N fractionation, degradability, intestinal digestibility, and adequacy for ruminal microbial activity of cultivated legumes

M de J Marichal, M Carriquiry, L Astigarraga and A I Trujillo

Departamento de Producción Animal y Pasturas, Facultad de Agronomía,Universidad de la República, E. Garzón 780, 12900 Montevideo, Uruguay
marichal@fagro.edu.uy

Abstract

Differences in N fractionation, degradability of organic matter (OM) and N, forage adequacy for rumen microbial activity (estimated as the ratio between rumen available N and fermentable OM; RAN:FOM), and intestinal digestibility of rumen undegradable  protein (RUP) of birdsfoot trefoil, red clover, and alfalfa were evaluated. Within each legume specie, variations in phenological stages were studied. Nitrogen was partitioned into five fractions in accordance with  Cornell Net Carbohydrate and Protein System. Three fistulated cows were used to evaluate ruminal degradation (nylon bag technique), and RUP intestinal digestibility (mobile bag technique).

Legumes differed (P<0.004) in chemical composition. The potentially degradable fraction (PDF), degradation rate (kd), and effective degradability (ED) of OM and N were similar among species, and birdsfoot trefoil presented the smallest (P<0.05) soluble fraction (SF) of OM and N. The RAN: FOM ratio was similar in the three legumes, and  intestinal digestibility of N tended (P<0.06)  to be different among species. 

Within each specie, a phenological stage effect (P<0.05) in chemical composition was detected. In the phenological stages evaluated, degradation kinetics of OM and N of the three legumes was similar. In birdsfoot trefoil and alfalfa, the ED_OM and ED_N were greater  (P < 0.04) in plants in vegetative stage than in bloom; meanwhile, in red clover the ED_OM, but not ED_N, was greater (P=0.023)  in vegetative stage. Vegetative forages tended (P < 0.06) to present the greatest RAN: FOM, and no phenological stages effect was detected in intestinal digestibility of RUP.

Results provide useful data for estimating protein supply to grazing ruminants. Research is needed to expand databases on N and OM characteristics of forages, in order to use accurately current feeding standards in grazing production systems.

Key words: alfalfa, birdsfoot trefoil, chemical composition, red clover

Introduction

As stated by Taweel 2006 “Grazing and grass-feeding production systems are regaining popularity and importance in the western world due to consumers’ demand, and concern on product quality, animal well-being, and landscape improvements. To maximize animal production from pasture-based systems in a cost-effective manner while sustaining the health, welfare of the animals, and the environment, it is important to maximize the efficiency of utilization of pastures.”   To efficiently convert plant biomass to animal products it is of special importance to accurately estimate feeding values of native, and cultivated pastures and forages. In current feeding systems for ruminants (AFRC 1993; NRC 1996; NRC 2001), feed nutrients are fractionated considering its ruminal fermentation, and postruminal digestion of undegraded fractions, and information on kinetics and extent of degradation of N, and organic matter (OM) in the rumen is required. However, in grazing conditions, limitations to use these feeding systems arise because reliable information on protein, and OM degradation characteristics of forages is lacking (Fulkerson et al 2007). In these systems, goals of rumen nutrition are to efficiently incorporate rumen degradable protein (RDP) to microbial protein, and to avoid production of ammonia in excess to that assimilated by microorganisms (Bach et al 2005).  Efficiency of utilization of RDP will mainly depend on feed fermentable energy which is usually the first limiting nutrient for microbial growth in pasture fed animals (Fulkerson et al 2007). In forages, the amount of OM fermented in the rumen will define the energy availability to rumen microbia, and OM fermentability will mainly depend on amounts of cell content carbohydrates, which are easily degraded in the rumen, and polysaccharides associated with cell wall whose degradation will depend on cell wall chemistry, and structure (Wilson and Hatfield 1997; Tas et al 2006). Rumen undegradable protein (RUP) contributes to the metabolizable protein pool depending on proportion of the RUP digested postruminally. In the AFRC (1993) system, as well as in the Beef level 1 (1996) and NRC Dairy (2001) standards, in situ methodologies are the main procedures adopted to estimate RDP and RUP, and digestibility of RUP. In level 2 of Beef model (NRC 1996), the Cornell Net Carbohydrate and Protein System (CNCPS) was adopted, and chemically determined protein fractions are used. The sensitivity of the models to their respective inputs emphasizes the need to estimate protein characteristics of different species of pastures grown in different environments, and evaluated in various phenological stages, as those factors may determine wide variation in chemical composition, fermentation characteristics, extent of postruminal digestion, and would contribute to accurately predict or evaluate animal response (Kingston-Smith and Theodorou 2000; Schwab et al 2003; Tas et al 2006). The objectives of this study were to quantify differences in fractionation of N, degradability and degradation kinetics of OM and N, adequacy of forages for ruminal microbial activity, and intestinal digestibility of RUP of three cultivated legumes in different phenological stages.

