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In vitro microbial fermentation and protein utilisation of tropical forage legumes grown during the dry season
Animal Feed Science and Technology 95 (2002) 1±14
In vitro microbial fermentation and protein utilisation of tropical forage legumes grown during the dry season M. Fondevilaa,*, J.C.M. Nogueira-Filhob, A. Barrios-Urdanetac a
Departamento de ProduccioÂn Animal y Ciencia de los Alimentos, Universidad de Zaragoza, M. Servet 177, 50013 Zaragoza, Spain b Facultade de Zootecnia e Engenharia de Alimentos, Universidade de Sao Paulo, Pirassununga, Brazil c Facultad de AgronomõÂa, Universidad del Zulia, Maracaibo, Venezuela Received 6 November 2000; received in revised form 29 May 2001; accepted 10 October 2001
Abstract The nutritive value of four tropical forage legumes grown during the dry season was assessed by gas production, microbial adhesion and in vitro N degradation and intestinal digestion. Species tested were: Leucaena leucocephala (Ll), Cajanus cajan (Cc), Neonotonia wightii (Nw) and Stylosanthes macrocephala (Sm). Chemical analysis showed higher ligni®cation of Sm (187 g acid detergent lignin, ADL/kg dry matter, DM) compared with Nw (92 g ADL/kg DM), and a higher concentration of extractable and ®bre-bound condensed tannins (CT) in browse (Ll and Cc) than in herbaceous (Nw and Sm) legumes (82, 110, 6 and 6 g total CT/kg DM, respectively). A higher total gas volume occurred for Nw (188, 163, 146 and 130 ml gas/g DM for Nw, Ll, Cc and Sm, respectively; P < 0:001). Rates of fermentation at 24 h were 0.069, 0.027, 0.046 and 0.051 h 1 for Nw, Ll, Cc and Sm (P < 0:001). The DM degradation of the legume species ranked them similar to gas production. There were no differences among legumes in microbial adhesion patterns, nor major differences in enzymatic activity against structural polysaccharides until 32 h, but then Nw showed higher total cellulase (P < 0:001), xylanase (P < 0:001), b-glucosidase (P < 0:01) and b±xylosidase (P < 0:05) activities than Ll and Cc. In vitro nitrogen disappearance after 32 h incubation was higher for Nw, intermediate for Sm and lowest for Ll and Cc (P < 0:001), probably because of the high CT concentration in the latter. Intestinal digestion of Ll and Cc was also low, suggesting that CT may still be impeding digestion to a certain extent at this site. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Tropical forage legumes; Gas production; Microbial activity; Nitrogen digestion
* Corresponding author. Tel.: 34-976-761660; fax: 34-976-761590. E-mail address: [email protected] (M. Fondevila).
0377-8401/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 1 ) 0 0 3 1 5 - 7
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1. Introduction Beef production in central Brazil is based on pastures, mostly of grasses. During the April to September dry season scarce water availability causes a drop in the nutritional quality of forage as proportions of cell wall and lignin increase and that of protein falls. This leads to a de®cit in rumen fermentable N that may compromise microbial growth thereby reducing supplies of intestinally absorbable protein. As a result, ruminants offered these forages as the only feed have low-dry matter (DM) intake and growth rates. Feeding of forage legumes (Roberts, 1987; Humphreys, 1995) as a protein source with grasses has been proposed as an inexpensive option to increase N intake and diet digestibility during the dry season. Lascano and Estrada (1989) observed an increase in liveweight gains (from 252 to 487 g per day) through 9 consecutive years during the dry period in a Pueraria phaseoloides±Brachiaria decumbens co-culture, when compared to a B. decumbens pasture. Elliott and McMeniman (1987) suggested that, in general, bovine weight gains may be increased up to 400% in adequately fertilised mixed grass-legume tropical pastures versus pastures of grasses alone. Since forage legumes are voluminous and take up space in the rumen, those species that are more rapidly degraded will elicit increased DM intake of grass forages by alleviating nutritional de®ciencies, and by disappearing faster from the rumen. However, tropical legumes at advanced stages of maturity, especially browse species, have a considerable proportion of condensed tannins (Nsahlai et al., 1994; Jackson et al., 1996), that may form complexes with proteins and carbohydrates, reducing their ruminal degradability. Whereas this generally increases ef®ciency of rumen N utilisation and intestinal input of feed N, it can restrict ®bre digestion in the rumen, resulting in unsynchronised availability of N and energy to microbes (Butter et al., 1999), and reduced diet intake due to rumen ®lling. Condensed tannins can diminish rumen proteolysis, and the resulting lower rumen ammonia concentration may also limit microbial activity (Bae et al., 1993; McAllister et al., 1994). The objective was to compare the chemical composition, in vitro fermentation pattern and protein utilisation of four species of tropical legumes harvested during the advanced dry season in central Brazil, in order to determine the most suitable species as supplements for bovines held on pastures during the dry season. 2. Materials and methods 2.1. Feed samples The four legume species evaluated were grown on the Campus of the Facultade de Zootecnia e Engenheria de Alimentos of the University of Sao Paulo (Pirassununga, Sao Paulo, Brazil). Climate and soil characteristics were de®ned by Nogueira et al. (2000). Species tested were chosen because of their importance for ruminant nutrition in this region, and were: Leucaena leucocephala (Ll); Cajanus cajan (Cc); Neonotonia wightii (Nw) and Stylosanthes macrocephala (Sm). Browse legumes (Ll and Cc) were planted in October 1995 and their leaves and small stems (below 0.5 cm diameter) were clipped in
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September 1996. Herbaceous legumes (Nw and Sm) were cut 10 cm above the ground in June 1996, and harvested in September after 100 days of regrowth, with a total rainfall of 58 mm throughout the period. Samples were dried at 45 8C in an air-forced oven and ground through a 1 mm screen before in vitro trials and chemical analysis. 2.2. In vitro gas production The microbial fermentation pattern of forages was studied by measuring the volume of gas produced during the in vitro incubation of samples (200 mg DM per syringe) according to the technique of Menke and Steingass (1988), as described by GonzaÂlez Ronquillo et al. (1998), in three fermentation series with duplicate syringes for each substrate on each series. Rumen contents were obtained from three adult sheep via rumen cannula just before the morning feeding (900 g of a 50:50 barley straw and alfalfa hay diet enriched with 20 g of a vitamin±mineral mixture), pooled and used as the main portion of the inoculum solution (Menke and Steingass, 1988). The volume of gas produced was recorded after 2, 4, 6, 8, 12, 14, 21, 24, 30, 36, 48, 60, 72 and 96 h of incubation. After the incubation period, fermentation residues of each syringe were recovered and washed with distilled water, ®ltered (40±100 mm pore size) dried at 60 8C for 48 h to estimate the proportion of DM that disappeared (DMd) and the relative gas yield (RGY: ml of gas after 96 h/g of DMd) as in GonzaÂlez Ronquillo et al. (1998). Apparent organic matter (OM) digestibility (OMD, %) of forages was estimated from the volume of gas produced after 24 h of incubation (GP, ml/ 200 mg DM) and the proportion of crude protein (CP, g/kg DM), following the equation of Menke and Steingass (1988): OMD 24:91 0:7222 GP 0:0815 CP;
n 185; R2 0:78
To estimate the pattern of microbial fermentation, cumulative gas production was ®tted iteratively to the model proposed by France et al. (1993): p p y Af1 exp b t T c t T g where y represents the cumulative gas production (ml), t is incubation time (h), A is asymptote (total gas; ml), T is lag time (h) and b and c are rate constants (h 1 and h 1/2). The fractional degradation rate (m, h 1) was considered to vary with time according to: c m b p ; t T 2 t 2.3. Microbial activity A subsequent experiment was carried out in vitro to measure bacterial adhesion and enzymatic activity over three species (Ll, Cc and Nw) chosen according to previous results. Incubation and sampling procedures have been described (Nogueira et al., 2000). In brief, two series of incubations were carried out, with duplicate 50 ml tubes per sample (500 mg DM) provided with a Bunsen-type valve, that were sampled after 4, 8, 12, 24 or 32 h. After each incubation series, residues from the two tubes of the same treatment were pooled, and half (500 mg, about 125±150 mg DM) was frozen at 20 8C for further enzymatic
