesis of compounds needed for microbial adhesion on fibre and (or) enzymes to start fibre degradation. These results also emphasize that the rumen degradation is a second order kinetics process (Detmann et al., 2008).
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The divergence between RAN concentrations for optimizing NDF degradation (8 mg/dL) and intake (15 mg/dL) represents an apparent nutritional contradiction, because intake of insoluble fibrous compounds is intimately associated with the rumen fill effect of these compounds (Lazzarini, 2007; Sampaio, 2007), which is determined, in part, by velocity of NDF degradation (Paulino et al., 2006). A similar pattern was reported by Leng (1990), who suggested concentrations of 10 and 20 mg RAN/dL to optimize degradation in the rumen and voluntary intake under tropical conditions, respectively. However, this divergence seems to represent differences in nitrogenous compounds requirements between microorganisms and the host animal (Van Soest, 1994). Thus, RAN concentration of 8 mg/dL seems enough to meet the nitrogen demand of fibrolytic microorganisms, although the animal maximum intake is only reached at higher RAN concentrations (Fig. 2). In this way, even with a lower efficiency of microbial assimilation of nitrogenous compounds in the rumen (Fig. 1), the animal voluntary NDF intake continues to be stimulated (Fig. 2). This could indicate that the animal is trying to meet its requirements for metabolizable protein, as observed under marginal deficiency conditions (Forbes, 1995). Alternatively, when there is a demand for metabolic adaptation it can alter the metabolizable energy (ME):metabolizable protein (MP) ratio in its metabolism (Illius and Jessop, 1996). However, the attempt of the animal to supply the requirements for nitrogenous compounds is associated with a harmonic balance between degradation and intake. The increase on intake is related to an increase on passage (Figs. 2 and 3). When the passage increases, the time available for microorganisms to act on NDF is decreased (Detmann et al., 2008). If these activities are perfectly coordinated, and a constant velocity of enzyme action is considered, a decrease on ED estimates would be observed, which could compromise the availability of energy to the animal. However, this effect was not verified in our experiments (Fig. 4). Thereby, the higher NDF intake on RAN concentrations greater than the demand to optimize degradation (Fig. 2) seems to constitute a compensatory mechanism to equilibrate the ME:MP ratio. If a direct and positive association occur between kd and RAN concentration (Fig. 7), the increase on passage would be compensated by a higher enzymatic
Fig. 8. Classic schematic representation of the Michaelis–Menten model.
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velocity when the RAN levels reach values above the optimum for fibre degradation. This process would lead to the stability of ED estimates. Thereby, the energy extracted from lowquality NDF would become unchangeable (Fig. 4). Thus, the increase on intake would imply a higher supply of MP from microbial protein (Fig. 6) and, simultaneously, would result in a better adequacy of the animal metabolism. This pattern seems to be plausible under the point of view of a multifactorial intake control. Assuming ME:MP ratio as one factor to affect intake, the adjustment of this by the animal, instead optimal fibre degradation, indicates an attempt to minimize “discomfort” (e.g. heat body production) and optimize the intake (Forbes, 2003). In a simple way, the kinetics of fibrolytic enzymes in the rumen could be interpreted as: k1
k3
E + S ⇌ ES⇀E + P; k2
ð14Þ
where E is the enzyme, S is the substrate, ES is the enzyme– substrate complex, P is the product, and k1, k2 and k3 are the constants associated with the velocities of chemical transformations. In the rumen, the components of Eq. (14) must have a complex interpretation, since none of it is homogeneous. Thereby, in the rumen perspective, those components can be described as follow: E, the fibrolytic enzymatic systems; S, the basal substrate represented by potentially degradable fraction of NDF; and P, the final products of NDF degradation (volatile fatty acids, VFA). In this way, the rate of NDF degradation can be expressed as: kd = f ½ðE : SÞ; k1; k2; k3;
ð15Þ
where E:S is the enzyme:substrate ratio. According to the classic interpretation of the Michaelis– Menten kinetics (Fig. 8), when the enzyme concentration overcomes the substrate concentration (or linkage sites), the formation of final products would be only dependent on substrate (first order reaction). However, as the substrate concentration increases and becomes enough to sustain all enzymes as an ES complex, the product formation becomes dependent on enzyme availability (zero order reactions) and is regulated by the k3 constant Eq. (14)). Thereby, the reaction pattern is determined by E:S ratio and consists of a second order kinetics process (Mertens, 2005; Nelson and Cox, 1999; Voet and Voet, 2006). In a general way, the evaluation of fibrolytic activity in the rumen using classic Michaelis–Menten model does not make biological sense, since situations of substrate limitation (such as NDF) are not expected. Therefore, changes in fibre degradation velocity would be caused by alterations on enzyme:substrate ratio, and affected by fluctuations on enzymes concentration (Detmann et al., 2005, 2008; Lazzarini, 2007; Sampaio, 2007). Although the microbial enzyme synthesis in the rumen results from a synchronized availability of many substrates (e.g. nitrogen, carbon chains, energy, minerals, etc.), the deficiency of nitrogenous compounds has been reported as the main limiting step for microbial synthesis. This idea has been supported by results showing that supplementation
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Fig. 9. Estimates of relative fractional degradation rate of NDF (relative kd), expressed as fraction of maximum degradation rate (kdmax), as a function of rumen ammonia nitrogen (RAN) concentration according to the Michaelis– Menten kinetics.