Materials and methods 

All procedures in this study were conducted according to the Good Practice Guide for the Use of Animals in Research, Testing and Teaching of the Universidad de la República of Uruguay (Diario Oficial N° 25.467  2000).

Forages

Three legumes from a Research Project on Nutritional Evaluation of Grazed Forages were evaluated. Samples were obtained from experimental plots (0.75 ha) located in the Centro Regional Sur (34.5°S, 56°W) of the Facultad de Agronomía (Universidad de la República, Uruguay). Legumes were Lotus corniculatus, va. San Gabriel (birdsfoot trefoil), Trifolium pratense, va. LE 116 (red clover), and Medicago sativa, cv. Estanzuela Chaná (alfalfa), respectively,  seeded in may at rates of  17, 10, and 19 kg/ha,  fertilized at sow  with 30, 30 and 50  kg of P / ha,   and annually refertilized with 20 kg of P  /ha.  Monthly rainfall , and maximum and minimum temperatures for the experimental site during the period when legume samples were taken for nutrient analysis, are shown in Figure 1.  During three consecutive years, pastures  were harvested once they reached the grazing height (average 22 cm; range: 17 to 28 cm); as a result of this harvesting schedule,  10, 9 and 12 samples of birdsfoot trefoil, red clover, and alfalfa were obtained.

Individual samples evaluated here were composited samples  obtained from daily cuts (7 d, mowing machine, approximately 5 cm above the soil level) of forages utilized in digestion, and intake trials with wethers (data not shown). Samples were dried at 60^oC (48 h), ground in a Willey mill through a 2 mm screen, and stored for chemical analysis, and degradability and intestinal digestibility trials. At each harvest date, and previous to harvest, in each legume 50 stems were randomly selected to define the average phenological stage of herbage. Stems were cut with a manual scissor and  individually classified in a four stage numerical system. Maturity stages was assigned as follows:  maturity 1 =  no visible green buds, flowers or seed pods, maturity 2 = visible green buds but not flowers or seed pods, maturity 3 = visible flowers but no seed pods, and maturity 4 = visible green seed pods. Amount of stems in each stage was counted, and herbages were scored for phenological stage using the mean by count index (Kalu and Fick 1981). General descriptive maturity classifications were as follows: maturity 1 = vegetative; maturity 2 =  pre bloom; maturity 3 = bloom, and maturity 4 = seed pod.   Year and month of harvest, and phenological stages of pastures evaluated are presented in Table 1.

Chemical analysis

Dry matter (DM), ash, and nitrogen (Kjeldahl, lysine as standard, crude protein CP, N x 6.25) were determined according to AOAC (2007). Neutral detergent fiber (NDF), acid detergent fiber (ADF) (both expressed in ash-free basis), and sulphuric acid lignin (lignin) were determined sequentially (Van Soest et al 1991). Neither sodium sulphite nor heat stable α-amylase were used in the neutral detergent solution. Non-fiber carbohydrates (NFC) were calculated by difference (NRC 2001).  Nitrogen was partitioned into five fractions (fractions A, B1, B2, B3, and C) in accordance with  Cornell Net Carbohydrate and Protein System (Sniffen et al 1992), and neutral detergent insoluble N (NDIN) were determined, using the procedures described by Licitra et al (1996) and Van Soest et al (1991) omitting sodium sulfite, and heat/stable α-amylase from the neutral detergent solution. Fraction A is non protein N (NPN), fraction B₁ is rapidly degraded true protein, fraction B₂  estimates the true protein with an intermediate degradation rate, and fractions B₃ and C (acid detergent insoluble N, ADIN) represent, respectively, slowly degraded true protein and unavailable protein bound to cell wall.