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analyses. The enzymatic extract was obtained from the thawed subsample of the fermentation residue after its hydrolysis with lysozyme (Nogueira et al., 2000). Another part of the initial pooled residue (350 mg, about 90±110 mg DM) was kept at 60 8C in 3 ml 1N KOH for 30 min, and then freeze dried for determination of 15 N in the residue, as an index of bacterial adhesion. An extra tube with Ll as substrate was added to each series of incubation and removed at 24 h, for determining the 15 N enrichment in pure bacteria after detaching with a 0.1% methylcellulose solution and then processed for 15 N analysis, as referred in Nogueira et al. (2000). Finally, a third subsample was dried at 60 8C for 48 h for determining the residual DM content. 2.4. Laboratory and statistical analyses Plant samples were analysed for their DM by drying at 105 8C for 24 h, and organic matter (OM) by ashing at 550 8C for 6 h. Kjeldahl N was determined in a Kjeltec 1030 apparatus (Tecator), using. Se as catalyst and 1% boric acid in titration. Neutral detergent ®bre (NDF), acid detergent ®bre (ADF) and acid detergent lignin (ADL) were determined sequentially by the procedures proposed by Van Soest et al. (1991), without using sodium sulphite and alpha-amylase, and expressed without residual ashes. The acid detergent insoluble N (ADIN) was also measured according to Van Soest et al. (1991). Condensed tannins (CT) were extracted and analysed by the procedure proposed by Terrill et al. (1992), as modi®ed by PeÂrez Maldonado and Norton (1996), with three replicates per sample. Once determined the absorbance (550 nm), extractable, protein-bound and ®brebound CT values were adjusted to a standard curve using puri®ed quebracho tannins as standard. The intestinal digestibility of protein was determined according to Calsamiglia and Stern (1995). Forage samples (0.5 g) were incubated in vitro in quadruplicate tubes for 32 h, in each of two consecutive runs. The residue from one of the tubes was used for determining microbial non-degradable N, and the remaining three residues were pooled and dried at 60 8C for 48 h. Then, residues were digested in vitro with HCl-pepsin at pH 1.9 for 1 h and after with pancreatin at pH 7.8 for 24 h. Before calculations, the nitrogen contribution of pepsin-pancreatin solution (blank) was substracted from soluble N for correction purposes. Polysaccharidase activity of the enzymatic extract was tested colorimetrically against carboxymethylcellulose (CMC, Sigma) and xylan (Sigma) by the Nelson±Somogyi method, and b-glycosidase (b-D-glucosidase and b-D-xylosidase) activities were determined from the corresponding p-nitrophenyl compound (Sigma) as referred by Nogueira et al. (2000). Polysaccharidase (mmoles of sugar released per ml of extract and per min) and b-glycosidase (nmoles p-nitrophenol released per ml and per min) activities were expressed by mg of dry residue of fermentation (total activity) or by mg 15 N in the residue (bacterial activity). For bacterial adhesion studies, the inoculum was previously labelled with 15 N (52 mg 15 N per tube, using ammonium sulphate, 14 atom 15 N, ISOTEC Inc., OH, USA). Enrichment was determined using a mass spectrophotometer hooked in series to a DUMAS-style N analyser, and the calculations of bacterial concentration were performed as in Nogueira et al. (2000). All analyses were carried out in duplicate.
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For the statistical analysis of differences in gas production results, the mean of the two syringes of each sample in each series of incubation were used as the experimental unit. Differences among species by time of incubation were contrasted by ANOVA, with the interaction series of incubation species as the error term (8 d.f.). When microbial activity (results of adhesion and enzymatic activity) was studied, the main effects substrate, time of incubation and series were considered, and these and the interaction substrate time were contrasted with the residual error (14 d.f.). The N degradability and intestinal digestibility were also analysed by ANOVA, considering species as main factor and incubation run as factor, contrasted against an error term with 24 and 3 d.f., respectively. For all cases, differences among treatment means were identi®ed by the Tukey's test at P < 0:05. 3. Results Chemical analyses were performed on only one sample for each forage, and consequently were not compared statistically. However, among browse species Ll showed numerically lower NDF, ADF and ADL content than Cc (Table 1). Among herbaceous species, Nw gave lower proportions of these constituents than Sm, with an apparently larger amount of cell wall components in the latter than in both Ll and Cc. Total N content was very similar for Ll, Cc and Nw, but it was lower for Sm. The ADIN proportion was numerically higher for Sm, intermediate for Ll and Cc and lower for Nw. Total CT were markedly higher in browse than in herbaceous legumes, especially extractable and ®brebound CT. Protein-bound CT were 10-fold higher in Cc than in the other three legume species. Fig. 1 shows the average pattern of gas production from the legumes, as an index of microbial fermentation. Coef®cients of the equations obtained when those curves were Table 1 Chemical composition (g/kg dry matter) of the tropical legumesa Browse legumes
OM NDF ADF ADL N ADIN Extractable CT Fibre-bound CT Protein-bound CT Total CT
Herbaceous legumes
Ll
Cc
Nw
Sm
897 390 198 88 33.7 3.3 73.0 6.2 2.7 81.9
945 523 328 147 30.2 3.5 79.0 6.0 24.6 109.6
911 472 330 92 32.7 2.5 1.2 2.2 2.7 6.1
962 690 549 187 18.8 4.3 1.3 1.3 3.5 6.1
a Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii; Sm: S. macrocephala. OM: organic matter; N: nitrogen; NDF: neutral detergent fibre; ADF: acid detergent fibre; ADL: acid detergent lignin; ADIN: acid detergentinsoluble nitrogen; CT: condensed tannins.
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Fig. 1. Pattern of the volume of gas produced by in vitro microbial fermentation of L. leucocephala (*), C. cajan (&), N. wightii (~) and S. macrocephala (*). Upper vertical bars show standard errors of means.
adjusted to the model proposed by France et al. (1993), together with the average DMd, RGY and the estimated OMD of the species are in Table 2. From 12 h onwards, the volume of gas produced by fermentation of Nw was higher than that from Ll, Cc and Sm (P < 0:05 from 12 to 30 h and at 60 h; P < 0:01 at 36, 48, 72 and 96 h). There were no differences among the other three species at any incubation time. Estimated total gas production (Table 2) showed differences (P < 0:001), with Nw being the most, and Sm the least, fermentable species. There were no differences in lag time T. Both rate constants b and c showed differences among species (P < 0:001), and so the calculated fractional degradation rates were faster for Nw and slower for Ll, while Sm and Cc were intermediate. The DMd at the end of the experimental period showed differences (P < 0:001) that agreed with total gas production results. However, treatment comparison in RGYonly tended to be signi®cant (P < 0:10), with higher values for Sm and Cc than for Ll and Nw. Estimated OMD gave a higher value for Nw, intermediate for Ll and Cc and lower for Sm. Microbial adhesion to solid particles, estimated by 15 N in the second experiment comparing Ll, Cc and Nw up to 32 h incubation, is shown in Fig. 2. Among incubation times, adhesion at 4 h was less than from 12 to 32 h (P < 0:001), but there were no differences in microbial adhesion among legume species (P > 0:10) throughout. Since maximum differences in enzymatic activity occurred at the end of the studied period, only total (per unit of residue weight) and bacterial (per unit of adhered bacteria) enzymatic activities against carbohydrates of the extracts from the residues at 24 and 32 h of incubation are in Table 3. The pattern of total and bacterial polysaccharidases and b-glycosidases activities are shown in Figs. 3 and 4, respectively. Overall total cellulase
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Table 2 Parameters of curves fitted from in vitro gas production results, dry matter disappeared (DMd, mg/g), relative gas yielded (RGY, ml gas/g DM disappeared) and estimated organic matter digestibility (OMD, g/kg) of legumesa,b Browse legumes Ll c,d
A b c T m6 m12 m24 m48 DMd RGY OMD
163 ab 0.021 0.064 0.10 0.034 0.030 0.027 0.025 473 b 313 556
Herbaceous legumes Cc
c a b c c c
146 bc 0.044 0.021 0.44 0.048 0.047 0.046 0.045 412 bc 359 550
Nw b ab ab bc b b
188 a 0.077 a 0.078 c 0.70 0.061 a 0.066 a 0.069 a 0.072 a 634 a 306 628
S.E.M. Sm 130 c 0.054 b 0.028 bc 0.43 0.048 ab 0.050 ab 0.051 b 0.052 b 357 c 380 473
6.1 0.0036 0.0119 0.577 0.0038 0.0036 0.0035 0.0035 1.3 17.2 ±
a
Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii; Sm: S. macrocephala. Different letters (a, b, c) within rows indicate differences (P < 0:05). c Parameters: A: total gas production (ml/g incubated DM); b: fermentation rate (h 1); c: fermentation rate (h 1/2); T: lag time (h); m6, m12, m24, m48: fractional fermentation rates at different incubation times (h 1), estimated from the rate constants b and c. d There were differences (P < 0:001) in all parameters except for T (P > 0:05) and RGY (P < 0:10). OMD was not compared statistically. b
Fig. 2. Microbial adhesion to the substrate (L. leucocephala (*); C. cajan (&); N. wightii (~)), estimated by labelling mixed ruminal bacteria in the dry residue of fermentation with 15 N (S:E:M: 1:398).