increases the utilization of basal low-quality substrate and, simultaneously, the energy available for microbial growth (Paulino et al., 2006). For fibrolytic bacteria, the nitrogen availability can be evaluated through RAN concentration, which is the preferential nitrogen source used by these microorganisms for growth (Russell, 2002). Thus, the sequence of reactions expressed in Eq. (14) can be redefined as: k1
k2
k4
RAN + CC→E→E + S⇌ES⇀E + P; k3
ð16Þ
where RAN is the rumen ammonia nitrogen concentration, CC is the availability of carbon chains in the rumen that can be used for amino acids synthesis by microorganisms, k1 is the constant related to the synthesis of fibrolytic enzymes, and k2, k3, k4 are the constants associated with the velocities of chemical transformations on NDF. In this way, the nitrogen concentration represents a limiting step (k1 constant; Eq. (16) for the synthesis of enzymes involved in fibre degradation. Assuming a steady-state condition for NDF mass in the rumen, the variation on mass of products formed would be defined by changes in the quantity of enzymes. This condition would change the independent variable of the classic Michaelis–Menten model, from substrate Eq. (12), Fig. 8 to enzymes. The concentration of enzymes (or fibrolytic complexes) in the rumen is hard to be measured. Since RAN is a potential precursor for enzyme synthesis (Eq. 16), and its concentration is easily measured, it can be used as an indirect indicator of enzymatic activity in the rumen. This represents the first adaptation of the Michaelis–Menten model in Eqs. (12) and (13). On the other hand, the parameters Vmax and V0 of the classical Michaelis–Menten model are used to express enzymatic activity based on mass of products produced by time (Bracht et al., 2003; Voet and Voet, 2006). However, the measurement of quantity or mass (e.g. mmol, mg, etc.) of VFA produced from NDF is hard to be performed. Alternatively, one can assume a steady-state condition for NDF mass in the rumen. In this case, the fractional rate of NDF degradation (kd − h− 1)
could indicate the fibre mass converted to products per unit of time, justifying the second adaptation of Eqs. (12) and (13). The kinetic representation of the Michaelis–Menten model using the relationship between RAN and kd is presented in Fig. 9. It should be noted that alterations in RAN concentration change the sequence of order reactions when compared to the classic model (Figs. 8 and 9). If we consider the RAN concentration of 8 mg/dL as the optimizing point for NDF degradation (Fig. 4), it can be concluded that RAN concentrations below that value would be the main limiting factor for fibre degradation due to enzymes deficiency in the medium (predominance of zero order reactions). As the RAN concentration increases above 8 mg/dL, the synthesis of hydrolytic enzymes is not limited by nitrogen precursors and the substrate (NDF) becomes the main limiting factor for degradation (predominance of first order reactions) (Fig. 9). It must be emphasized that substrate (NDF) limitation in the rumen is not due to concentration, but enzymes might be restricted to access the target, as the process of fibre degradation involves some physical aspects (e.g. hydration, mastication, etc.). This observation emphasizes the significance of the transitory phase for enzyme activity in the rumen. The Michaelis–Menten constant (km) is characteristic for each enzyme and represents, in the classical model, the substrate concentration equivalent to the half of the maximum velocity (Nelson and Cox, 1999; Plowman, 1972; Voet and Voet, 2006). Thereby, according to the adapted model, km would represent the RAN concentration where half of the kdmax would be observed. However, the km values estimated in this study (2.21 mg RAN/dL) (Fig. 7; Table 2) indicate that this parameter can only be considered a theoretical reference. Under tropical conditions, concentrations of 5 mg RAN/dL are considered limiting to maintain continuous microbial growth in the rumen and to equalize nitrogen intake and flow of microbial nitrogen into the small intestine (Sampaio, 2007). Under the context of optimizing NDF degradation and intake (8 and 15 mg RAN/dL, respectively), it were defined the concepts of constants for optimizing rumen degradation (kdeg) and voluntary intake (ki). These constants were defined from Hanes–Woolf plot (Fig. 7) and are associated with 0.78kdmax and 0.87kdmax, respectively (Table 2). According to McAllan and Smith (1983), the RAN concentration demanded by rumen microorganisms adhered to fibre could be higher compared to microorganisms from the rumen fluid. Thus, the RAN concentration used to optimize fibre utilization seems to be enough to optimize the use of other diet components in the rumen, as such nonfibrous carbohydrates.
Table 2 Parameterization of kinetics constants of fibrolitic enzymatic complex and their relative degradation rates (relative kd) as a function of rumen ammonia nitrogen (RAN) concentration. Kinetics reference
Acronimous
RAN a
Relative kd b
½ kdmax Rumen NDF degradation NDF intake
km kdeg ki
2.21 8.00 15.00
0.50 0.78 0.87
a b
mg/dL of rumen fluid. Calculated as ratio on kdmax.
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