In situ procedures: ruminal and intestinal incubations

Three dry Holstein cows with permanent rumen and T duodenal cannulas housed in individual  stalls were used to evaluate in situ disappearance kinetics of N and OM, and intestinal digestibility of undegraded protein. Animals were fed 10 kg DM of alfalfa hay (170 g CP/kg DM, 420 g NDF/ kg DM) in two meals (8 and 17 h), with free access to water and mineral salts (950g minerals /  kg, 125 g Ca / kg, 450 g NaCl/ kg, 4.0 ppm F, Ca:P = 2:1). Ruminal degradation kinetics of N and OM was studied using the nylon bag technique  (Ørskov and McDonald 1979). Polyester bags (14 × 9 cm; 50 ± 15 μm pore size; ANKOM Technology, Macedon, NY) with 3 g dried forage were incubated during  2, 4, 8, 12, 24, 48, and 72 h in the rumen of each animal in two consecutive periods. Previous to incubation, bags were soaked 15 min in warm (39^oC) water. Bags were placed simultaneously in the rumen immediately after the morning meal, and removed sequentially at designated times. After collected from rumen, bags were rinsed with cold water, and stored at -20^oC. When thawed, bags were washed three times in washing machine (30 L, 30 bags per washing batch, 3 min), dried in a forced-air oven (60^oC, 48h), and weighted. Six bags per forage sample were not incubated in the rumen, and manipulated as the incubated ones to obtain the zero h value. Residues of replicates per time within cow were pooled for N and ash analysis (AOAC 2007). Intestinal digestibility of rumen undegradable protein (RUP) was estimated by the mobile bag technique (Peyraud et al 1988). Fifteen nylon bags (6 x 7 cm, pore size 45 µm) per forage filled with 1.5 g, were soaked in warm water (39^oC, 15 min), and incubated by 16 h in the rumen (30 bags/cow/day). After incubation, bags were placed (2.5 h) in acid pepsin-HCl solution (pH 2; 3 g pepsin/ L 0.1 N HCl) in water bath (39 ^oC), and shaked every 5 min. Afterwards, bags were introduced into the small intestine during the evening meal (17 h, 15 bags/cow/day,); each bag was carefully washed by chyme before introducing the following one. Bags were recovered from faeces the following day from 8 to 17 h, and bags appearing after 17 h were discarded. Recovered bags were rinsed under cold tap water, and frozen (-20 ^oC). Six bags per forage, previously soaked in warm water (39ºC), were incubated 16 h in cows rumen to estimate nitrogen available for postruminal digestion. Bags characteristics and manipulation were the same as described before. When thawed, bags were machine-washed (30 L, 60 bags/ washing batch, 3 cycles, 3 min), dried (60ºC, 48 h), weighted and stored for N analysis (AOAC 2007).

Calculations

The kinetics of N and OM disappearance from incubated bags with rumen incubation time (t; including zero values) was described for each animal using models of Ørskov and McDonald (1979) (Model 1,without lag time), and Dhanoa (1988) (Model 2,with lag time).

Model 1:   Disappearance (g/kg N or OM) = a + b x (1 – e^–kdt)

Model 2:  Disappearance (g/ kg N or OM) = a + b x (1 – e^–kd(t-L))

Constants a and b represent, respectively, the soluble fraction (SF, g/ kg N or OM) and the potentially degradable fraction (PDF, g/ kg N or OM) which disappears at a constant fractional rate (kd, /h) per unit of time (t), and L is a discrete digestion lag time (h). Degradation parameters were calculated using the no linear procedure with Marquardt method (PROC NLIN) (SAS Institute, Inc., Cary, NC). The non linear parameters a, b, kd, and L estimated by the model with the largest r², were reported. Effective degradability (ED) was calculated using the following  equation:  

ED (g / kg  N or OM) = a + [b x kd/(kd + kp)] x e^-kpL

where kp is the passage rate (0.06/ h).