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Table 3 Total (per g dry matter residue per min) and bacterial (per mg 15 N per min) polysaccharidase (CMCase or xylanase; mmoles sugar released), or b-glycosidase (b-glucosidase or b-xylosidase; nmoles p-nitrophenol released) activities of extracts from the solid fermentation residue of the legumes after 24 and 32 h of incubationa,b 24 h
Total CMCase Xylanase b-Glucosidase b-Xylosidase Bacterial CMCase Xylanase b-Glucosidase b-Xylosidase a b
32 h
S.E.M.
Ll
Cc
Nw
Ll
Cc
Nw
6.34 a 3.07 144.9 135.5 a
1.87 b 2.98 88.6 57.8 b
2.73 b 2.56 156.8 72.6 ab
2.41 b 0.98 b 52.1 b 45.6 b
1.15 b 1.46 b 90.8 b 75.4 b
7.08 a 6.48 a 173.3 a 150.3 a
0.844 a 0.405 19.1 18.2
0.134 b 0.213 6.4 4.2
0.198 b 0.184 12.3 5.5
0.183 ab 0.078 b 4.1 3.4
0.095 b 0.120 b 7.5 6.2
0.526 a 0.479 a 12.7 11.1
0.429 0.510 20.96 18.75 0.1150 0.0644 4.47 4.26
Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii. Different letters (a, b) within rows indicate differences (P < 0:05).
(against CMC) activity was higher (P < 0:001) for Ll and Nw than for Cc (2.58, 2.50 and 1.55 mmoles glucose/g DM), but these differences were only signi®cant (P < 0:001) between Ll and the other species at 24 h, and between Nw and the browse legumes at 32 h (Table 3 and Fig. 3). However, activity for Nw started later than for the browse legumes, and the maximum for Ll occurred at 24 h, whereas Nw peaked at 32 h. A similar pattern can be observed for total xylanase activity, but in this case there were no differences between the browse species, and activity over Nw was higher only at 32 h. Total bglucosidase activity was higher over Nw at 12 and 32 h (P < 0:001) and, whereas it plateaued from 4 h onwards for both browse legumes, it peaked at 12 h for Nw. The observed pattern of total b-xylosidase activity was variable throughout the control period, but differences (P < 0:005) occurred at 12, 24 and 32 h of incubation, and overall Nw was higher than Cc (P < 0:05). When expressed in bacterial concentration basis, differences between legumes in the pattern of enzymatic activity (Fig. 4) were smaller than those in total activity. Bacterial cellulase activity was higher in Ll at 24 h, and lower in Cc at 32 h (P < 0:05). Bacterial xylanase activity started earlier in Cc (P < 0:05 at 4 h), but at 32 h that from Nw was higher (P < 0:05) than that from the browse species. A higher bacterial b-glucosidase activity at 4 h was observed with Ll (P < 0:05), but no differences were observed afterwards. Nitrogen degradability after 32 h of in vitro microbial incubation, together with the intestinal digestion estimated enzymatically, are in Table 4. Degradability of the nitrogenous fraction were higher for Nw, intermediate for Sm, and gave similar low values for both browse species (P < 0:001). The intestinal digestion of the N undegraded after microbial fermentation was ranked as: Nw > Sm > Cc > Ll, with differences (P < 0:05) between Nw and Ll only.
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Fig. 3. Total polysaccharidase (mmol sugar released/g DM) activity (CMCase and xylanase), and bglycosidase (nmoles p-nitrophenol released/g DM) activity (b-glucosidase and b-xylosidase) of extracts from the solid fermentation residue of L. leucocephala (*), C. cajan (&) and N. wightii (~) at different incubation times.
Table 4 Microbial nitrogen degradation after 32 h of in vitro incubation and in vitro intestinal nitrogen digestion of the legumesa,b Browse legumes
Total N (g/kg DM) N degradation (g/g N) Intestinal N digestion g/g N at the duodenum g/g total N a b
Herbaceous legumes
Ll
Cc
Nw
Sm
33.7 0.316 c
30.2 0.315 c
32.7 0.728 a
18.8 0.548 b
0.442 b 0.303
0.545 ab 0.373
0.823 a 0.225
Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii; Sm: S. macrocephala. Different letters (a, b, c) within rows show significant differences (P < 0:05).
0.670 ab 0.303
S.E.M.
± 0.0178 0.0301 0.0273
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Fig. 4. Bacterial polysaccharidase (mmol sugar released/mg 15 N) activity (CMCase and xylanase), and bglycosidase (nmoles p-nitrophenol released/mg 15 N) activity (b-glucosidase and b-xylosidase) of extracts from the solid fermentation residue of L. leucocephala (*), C. cajan (&) and N. wightii (~) at different incubation times.
4. Discussion It has been speculated that the gas production technique may not be suitable for evaluating high-protein feeds because released ammonia may react with CO2 and precipitate, thus reducing gas volume (Menke and Steingass, 1988), and an inverse relationship has been observed between protein content of the test food and gas volume (Krishnamoorthy et al., 1995; GonzaÂlez Ronquillo et al., 1998). In our case, in vitro 32 h N degradation showed higher results for Nw, indicating that, although an underestimation of DM fermentation could have occurred because of a higher NH3 production (which cannot be veri®ed in our results), differences between Nw and the other species still remained. It is also worth noting that, as previously observed (Sileshi et al., 1996; Nogueira et al., 2000), DMd results which cannot be affected by ammonia concentration, are in agreement with those from gas production.
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4.1. Microbial fermentation Chemical comparison of the legumes established a clear difference between browse and herbaceous legume species, with the former having a much higher content of condensed tannins. The major differences are in extractable CT, which, according to Longland et al. (1995), are less involved in the restriction of microbial fermentation than bound CT. However, ®bre-bound CT are higher in browse species, suggesting a possible lower microbial utilisation of carbohydrates. Among the herbaceous species, Nw apparently had a lower NDF content than Sm. This can affect plant fermentation patterns, and the higher ADL content of Sm may explain its lower total gas production, slower fermentation rates and lower DMd compared with Nw. Gas production evolved similarly for browse species (Ll and Cc) throughout the control period, except for the faster rate with Cc, and there were no differences in total gas production. Other gas production trials in literature, (Nw versus Ll, Krishnamoorthy et al., 1995, Cc versus Ll, Siaw et al., 1993), ranked the species similarly, despite possible differences in growth conditions. Complexes with protein and carbohydrates are formed in tannin-rich plants, which restrict fermentation of cell walls by rumen microorganisms. Bae et al. (1993) showed that in vitro cellulose digestion by Fibrobacter succinogenes was affected when condensed tannins from Lotus corniculatus were added and a reduction in volatile fatty acid production in vitro has also been observed (Makkar et al., 1995). It has been suggested that tannins may prevent colonisation of leaf material (Chiquette et al., 1988), and McAllister et al. (1994) showed visual evidence of this effect on F. succinogenes colonisation of ®lter paper. However, this effect is strongly related with the type and concentration of tannins, and Makkar et al. (1989) reported that the plants with the higher tannin content were not necessarily those that support lower adhesion. Makkar et al. (1988) observed a reduction in microbial cellulases associated with increased CT concentration. Although microbial adhesion on Nw was numerically higher at 12 h than on Ll and Cc (15.0 versus 12.2 and 12.3 mg 15 N/g DM), and differences partly remained at 24 h (14.3 versus 9.9 mg 15 N/g DM on Nw and Ll), there were no differences in attachment of mixed rumen organisms to plant particles. Total and bacterial enzymatic activities against structural polysaccharides did not substantially differ greatly until 32 h incubation, but at this time total cellulase and xylanase activities over Nw were three-fold higher than over the browse legumes (Table 3). This may explain, at least in part, differences in gas production pattern among these species. However, the magnitude of existing differences between Nw and the tannin-rich Ll and Cc cannot be totally associated with a negative effect of the CT over microbial action either by adhesion or enzymatic activity, against cell wall structures. Thus, the lower fermentability of Ll and Cc compared with Nw may also be related to an effect of CT over other plant carbohydrates (Jansman and Longstaff, 1993) or to other factors than CT. 4.2. N utilisation In addition to their utility as energy sources for rumen microbes either as a complement or substitutes of grasses during the dry season, the potential as N sources of these legumes was also evaluated. Despite of water de®cit during growth, total N content of the species
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(except for Sm) was high. The N degradation after 32 h fermentation assuming a rumen rate of passage of 0.03 h 1 for these forages was low in browse legumes, presumably because of their high CT content. Poppi et al. (1999) observed higher extent and rate of protein degradation of Stylosanthes spp. than C. cajan or L. leucocephala, and Krishnamoorthy et al. (1995) showed a N degradation of 0.67 for N. wightii, whereas that for two L. leucocephala varieties was 0.24 and 0.51. However, variation in protein-bound CT proportions among species do not support such large differences in N degradation between Ll and the herbaceous legumes, and might indicate that other CT, either extractable or ®brebound, might be affecting the response. It is worth mentioning that the high CT content observed in Cc, especially the differences with Ll in protein-bound CT, can only be attributable to a poorer adaptation to drought conditions. The higher protein-bound CT proportion was not re¯ected in differences in N degradability between the browse species. It has been reported that in vitro estimation of intestinal digestion of N gives lower values than the in vivo mobile bags (Calsamiglia and Stern, 1995), mainly because of the synergistic action of various enzymes in vivo. In our lab, evaluation of different protein supplements with both techniques after 16 h in situ rumen incubation showed, on average, a 13% lower digestibility in vitro, but ranking was the same. Hence, this technique has been proven to be a useful tool for comparing protein sources. From results in Table 4, it can be calculated that the rumen degradable N from feed origin is 11, 10, 24 and 10 g N/kg feed DM, and the amount of N digested in the intestines is 10, 11, 7 and 6 g/kg for Ll, Cc, Nw and Sm, respectively. In this way, the proportions of nondegraded, non-digested N are 0.38, 0.31, 0.05 and 0.15 for the same species. Therefore, in vitro results indicate that the herbaceous species are better utilised as N sources, and the browse legumes give a higher amount of both feed N reaching the duodenum and N digested in the intestine, although this parameter is lower than expected. The lower intestinal digestion with Sm, as well as its lower degradability, compared with Nw can be justi®ed by its higher ligni®cation and ADIN content. However, the low intestinal N digestion from Ll and Cc implies that some limitation of protein availability because of tannins remains. Butter et al. (1999) suggested that, although tannin binding is dissociated under pH 5.6 or over pH 7.0, it might be formed again as the digesta passes from the abomasum to the intestines, thus reducing amino acid absorption. Indeed, Wang et al. (1996) indicated that some tannin±protein complexes are still present at the terminal ileum. In their review, Jansman and Longstaff (1993) suggested that dietary tannins might inhibit various enzymes from the intestinal mucosa. In agreement with our results, GarcõÂa et al. (1996) reported a low rumen protein degradability of L. leucocephala (0.42), but also a post-ruminal digestion of undegradable protein of only 0.48. Jones and Palmer (2000), estimating N digestion of some shrub legumes by an acidpepsin digestion after in vitro microbial fermentation, observed a 0.10±0.15 positive response to polyethylene glycol addition (the difference is assumed to be the CT effect). This indicates that protein in some shrub legumes is over-protected by the tannins, and so may not be completely available in the small intestine. Considering the tannin content of the same samples from another study, (Jackson et al., 1996) the negative effect of tannins on protein digestion is apparently more related to the extractable, than to the proteinbound CT, which might explain the small differences between Ll and Cc in estimated N intestinal digestion.
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5. Conclusions The practical interest of using these legumes as feeds during the dry season in the tropics mainly relies in their value as protein and energy sources albeit in¯uenced by growth conditions. N. wightii is fermented by rumen microorganisms in a greater extent than the other species, and most of its N is utilised. However, the input of feed N to the intestines is comparatively low because of its high N degradability. S. macrocephala is notably affected by drought, and its low N and high ADL and ADIN contents limit its microbial fermentation and N utilisation. Tannin-rich shrubs are less fermented than N. wightii, but this effect does not seem to be related to a negative effect of condensed tannins on microbial adhesion or enzymatic activity against structural polysaccharides. Even though L. leucocephala and C. cajan had a low intestinal N digestion, their low degradability by rumen microbes makes them an important source of N digestible protein in the intestines. However, the protective effect of condensed tannins of protein may remain to a biologically signi®cant degree in the lower portions of the digestive tract. Acknowledgements The stage of Dr. J.C.M. Nogueira Filho and Dr. A. Barrios Urdaneta in the University of Zaragoza were supported by FAPESP (Brazil) and CONICIT (Venezuela), respectively. References Bae, H.D., McAllister, T.A., Yanke, J., Cheng, K.J., Muir, A.D., 1993. Effect of condensed tannins on endoglucanase activity and filter paper digestion by Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 59, 2132±2138. Butter, N.L., Dawson, J.M., Buttery, P.J., 1999. Effects of dietary tannins on ruminants. In: Caygill, J.C., Mueller-Harvey, I. (Eds.), Secondary Plant Products. Antinutritional and Beneficial Actions in Animal Feeding. Nottingham University Press, UK, pp. 51±70. Calsamiglia, S., Stern, M.D., 1995. A three-step in vitro procedure for estimating intestinal digestion of protein in ruminants. J. Anim. Sci. 73, 1459±1465. Chiquette, J., Cheng, K.J., Costerton, J.W., Mulligan, L.P., 1988. Effect of tannins on the digestibility of two isosynthetic strains of birdsfoot trefoil (Lotus corniculatus L.) using in vitro and in situ techniques. Can. J. Anim. Sci. 68, 751±760. Elliott, R., McMeniman, N.P., 1987. Supplementation of ruminant diets with forages. In: Hacker, J.B., Ternouth, J.H. (Eds.), The Nutrition of Herbivores. Academic Press, Merrickville, Australia, pp. 409±428. France, J., Dhanoa, M.S., Theodorou, M.K., Lister, S.J., Davies, D.R., Isac, D., 1993. A model to interpret gas accumulation profiles associated with in vitro degradation of ruminant feeds. J. Theoret. Biol. 163, 99±111. GarcõÂa, G.W., Ferguson, T.V., Neckles, F.A., Archibald, K.A.E., 1996. The nutritive value and forage productivity of Leucaena leucocephala. Anim. Feed Sci. Technol. 60, 29±41. GonzaÂlez Ronquillo, M., Fondevila, M., Barrios Urdaneta, A., Newman, Y., 1998. In vitro gas production from buffel grass (Cenchrus ciliaris L.) fermentation in relation to the cutting interval, the level of nitrogen fertilisation and the season of growth. Anim. Feed Sci. Technol. 72, 19±32. Humphreys, L.R., 1995. Diversity and productivity of tropical legumes. In: D'Mello, J.P.F., Devendra, C. (Eds.), Tropical Legumes in Animal Nutrition. CAB International, Wallingford, UK, pp. 1±21. Jackson, F.S., Arry, T.N., Lascano, C., Palmer, B., 1996. The extractable and bound condensed tannin content of leaves from tropical tree, shrub and forage legumes. J. Sci. Food Agric. 71, 103±110.