To evaluate the adequacy of forages for ruminal microbial activity, the supply of rumen available N (RAN) per kg of fermentable organic matter (FOM) was calculated. The  RAN  and FOM were calculated according to  Bach et al (2005), and NRC (2001) using the following equations:

RAN (g / kg N) = 1.1 x N in forage (g /kg DM) x ED of N (g/ kg N)

FOM (g/ kg OM) = OM in forage (g/ kg DM) x ED of OM (g/ kgOM)

The intestinal digestibility of  RUP was calculated as the amount of N lost from intestinal incubated bags divided by the amount of N in the bags before intestinal passage, and was expressed as proportion of RUP (g/ kg RUP).

Statistical analysis

Data were analyzed in a complete randomized (chemical composition data), or a randomized block design (degradability, forages adequacy for ruminal microbial activity, and digestibility data) in a nested mixed model using a mixed procedure (PROC MIXED) (SAS Institute., Inc., Cary, NC) with degrees of freedom adjusted by the Kenward - Roger method. The model included specie, and phenological stage nested within specie as main effects, and animal (if corresponded) as random effect. Means were considered to differ when P < 0.05, and differences among means with P values between 0.05 and 0.10 were accepted as representing tendencies to differences. Pearson correlation between proportion  of CP as  fraction C, and  intestinal digestibility of RUP, was computed using a correlation procedure (PROC CORR) (SAS Institute., Inc., Cary, NC). Correlations were considered significant at P < 0.05.

Results  

Legumes

Chemical composition of legumes is presented in Tables 2.

Differences (P<0.004) in ADF, lignin, and proportions of CP as NDIN, fractions B3 and C, were detected among species. Birdsfoot trefoil registered the greatest (P<0.04) ADF and lignin, and alfalfa presented the lowest (P<0.03) proportions of N as NDIN, fraction B3 and C. The three legumes presented most of its N as fractions associated with cell content proteins (fractions A + B1 + B2; 600, 550 and 710 g/kg N, for birdsfoot trefoil, red clover and alfalfa, respectively), presenting alfalfa the greatest value (P<0.002), and similar proportions of N as soluble N (fractions A + B1; 250 g/kg N, respectively). However, birdsfoot trefoil tended (P=0.075) to register the lowest NNP (fraction A) in soluble N (510, 640 and 660g/kg soluble N, for birdsfoot trefoil, red clover, and alfalfa, respectively).

Disappearance data of OM, and N did not show evidence of lag time and fitted t SEQ CHAPTER \h \r 1he model of Ørskov and McDonald (1979) with r² that exceeded 0.70 for all individual sample-cow curves. The PDF, kd , and ED of OM and N (Table 3) did not differ among species, and birdsfoot trefoil presented the smallest (P<0.05) SF of OM and N .

Considering all individual samples, ranges of  ED_OM and ED_N  were, respectively  from 390 to 690, and 600 to 840 g/ kg, presenting all individual samples ED_N greater  than 590 g/kg N,  but only 25% of them with ED_OM greater than 600 g/kg OM. The forage adequacy for rumen microbial activity, estimated by the RAN: FOM ratio was similar in the three legumes (Table 3), and in most (87%) individual samples, values were greater than 42 g RAN/kg FOM. Considerable variation in the relationship between the RAN and FOM was observed when individual samples of the three legumes were considered (430 to 910, 310 to 630, and 440 to 740 g RAN / kg FOM in birdsfoot trefoil, red clover and alfalfa respectively). The intestinal digestibility of RUP of the red clover and alfalfa was similar, and tended  (P<0.06) to be greater than in birdsfoot trefoil (Table 3).  In alfalfa and red clover, 50 and 67%, of respectively individual samples presented intestinal digestibilities between 500 and 700 g/ kg RUP, whereas in birdsfoot trefoil, 60% of the pastures resulted in intestinal digestibilities lower than 500 g/ kg RUP. All samples considered, a significant and low negative correlation (r = - 0.35; n = 39; P<0.05) was established  between the intestinal digestibility of RUP and fraction C, explaining this fraction  12% of the total variation observed.