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Jansman, A.J.M., Longstaff, M., 1993. Nutritional effects of tannins and vicine/convicine in legume seeds. In: van der Poel, A.F.B., Huisman, J., Saini, H.S. (Eds.), Recent Advances of Research in Antinutritional Factors in Legume Seeds. EAAP Publ. no. 70, Wageningen Pers, The Netherlands. Jones, R.J.Y., Palmer, B., 2000. In vitro digestion studies using 14 C-labelled polyethylene glycol (PEG) 4000: comparison of six tanniferous shrub legumes and the grass Pannicum maximum. Anim. Feed Sci. Technol. 85, 215±221. Krishnamoorthy, U., Soller, H., Steingass, H., Menke, K.H., 1995. Energy and protein evaluation of tropical feedstuffs for whole tract and ruminal digestion by chemical analysis and rumen inoculum studies in vitro. Anim. Feed Sci. Technol. 52, 177±188. Lascano, C., Estrada, J., 1989. Long-term productivity of legume-based and pure pastures in the eastern plains of Columbia. In: Proceedings of the 16th International Grassland Congress, Nice, France, pp. 1179±1180. Longland, A.C., Theodorou, M.K., Sanderson, R., Lister, S.J., Powell, C.J., Morris, P., 1995. Non-starch polysaccharide composition and in vitro fermentability of tropical forage legumes varying in phenolic content. Anim. Feed Sci. Technol. 55, 161±177. Makkar, H.P.S., Singh, B., Dawra, R.K., 1988. Effect of tannin-rich leaves of oak on various microbial activities of the rumen. Br. J. Nutr. 60, 287±296. Makkar, H.P.S., Singh, B., Negi, S.S., 1989. Relationship of rumen degradability with microbial colonization, cell wall constituents and tannin levels in some tree leaves. Anim. Production 49, 299±303. Makkar, H.P.S., Blummel, M., Becker, K., 1995. In vitro effects of and interaction between tannins and saponins and fate of tannins in the rumen. J. Sci. Food Agric. 69, 481±493. McAllister, T.A., Bae, H.D., Jones, G.A., Cheng, K.J., 1994. Microial attachment and feed digestion in the rumen. J. Anim. Sci. 72, 3004±3018. Menke, K.H., Steingass, H., 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Develop. 28, 7±55. Nogueira, J.C.M., Fondevila, M., Barrios-Urdaneta, A., GonzaÂlez Ronquillo, M., 2000. In vitro microbial fermentation of tropical grasses at an advanced maturity stage. Anim. Feed Sci. Technol. 83, 145±157. Nsahlai, I.V., Siaw, D.E.K.A., Osuji, P.O., 1994. The relationships between gas production and chemical composition of 23 browses of the genus Sesbania. J. Sci. Food Agric. 65, 13±20. PeÂrez Maldonado, R.A., Norton, B.W., 1996. The effect of condensed tannins from Desmodium intortum and Calliandria calothyrsus on protein and carbohydrate digestion in sheep and goats. Br. J. Nutr. 76, 515±533. Poppi, D.P., McLennan, S.R., Bediye, S., de Vega, A., Zorrilla-Rios, J., 1999. Forage quality: strategies for increasing nutritive value of forages. In: Buchanan-Smith, J.G., Bailey, L.D., McCaughey, P. (Eds.), Proceedings of the XVIII International Grassland Congress, Winipeg, Canada, 1997, pp. 307±322. Roberts, B.R., 1987. The availability of herbage. In: Hacker, J.B., Ternouth, J.H. (Eds.), The Nutrition of Herbivores. Academic Press, Merrickville, Australia, pp. 47±63. Siaw, D.E.K.A., Osuji, P.O., Nsahlai, I.V., 1993. Evaluation of multipurpose tree germplasm: the use of gas production and rumen degradation characteristics. J. Agric. Sci., Cambridge 120, 319±330. Sileshi, Z., Owen, E., Dhanoa, M.S., Theodorou, M.K., 1996. Prediction of in situ rumen dry matter disappearance of Ethiopian forages from an in vitro gas production technique using a pressure transducer, chemical analysis or in vitro digestibility. Anim. Feed Sci. Technol. 61, 73±87. Terrill, T.H., Rowan, A.M., Douglas, G.B., Barry, T.N., 1992. Determination of extractable and bound condensed tannin concentration in forage plants, protein concentrate meals and cereal grains. J. Sci. Food Agric. 58, 321±329. Wang, Y., Douglas, G.C., Waghorn, G.C., Barry, T.N., Foote, A.G., Purchas, R.W., 1996. Effect of condensed tannins upon the performance of lambs grazing Lotus corniculatus and lucerne (Medicago sativa). J. Agric. Sci., Cambridge 126, 87±98. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583±3597.
In vitro microbial fermentation and protein utilisation of tropical forage legumes grown during the dry season M. Fondevilaa,*, J.C.M. Nogueira-Filhob, A. Barrios-Urdanetac a
Departamento de ProduccioÂn Animal y Ciencia de los Alimentos, Universidad de Zaragoza, M. Servet 177, 50013 Zaragoza, Spain b Facultade de Zootecnia e Engenharia de Alimentos, Universidade de Sao Paulo, Pirassununga, Brazil c Facultad de AgronomõÂa, Universidad del Zulia, Maracaibo, Venezuela Received 6 November 2000; received in revised form 29 May 2001; accepted 10 October 2001
Abstract The nutritive value of four tropical forage legumes grown during the dry season was assessed by gas production, microbial adhesion and in vitro N degradation and intestinal digestion. Species tested were: Leucaena leucocephala (Ll), Cajanus cajan (Cc), Neonotonia wightii (Nw) and Stylosanthes macrocephala (Sm). Chemical analysis showed higher ligni®cation of Sm (187 g acid detergent lignin, ADL/kg dry matter, DM) compared with Nw (92 g ADL/kg DM), and a higher concentration of extractable and ®bre-bound condensed tannins (CT) in browse (Ll and Cc) than in herbaceous (Nw and Sm) legumes (82, 110, 6 and 6 g total CT/kg DM, respectively). A higher total gas volume occurred for Nw (188, 163, 146 and 130 ml gas/g DM for Nw, Ll, Cc and Sm, respectively; P < 0:001). Rates of fermentation at 24 h were 0.069, 0.027, 0.046 and 0.051 h 1 for Nw, Ll, Cc and Sm (P < 0:001). The DM degradation of the legume species ranked them similar to gas production. There were no differences among legumes in microbial adhesion patterns, nor major differences in enzymatic activity against structural polysaccharides until 32 h, but then Nw showed higher total cellulase (P < 0:001), xylanase (P < 0:001), b-glucosidase (P < 0:01) and b±xylosidase (P < 0:05) activities than Ll and Cc. In vitro nitrogen disappearance after 32 h incubation was higher for Nw, intermediate for Sm and lowest for Ll and Cc (P < 0:001), probably because of the high CT concentration in the latter. Intestinal digestion of Ll and Cc was also low, suggesting that CT may still be impeding digestion to a certain extent at this site. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Tropical forage legumes; Gas production; Microbial activity; Nitrogen digestion
* Corresponding author. Tel.: 34-976-761660; fax: 34-976-761590. E-mail address: [email protected] (M. Fondevila).
0377-8401/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 1 ) 0 0 3 1 5 - 7
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1. Introduction Beef production in central Brazil is based on pastures, mostly of grasses. During the April to September dry season scarce water availability causes a drop in the nutritional quality of forage as proportions of cell wall and lignin increase and that of protein falls. This leads to a de®cit in rumen fermentable N that may compromise microbial growth thereby reducing supplies of intestinally absorbable protein. As a result, ruminants offered these forages as the only feed have low-dry matter (DM) intake and growth rates. Feeding of forage legumes (Roberts, 1987; Humphreys, 1995) as a protein source with grasses has been proposed as an inexpensive option to increase N intake and diet digestibility during the dry season. Lascano and Estrada (1989) observed an increase in liveweight gains (from 252 to 487 g per day) through 9 consecutive years during the dry period in a Pueraria phaseoloides±Brachiaria decumbens co-culture, when compared to a B. decumbens pasture. Elliott and McMeniman (1987) suggested that, in general, bovine weight gains may be increased up to 400% in adequately fertilised mixed grass-legume tropical pastures versus pastures of grasses alone. Since forage legumes are voluminous and take up space in the rumen, those species that are more rapidly degraded will elicit increased DM intake of grass forages by alleviating nutritional de®ciencies, and by disappearing faster from the rumen. However, tropical legumes at advanced stages of maturity, especially browse species, have a considerable proportion of condensed tannins (Nsahlai et al., 1994; Jackson et al., 1996), that may form complexes with proteins and carbohydrates, reducing their ruminal degradability. Whereas this generally increases ef®ciency of rumen N utilisation and intestinal input of feed N, it can restrict ®bre digestion in the rumen, resulting in unsynchronised availability of N and energy to microbes (Butter et al., 1999), and reduced diet intake due to rumen ®lling. Condensed tannins can diminish rumen proteolysis, and the resulting lower rumen ammonia concentration may also limit microbial activity (Bae et al., 1993; McAllister et al., 1994). The objective was to compare the chemical composition, in vitro fermentation pattern and protein utilisation of four species of tropical legumes harvested during the advanced dry season in central Brazil, in order to determine the most suitable species as supplements for bovines held on pastures during the dry season. 2. Materials and methods 2.1. Feed samples The four legume species evaluated were grown on the Campus of the Facultade de Zootecnia e Engenheria de Alimentos of the University of Sao Paulo (Pirassununga, Sao Paulo, Brazil). Climate and soil characteristics were de®ned by Nogueira et al. (2000). Species tested were chosen because of their importance for ruminant nutrition in this region, and were: Leucaena leucocephala (Ll); Cajanus cajan (Cc); Neonotonia wightii (Nw) and Stylosanthes macrocephala (Sm). Browse legumes (Ll and Cc) were planted in October 1995 and their leaves and small stems (below 0.5 cm diameter) were clipped in
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September 1996. Herbaceous legumes (Nw and Sm) were cut 10 cm above the ground in June 1996, and harvested in September after 100 days of regrowth, with a total rainfall of 58 mm throughout the period. Samples were dried at 45 8C in an air-forced oven and ground through a 1 mm screen before in vitro trials and chemical analysis. 2.2. In vitro gas production The microbial fermentation pattern of forages was studied by measuring the volume of gas produced during the in vitro incubation of samples (200 mg DM per syringe) according to the technique of Menke and Steingass (1988), as described by GonzaÂlez Ronquillo et al. (1998), in three fermentation series with duplicate syringes for each substrate on each series. Rumen contents were obtained from three adult sheep via rumen cannula just before the morning feeding (900 g of a 50:50 barley straw and alfalfa hay diet enriched with 20 g of a vitamin±mineral mixture), pooled and used as the main portion of the inoculum solution (Menke and Steingass, 1988). The volume of gas produced was recorded after 2, 4, 6, 8, 12, 14, 21, 24, 30, 36, 48, 60, 72 and 96 h of incubation. After the incubation period, fermentation residues of each syringe were recovered and washed with distilled water, ®ltered (40±100 mm pore size) dried at 60 8C for 48 h to estimate the proportion of DM that disappeared (DMd) and the relative gas yield (RGY: ml of gas after 96 h/g of DMd) as in GonzaÂlez Ronquillo et al. (1998). Apparent organic matter (OM) digestibility (OMD, %) of forages was estimated from the volume of gas produced after 24 h of incubation (GP, ml/ 200 mg DM) and the proportion of crude protein (CP, g/kg DM), following the equation of Menke and Steingass (1988): OMD 24:91 0:7222 GP 0:0815 CP;
n 185; R2 0:78
To estimate the pattern of microbial fermentation, cumulative gas production was ®tted iteratively to the model proposed by France et al. (1993): p p y Af1 exp b t T c t T g where y represents the cumulative gas production (ml), t is incubation time (h), A is asymptote (total gas; ml), T is lag time (h) and b and c are rate constants (h 1 and h 1/2). The fractional degradation rate (m, h 1) was considered to vary with time according to: c m b p ; t T 2 t 2.3. Microbial activity A subsequent experiment was carried out in vitro to measure bacterial adhesion and enzymatic activity over three species (Ll, Cc and Nw) chosen according to previous results. Incubation and sampling procedures have been described (Nogueira et al., 2000). In brief, two series of incubations were carried out, with duplicate 50 ml tubes per sample (500 mg DM) provided with a Bunsen-type valve, that were sampled after 4, 8, 12, 24 or 32 h. After each incubation series, residues from the two tubes of the same treatment were pooled, and half (500 mg, about 125±150 mg DM) was frozen at 20 8C for further enzymatic
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analyses. The enzymatic extract was obtained from the thawed subsample of the fermentation residue after its hydrolysis with lysozyme (Nogueira et al., 2000). Another part of the initial pooled residue (350 mg, about 90±110 mg DM) was kept at 60 8C in 3 ml 1N KOH for 30 min, and then freeze dried for determination of 15 N in the residue, as an index of bacterial adhesion. An extra tube with Ll as substrate was added to each series of incubation and removed at 24 h, for determining the 15 N enrichment in pure bacteria after detaching with a 0.1% methylcellulose solution and then processed for 15 N analysis, as referred in Nogueira et al. (2000). Finally, a third subsample was dried at 60 8C for 48 h for determining the residual DM content. 2.4. Laboratory and statistical analyses Plant samples were analysed for their DM by drying at 105 8C for 24 h, and organic matter (OM) by ashing at 550 8C for 6 h. Kjeldahl N was determined in a Kjeltec 1030 apparatus (Tecator), using. Se as catalyst and 1% boric acid in titration. Neutral detergent ®bre (NDF), acid detergent ®bre (ADF) and acid detergent lignin (ADL) were determined sequentially by the procedures proposed by Van Soest et al. (1991), without using sodium sulphite and alpha-amylase, and expressed without residual ashes. The acid detergent insoluble N (ADIN) was also measured according to Van Soest et al. (1991). Condensed tannins (CT) were extracted and analysed by the procedure proposed by Terrill et al. (1992), as modi®ed by PeÂrez Maldonado and Norton (1996), with three replicates per sample. Once determined the absorbance (550 nm), extractable, protein-bound and ®brebound CT values were adjusted to a standard curve using puri®ed quebracho tannins as standard. The intestinal digestibility of protein was determined according to Calsamiglia and Stern (1995). Forage samples (0.5 g) were incubated in vitro in quadruplicate tubes for 32 h, in each of two consecutive runs. The residue from one of the tubes was used for determining microbial non-degradable N, and the remaining three residues were pooled and dried at 60 8C for 48 h. Then, residues were digested in vitro with HCl-pepsin at pH 1.9 for 1 h and after with pancreatin at pH 7.8 for 24 h. Before calculations, the nitrogen contribution of pepsin-pancreatin solution (blank) was substracted from soluble N for correction purposes. Polysaccharidase activity of the enzymatic extract was tested colorimetrically against carboxymethylcellulose (CMC, Sigma) and xylan (Sigma) by the Nelson±Somogyi method, and b-glycosidase (b-D-glucosidase and b-D-xylosidase) activities were determined from the corresponding p-nitrophenyl compound (Sigma) as referred by Nogueira et al. (2000). Polysaccharidase (mmoles of sugar released per ml of extract and per min) and b-glycosidase (nmoles p-nitrophenol released per ml and per min) activities were expressed by mg of dry residue of fermentation (total activity) or by mg 15 N in the residue (bacterial activity). For bacterial adhesion studies, the inoculum was previously labelled with 15 N (52 mg 15 N per tube, using ammonium sulphate, 14 atom 15 N, ISOTEC Inc., OH, USA). Enrichment was determined using a mass spectrophotometer hooked in series to a DUMAS-style N analyser, and the calculations of bacterial concentration were performed as in Nogueira et al. (2000). All analyses were carried out in duplicate.
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For the statistical analysis of differences in gas production results, the mean of the two syringes of each sample in each series of incubation were used as the experimental unit. Differences among species by time of incubation were contrasted by ANOVA, with the interaction series of incubation species as the error term (8 d.f.). When microbial activity (results of adhesion and enzymatic activity) was studied, the main effects substrate, time of incubation and series were considered, and these and the interaction substrate time were contrasted with the residual error (14 d.f.). The N degradability and intestinal digestibility were also analysed by ANOVA, considering species as main factor and incubation run as factor, contrasted against an error term with 24 and 3 d.f., respectively. For all cases, differences among treatment means were identi®ed by the Tukey's test at P < 0:05. 3. Results Chemical analyses were performed on only one sample for each forage, and consequently were not compared statistically. However, among browse species Ll showed numerically lower NDF, ADF and ADL content than Cc (Table 1). Among herbaceous species, Nw gave lower proportions of these constituents than Sm, with an apparently larger amount of cell wall components in the latter than in both Ll and Cc. Total N content was very similar for Ll, Cc and Nw, but it was lower for Sm. The ADIN proportion was numerically higher for Sm, intermediate for Ll and Cc and lower for Nw. Total CT were markedly higher in browse than in herbaceous legumes, especially extractable and ®brebound CT. Protein-bound CT were 10-fold higher in Cc than in the other three legume species. Fig. 1 shows the average pattern of gas production from the legumes, as an index of microbial fermentation. Coef®cients of the equations obtained when those curves were Table 1 Chemical composition (g/kg dry matter) of the tropical legumesa Browse legumes
OM NDF ADF ADL N ADIN Extractable CT Fibre-bound CT Protein-bound CT Total CT
Herbaceous legumes
Ll
Cc
Nw
Sm
897 390 198 88 33.7 3.3 73.0 6.2 2.7 81.9
945 523 328 147 30.2 3.5 79.0 6.0 24.6 109.6
911 472 330 92 32.7 2.5 1.2 2.2 2.7 6.1
962 690 549 187 18.8 4.3 1.3 1.3 3.5 6.1
a Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii; Sm: S. macrocephala. OM: organic matter; N: nitrogen; NDF: neutral detergent fibre; ADF: acid detergent fibre; ADL: acid detergent lignin; ADIN: acid detergentinsoluble nitrogen; CT: condensed tannins.
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Fig. 1. Pattern of the volume of gas produced by in vitro microbial fermentation of L. leucocephala (*), C. cajan (&), N. wightii (~) and S. macrocephala (*). Upper vertical bars show standard errors of means.