Phenological stage

In the three species, forages in vegetative stage presented the lowest (P<0.04) DM, ADF, and the greatest (P<0.03) CP. Additionally, in birdsfoot trefoil and alfalfa, forages in bloom presented the greatest (P<0.04) NDF, NDIN and fraction C (Table 4).

Within each legume specie, parameters of degradation of OM and N were similar in the different phenological stages (Tables 5).

In birdsfoot trefoil and alfalfa, the ED of OM and N of plants in vegetative stage was greater  (P<0.04) than  forages in bloom; meanwhile, in red clover the ED of OM, but not of N, was greater (P=0.02) in the vegetative than in the prebloom stage. A trend (P<0.06) for phenological stages to affect RAN: FOM ratio was detected in birdsfoot trefoil and alfalfa (vegetative vs bloom), and red clover (vegetative vs prebloom) (Table 5). Phenological stages did not resulted in differences in intestinal digestibility of RUP (Table 5).

Discussion 

Legumes

The NDF, ADF, and lignin contents of the three legumes were in the range of values reported by Hoffman et al (1993), Mieres (2004), and Fulkerson et al (2007). Similar cell wall content was observed in legumes species with birdsfoot trefoil presenting the greatest lignification. The greatest ADF in birdsfoot trefoil may reflect the presence of tannins, because species of the Lotus genus produce condensed tannins, which may appear in the ADF as a result of oxidative polymerization during the drying of NDF residue in sequential fiber determination (Van Soest 1994). As the three legumes presented similar CP, the smaller NDIN in alfalfa than in birsdsfoot trefoil and red clover, suggested that this specie could present greater proportions of the most degradadable proteins (proteins of cell contents). This difference in NDIN between alfalfa and red clover  was also reported by Sanderson and Wedin (1989). The proportion of N as NDIN of the three species was greater than values reported by Sanderson and Wedin (1989), Coblentz et al (1998), Tedeschi et al (2001), and Faría-Mármol et al (2002). This may be partially explained by differences in drying procedure of forages, and the presence of α-amylase in the neutral detergent solution. It has been reported, (Valk et al 1996; NRC 2001) that oven drying of forages (as performed in this study) may increase NDIN of pastures (without affecting ADIN) as compared with freeze drying, and that the inclusion α-amylase (as reported by Sanderson and Wedin 1989, Coblentz et al 1998, and Faría-Mármol et al 2002) may have contributed to explain NDIN differences because the  inclusion of the enzyme in the neutral solution may decrease this fraction. In the cell wall of the three legumes, fraction C was smaller than fraction B₃ suggesting that functional proteins predominated over structural ones. The greater fraction C in alfalfa than in birdsfoot trefoil and red clover could be associated with the lower lignin in alfalfa because fraction C is assumed to be structural N associated with lignin or lignin artifacts, reflecting this fraction differences in lignin concentration among species (Van Soest, 1994). 

In legumes,  SF, PDF, kd, and ED of OM  and N presented in this study were in good agreement with values reported elsewhere (Hoffman et al 1993; Niwinska et al 2005; Fulkerson et al 2007, 2008). The extent, and kinetics of OM degradation of forages in the rumen, will depend on content of NFC (which are almost instantly and completely degraded in the rumen), the rate and extent of degradation of cell wall polysaccharides (which depend on wall chemistry and anatomical structure of cells and tissues), and amount, and degradation kinetics of forage proteins (Wilson and Hatfield 1997; Tas et al 2006).  In this work, differences in ED_OM and similarities in SF, PDF and  kd  of OM detected among legumes, seemed to be associated to kinetics of N degradation and not to variations in NFC or cell wall content and composition, because the same patterns in SF, PDF, kd, and ED of OM and N were observed. The greater SF_N in alfalfa than in birdsfoot trefoil, may have responded to the greater proportion of N in cell content , and NPN in soluble protein in alfalfa, because the proportion of NPN and true protein in total N are factors associated with the susceptibility and accessibility of peptide bonds to microbial proteases (NRC 2001; Bach et al 2005).