adjusted to the model proposed by France et al. (1993), together with the average DMd, RGY and the estimated OMD of the species are in Table 2. From 12 h onwards, the volume of gas produced by fermentation of Nw was higher than that from Ll, Cc and Sm (P < 0:05 from 12 to 30 h and at 60 h; P < 0:01 at 36, 48, 72 and 96 h). There were no differences among the other three species at any incubation time. Estimated total gas production (Table 2) showed differences (P < 0:001), with Nw being the most, and Sm the least, fermentable species. There were no differences in lag time T. Both rate constants b and c showed differences among species (P < 0:001), and so the calculated fractional degradation rates were faster for Nw and slower for Ll, while Sm and Cc were intermediate. The DMd at the end of the experimental period showed differences (P < 0:001) that agreed with total gas production results. However, treatment comparison in RGYonly tended to be signi®cant (P < 0:10), with higher values for Sm and Cc than for Ll and Nw. Estimated OMD gave a higher value for Nw, intermediate for Ll and Cc and lower for Sm. Microbial adhesion to solid particles, estimated by 15 N in the second experiment comparing Ll, Cc and Nw up to 32 h incubation, is shown in Fig. 2. Among incubation times, adhesion at 4 h was less than from 12 to 32 h (P < 0:001), but there were no differences in microbial adhesion among legume species (P > 0:10) throughout. Since maximum differences in enzymatic activity occurred at the end of the studied period, only total (per unit of residue weight) and bacterial (per unit of adhered bacteria) enzymatic activities against carbohydrates of the extracts from the residues at 24 and 32 h of incubation are in Table 3. The pattern of total and bacterial polysaccharidases and b-glycosidases activities are shown in Figs. 3 and 4, respectively. Overall total cellulase
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Table 2 Parameters of curves fitted from in vitro gas production results, dry matter disappeared (DMd, mg/g), relative gas yielded (RGY, ml gas/g DM disappeared) and estimated organic matter digestibility (OMD, g/kg) of legumesa,b Browse legumes Ll c,d
A b c T m6 m12 m24 m48 DMd RGY OMD
163 ab 0.021 0.064 0.10 0.034 0.030 0.027 0.025 473 b 313 556
Herbaceous legumes Cc
c a b c c c
146 bc 0.044 0.021 0.44 0.048 0.047 0.046 0.045 412 bc 359 550
Nw b ab ab bc b b
188 a 0.077 a 0.078 c 0.70 0.061 a 0.066 a 0.069 a 0.072 a 634 a 306 628
S.E.M. Sm 130 c 0.054 b 0.028 bc 0.43 0.048 ab 0.050 ab 0.051 b 0.052 b 357 c 380 473
6.1 0.0036 0.0119 0.577 0.0038 0.0036 0.0035 0.0035 1.3 17.2 ±
a
Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii; Sm: S. macrocephala. Different letters (a, b, c) within rows indicate differences (P < 0:05). c Parameters: A: total gas production (ml/g incubated DM); b: fermentation rate (h 1); c: fermentation rate (h 1/2); T: lag time (h); m6, m12, m24, m48: fractional fermentation rates at different incubation times (h 1), estimated from the rate constants b and c. d There were differences (P < 0:001) in all parameters except for T (P > 0:05) and RGY (P < 0:10). OMD was not compared statistically. b
Fig. 2. Microbial adhesion to the substrate (L. leucocephala (*); C. cajan (&); N. wightii (~)), estimated by labelling mixed ruminal bacteria in the dry residue of fermentation with 15 N (S:E:M: 1:398).
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Table 3 Total (per g dry matter residue per min) and bacterial (per mg 15 N per min) polysaccharidase (CMCase or xylanase; mmoles sugar released), or b-glycosidase (b-glucosidase or b-xylosidase; nmoles p-nitrophenol released) activities of extracts from the solid fermentation residue of the legumes after 24 and 32 h of incubationa,b 24 h
Total CMCase Xylanase b-Glucosidase b-Xylosidase Bacterial CMCase Xylanase b-Glucosidase b-Xylosidase a b
32 h
S.E.M.
Ll
Cc
Nw
Ll
Cc
Nw
6.34 a 3.07 144.9 135.5 a
1.87 b 2.98 88.6 57.8 b
2.73 b 2.56 156.8 72.6 ab
2.41 b 0.98 b 52.1 b 45.6 b
1.15 b 1.46 b 90.8 b 75.4 b
7.08 a 6.48 a 173.3 a 150.3 a
0.844 a 0.405 19.1 18.2
0.134 b 0.213 6.4 4.2
0.198 b 0.184 12.3 5.5
0.183 ab 0.078 b 4.1 3.4
0.095 b 0.120 b 7.5 6.2
0.526 a 0.479 a 12.7 11.1
0.429 0.510 20.96 18.75 0.1150 0.0644 4.47 4.26
Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii. Different letters (a, b) within rows indicate differences (P < 0:05).
(against CMC) activity was higher (P < 0:001) for Ll and Nw than for Cc (2.58, 2.50 and 1.55 mmoles glucose/g DM), but these differences were only signi®cant (P < 0:001) between Ll and the other species at 24 h, and between Nw and the browse legumes at 32 h (Table 3 and Fig. 3). However, activity for Nw started later than for the browse legumes, and the maximum for Ll occurred at 24 h, whereas Nw peaked at 32 h. A similar pattern can be observed for total xylanase activity, but in this case there were no differences between the browse species, and activity over Nw was higher only at 32 h. Total bglucosidase activity was higher over Nw at 12 and 32 h (P < 0:001) and, whereas it plateaued from 4 h onwards for both browse legumes, it peaked at 12 h for Nw. The observed pattern of total b-xylosidase activity was variable throughout the control period, but differences (P < 0:005) occurred at 12, 24 and 32 h of incubation, and overall Nw was higher than Cc (P < 0:05). When expressed in bacterial concentration basis, differences between legumes in the pattern of enzymatic activity (Fig. 4) were smaller than those in total activity. Bacterial cellulase activity was higher in Ll at 24 h, and lower in Cc at 32 h (P < 0:05). Bacterial xylanase activity started earlier in Cc (P < 0:05 at 4 h), but at 32 h that from Nw was higher (P < 0:05) than that from the browse species. A higher bacterial b-glucosidase activity at 4 h was observed with Ll (P < 0:05), but no differences were observed afterwards. Nitrogen degradability after 32 h of in vitro microbial incubation, together with the intestinal digestion estimated enzymatically, are in Table 4. Degradability of the nitrogenous fraction were higher for Nw, intermediate for Sm, and gave similar low values for both browse species (P < 0:001). The intestinal digestion of the N undegraded after microbial fermentation was ranked as: Nw > Sm > Cc > Ll, with differences (P < 0:05) between Nw and Ll only.
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Fig. 3. Total polysaccharidase (mmol sugar released/g DM) activity (CMCase and xylanase), and bglycosidase (nmoles p-nitrophenol released/g DM) activity (b-glucosidase and b-xylosidase) of extracts from the solid fermentation residue of L. leucocephala (*), C. cajan (&) and N. wightii (~) at different incubation times.
Table 4 Microbial nitrogen degradation after 32 h of in vitro incubation and in vitro intestinal nitrogen digestion of the legumesa,b Browse legumes
Total N (g/kg DM) N degradation (g/g N) Intestinal N digestion g/g N at the duodenum g/g total N a b
Herbaceous legumes
Ll
Cc
Nw
Sm
33.7 0.316 c
30.2 0.315 c
32.7 0.728 a
18.8 0.548 b
0.442 b 0.303
0.545 ab 0.373
0.823 a 0.225
Ll: L. leucocephala; Cc: C. cajan; Nw: N. wightii; Sm: S. macrocephala. Different letters (a, b, c) within rows show significant differences (P < 0:05).
0.670 ab 0.303
S.E.M.
± 0.0178 0.0301 0.0273
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Fig. 4. Bacterial polysaccharidase (mmol sugar released/mg 15 N) activity (CMCase and xylanase), and bglycosidase (nmoles p-nitrophenol released/mg 15 N) activity (b-glucosidase and b-xylosidase) of extracts from the solid fermentation residue of L. leucocephala (*), C. cajan (&) and N. wightii (~) at different incubation times.
4. Discussion It has been speculated that the gas production technique may not be suitable for evaluating high-protein feeds because released ammonia may react with CO2 and precipitate, thus reducing gas volume (Menke and Steingass, 1988), and an inverse relationship has been observed between protein content of the test food and gas volume (Krishnamoorthy et al., 1995; GonzaÂlez Ronquillo et al., 1998). In our case, in vitro 32 h N degradation showed higher results for Nw, indicating that, although an underestimation of DM fermentation could have occurred because of a higher NH3 production (which cannot be veri®ed in our results), differences between Nw and the other species still remained. It is also worth noting that, as previously observed (Sileshi et al., 1996; Nogueira et al., 2000), DMd results which cannot be affected by ammonia concentration, are in agreement with those from gas production.
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4.1. Microbial fermentation Chemical comparison of the legumes established a clear difference between browse and herbaceous legume species, with the former having a much higher content of condensed tannins. The major differences are in extractable CT, which, according to Longland et al. (1995), are less involved in the restriction of microbial fermentation than bound CT. However, ®bre-bound CT are higher in browse species, suggesting a possible lower microbial utilisation of carbohydrates. Among the herbaceous species, Nw apparently had a lower NDF content than Sm. This can affect plant fermentation patterns, and the higher ADL content of Sm may explain its lower total gas production, slower fermentation rates and lower DMd compared with Nw. Gas production evolved similarly for browse species (Ll and Cc) throughout the control period, except for the faster rate with Cc, and there were no differences in total gas production. Other gas production trials in literature, (Nw versus Ll, Krishnamoorthy et al., 1995, Cc versus Ll, Siaw et al., 1993), ranked the species similarly, despite possible differences in growth conditions. Complexes with protein and carbohydrates are formed in tannin-rich plants, which restrict fermentation of cell walls by rumen microorganisms. Bae et al. (1993) showed that in vitro cellulose digestion by Fibrobacter succinogenes was affected when condensed tannins from Lotus corniculatus were added and a reduction in volatile fatty acid production in vitro has also been observed (Makkar et al., 1995). It has been suggested that tannins may prevent colonisation of leaf material (Chiquette et al., 1988), and McAllister et al. (1994) showed visual evidence of this effect on F. succinogenes colonisation of ®lter paper. However, this effect is strongly related with the type and concentration of tannins, and Makkar et al. (1989) reported that the plants with the higher tannin content were not necessarily those that support lower adhesion. Makkar et al. (1988) observed a reduction in microbial cellulases associated with increased CT concentration. Although microbial adhesion on Nw was numerically higher at 12 h than on Ll and Cc (15.0 versus 12.2 and 12.3 mg 15 N/g DM), and differences partly remained at 24 h (14.3 versus 9.9 mg 15 N/g DM on Nw and Ll), there were no differences in attachment of mixed rumen organisms to plant particles. Total and bacterial enzymatic activities against structural polysaccharides did not substantially differ greatly until 32 h incubation, but at this time total cellulase and xylanase activities over Nw were three-fold higher than over the browse legumes (Table 3). This may explain, at least in part, differences in gas production pattern among these species. However, the magnitude of existing differences between Nw and the tannin-rich Ll and Cc cannot be totally associated with a negative effect of the CT over microbial action either by adhesion or enzymatic activity, against cell wall structures. Thus, the lower fermentability of Ll and Cc compared with Nw may also be related to an effect of CT over other plant carbohydrates (Jansman and Longstaff, 1993) or to other factors than CT. 4.2. N utilisation In addition to their utility as energy sources for rumen microbes either as a complement or substitutes of grasses during the dry season, the potential as N sources of these legumes was also evaluated. Despite of water de®cit during growth, total N content of the species
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(except for Sm) was high. The N degradation after 32 h fermentation assuming a rumen rate of passage of 0.03 h 1 for these forages was low in browse legumes, presumably because of their high CT content. Poppi et al. (1999) observed higher extent and rate of protein degradation of Stylosanthes spp. than C. cajan or L. leucocephala, and Krishnamoorthy et al. (1995) showed a N degradation of 0.67 for N. wightii, whereas that for two L. leucocephala varieties was 0.24 and 0.51. However, variation in protein-bound CT proportions among species do not support such large differences in N degradation between Ll and the herbaceous legumes, and might indicate that other CT, either extractable or ®brebound, might be affecting the response. It is worth mentioning that the high CT content observed in Cc, especially the differences with Ll in protein-bound CT, can only be attributable to a poorer adaptation to drought conditions. The higher protein-bound CT proportion was not re¯ected in differences in N degradability between the browse species. It has been reported that in vitro estimation of intestinal digestion of N gives lower values than the in vivo mobile bags (Calsamiglia and Stern, 1995), mainly because of the synergistic action of various enzymes in vivo. In our lab, evaluation of different protein supplements with both techniques after 16 h in situ rumen incubation showed, on average, a 13% lower digestibility in vitro, but ranking was the same. Hence, this technique has been proven to be a useful tool for comparing protein sources. From results in Table 4, it can be calculated that the rumen degradable N from feed origin is 11, 10, 24 and 10 g N/kg feed DM, and the amount of N digested in the intestines is 10, 11, 7 and 6 g/kg for Ll, Cc, Nw and Sm, respectively. In this way, the proportions of nondegraded, non-digested N are 0.38, 0.31, 0.05 and 0.15 for the same species. Therefore, in vitro results indicate that the herbaceous species are better utilised as N sources, and the browse legumes give a higher amount of both feed N reaching the duodenum and N digested in the intestine, although this parameter is lower than expected. The lower intestinal digestion with Sm, as well as its lower degradability, compared with Nw can be justi®ed by its higher ligni®cation and ADIN content. However, the low intestinal N digestion from Ll and Cc implies that some limitation of protein availability because of tannins remains. Butter et al. (1999) suggested that, although tannin binding is dissociated under pH 5.6 or over pH 7.0, it might be formed again as the digesta passes from the abomasum to the intestines, thus reducing amino acid absorption. Indeed, Wang et al. (1996) indicated that some tannin±protein complexes are still present at the terminal ileum. In their review, Jansman and Longstaff (1993) suggested that dietary tannins might inhibit various enzymes from the intestinal mucosa. In agreement with our results, GarcõÂa et al. (1996) reported a low rumen protein degradability of L. leucocephala (0.42), but also a post-ruminal digestion of undegradable protein of only 0.48. Jones and Palmer (2000), estimating N digestion of some shrub legumes by an acidpepsin digestion after in vitro microbial fermentation, observed a 0.10±0.15 positive response to polyethylene glycol addition (the difference is assumed to be the CT effect). This indicates that protein in some shrub legumes is over-protected by the tannins, and so may not be completely available in the small intestine. Considering the tannin content of the same samples from another study, (Jackson et al., 1996) the negative effect of tannins on protein digestion is apparently more related to the extractable, than to the proteinbound CT, which might explain the small differences between Ll and Cc in estimated N intestinal digestion.
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5. Conclusions The practical interest of using these legumes as feeds during the dry season in the tropics mainly relies in their value as protein and energy sources albeit in¯uenced by growth conditions. N. wightii is fermented by rumen microorganisms in a greater extent than the other species, and most of its N is utilised. However, the input of feed N to the intestines is comparatively low because of its high N degradability. S. macrocephala is notably affected by drought, and its low N and high ADL and ADIN contents limit its microbial fermentation and N utilisation. Tannin-rich shrubs are less fermented than N. wightii, but this effect does not seem to be related to a negative effect of condensed tannins on microbial adhesion or enzymatic activity against structural polysaccharides. Even though L. leucocephala and C. cajan had a low intestinal N digestion, their low degradability by rumen microbes makes them an important source of N digestible protein in the intestines. However, the protective effect of condensed tannins of protein may remain to a biologically signi®cant degree in the lower portions of the digestive tract. Acknowledgements The stage of Dr. J.C.M. Nogueira Filho and Dr. A. Barrios Urdaneta in the University of Zaragoza were supported by FAPESP (Brazil) and CONICIT (Venezuela), respectively. References Bae, H.D., McAllister, T.A., Yanke, J., Cheng, K.J., Muir, A.D., 1993. Effect of condensed tannins on endoglucanase activity and filter paper digestion by Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 59, 2132±2138. Butter, N.L., Dawson, J.M., Buttery, P.J., 1999. Effects of dietary tannins on ruminants. In: Caygill, J.C., Mueller-Harvey, I. (Eds.), Secondary Plant Products. Antinutritional and Beneficial Actions in Animal Feeding. Nottingham University Press, UK, pp. 51±70. Calsamiglia, S., Stern, M.D., 1995. A three-step in vitro procedure for estimating intestinal digestion of protein in ruminants. J. Anim. Sci. 73, 1459±1465. Chiquette, J., Cheng, K.J., Costerton, J.W., Mulligan, L.P., 1988. Effect of tannins on the digestibility of two isosynthetic strains of birdsfoot trefoil (Lotus corniculatus L.) using in vitro and in situ techniques. Can. J. Anim. Sci. 68, 751±760. Elliott, R., McMeniman, N.P., 1987. Supplementation of ruminant diets with forages. In: Hacker, J.B., Ternouth, J.H. (Eds.), The Nutrition of Herbivores. Academic Press, Merrickville, Australia, pp. 409±428. France, J., Dhanoa, M.S., Theodorou, M.K., Lister, S.J., Davies, D.R., Isac, D., 1993. A model to interpret gas accumulation profiles associated with in vitro degradation of ruminant feeds. J. Theoret. Biol. 163, 99±111. GarcõÂa, G.W., Ferguson, T.V., Neckles, F.A., Archibald, K.A.E., 1996. The nutritive value and forage productivity of Leucaena leucocephala. Anim. Feed Sci. Technol. 60, 29±41. GonzaÂlez Ronquillo, M., Fondevila, M., Barrios Urdaneta, A., Newman, Y., 1998. In vitro gas production from buffel grass (Cenchrus ciliaris L.) fermentation in relation to the cutting interval, the level of nitrogen fertilisation and the season of growth. Anim. Feed Sci. Technol. 72, 19±32. Humphreys, L.R., 1995. Diversity and productivity of tropical legumes. In: D'Mello, J.P.F., Devendra, C. (Eds.), Tropical Legumes in Animal Nutrition. CAB International, Wallingford, UK, pp. 1±21. Jackson, F.S., Arry, T.N., Lascano, C., Palmer, B., 1996. The extractable and bound condensed tannin content of leaves from tropical tree, shrub and forage legumes. J. Sci. Food Agric. 71, 103±110.
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