The RAN:FOM ratio of most individual samples was greater than the optimal ratio (42 g of RAN/ Kg FOM)  proposed (Bach et al 2005) to achieve maximum efficiency of energy use, and maximum capture of N by rumen microorganisms. It could be expected than in the rumen of animals grazing these forages, there would be more N than the microbes could convert to protein, and ammonia in excess may be produced. The excess rumen N-NH3 may lead to reduce animal production because energy from forages will be used to convert ammonia to non toxic urea in the liver, and excrete it in the urine (Fulkerson et al 1998).

In this study, most (73%) of individual samples of the three legumes presented values of intestinal digestibility of RUP lower than tabular values (range: 650 to 700 g/ kg RUP; INRA, 1989; NRC, 2001), suggesting that tabular values could be overestimated. The trend for lower digestibility in birdsfoot trefoil than in alfalfa, could be associated to characteristics, and bioavailability  of N compounds escaping rumen fermentation. In this experiment birdsfoot trefoil presented greater fraction B₃ and C, which are the main fractions expected to escape rumen fermentation. Fraction B₃  estimates protein related to the  biosynthesis and modifications of cell wall components (functional proteins), and fraction C estimates proteins related to the architecture  of the cell wall (structural proteins) ,  being expected that structural proteins would be  less digestible than functional ones  (Kingston-Smith and Theodorou 2000; Cone et al 2006). Additionally, bioavailability of cell wall proteins will depend on their accessibility to digestive enzymes, what in turn will depend on amount, and characteristics of cell wall because these N compounds are embedded within the cellulose and hemicelluloses fibrils whose degree of lignification may vary. Then, the possible lower intestinal digestibility of RUP in birdsfoot trefoil than in alfalfa could have resulted from differences related to cell wall composition because birdsfoot trefoil presented similar NDF but greater ADF and lignin. Differences could also be associated to presence of tannins because Lotus genus are known for their high, and variable content of condensed tannins which interfere with digestibility of cell wall and protein, due to an incomplete release of protein bound to tannins in the lower gastrointestinal tract (Van Soest 1994; Marley et al 2005).

Phenological stage

The greater DM, NDF and ADF as though the less CP with greater proportion of N associated to cell wall (NDIN), particularly fraction C, between forages in vegetative, and pre bloom or bloom stages, reflected changes in cell metabolic activities, and amounts and characteristics of cell walls associated with advances in plants maturity (Jarrige et al 1995).

Phenological effects in ED of OM, and N appeared related to variations in content, and structure of cell walls, and fractions of N associated to cell wall, because the most mature forages presented the greatest NDF, ADF, NDIN, and fraction C. Similar differences in ED were reported in birdfoot trefoil, red clover, and alfalfa (Hoffman et al 1993; Elizalde et al 1999) although values observed in this study were lower than reported by these authors for forages (dried at 60^oC) in similar phenological stages. The similar OM concentration but lowest ED_OM  in the most  mature forages, suggested that animals grazing mature legumes would have less rumen fermentable energy than when grazing forages in vegetative stage; however, the lowest CP and ED_N in the most mature forages would determine these forages could be more adequate than plants in vegetative stage, for microbial activity as result of a more adequate RAN:FOM ratio.

Conclusions 

• Results of this study indicated that considerable variation exists in rumen degradability of N and OM, intestinal digestibility of RUP, and rumen adequacy for rumen microbial activity among individual samples of cultivated legumes. However, available data on these characteristics of grazing forages are scarce, therefore, it is clear that the application of current feeding standards may present limitations in grazing production systems. The use of average values without taking into consideration variability within and among species, and phenological stages may lead to inaccurate feed formulation with a major impact on production and feed cost. Information presented here is becoming increasingly important as environmental pressures increases to use efficiently N in animal rations in order to reduce overfeeding of protein which may lead to excessive N contamination. The results of this study suggests that further research should be conducted to expand the current database on N and OM characteristics of pastures, to accurate use the current feeding standards in grazing production systems.
   

Acknowledgments 

The authors wish to thank the R. Bentos, B.Cotro, P.de Oliveira, L.Dutra, J.Irigoyen, N.Machin, and S. Scremini for their assistance with measurements and care of experimental cows. This research was funded by the Comisión Sectorial de Investigación Científica (CSIC) of the Universidad de la República, Uruguay.

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Received 31 October 2009; Accepted 14 December 2009; Published 7 February 2010

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