Voluntary intake, apparent digestibility and nitrogen balance by sheep supplemented with Leucaena leucocephala

C Longo, E F Nozella, S L S Cabral Filho*, N Lavorenti**, D M S S Vitti and A L Abdalla 

Animal Nutrition laboratory, Centre for Nuclear Energy in Agriculture, University of São Paulo, CEP:13400-970,  Piracicaba, SP, Brazil

*University of Brasilia, Distrito Federal, Brazil

**São Carlos Federal University, São Carlos, São Paulo, Brazil

cibelelongo@gmail.com   ;   abdalla@cena.usp.br

Abstract 

This study aimed to evaluate supplementation of Leucaena leucocephala (leucaena) for ruminant and the effects of condensed tannin in rumen fermentation. Eight Santa Inês breed sheep (body weight = 35 ± 2.6 kg), were allocated to four diets mixed in a randomized block design. Animals were kept in individual pens for a seven-day feed adaptation followed by a seven-day intake trial where voluntary dry matter intake (VDMI) was estimated. The sheep were then moved into metabolic cages for a five-day digestibility trial to estimate apparent digestibility and nitrogen balance. The total experiment lasted 70 days divided into tree periods (blocks) of 21 days, leaving two days between periods for animal rest. Experimental diets were based on Tifton 85 hybrid Bermuda grass (Cynodon dactylon Pers. × Cynodon nlemfuensis Vanderyst) and supplemented with 20 (L20), 40 (L40) and 60% (L60) of leucaena hay related to diet dry matter. Control diet was composed by tifton hay and supplemented with mix protein concentrate. Chemical composition and condensed tannins determination were performed on offered and refused feed and excretion samples. Data were subject to analysis of variance using general procedure of the statistical software package. Means were compared by Student`t test (0.05) and linear and quadratic regressions were applied to evaluate the effect of leucaena level.

 

Supplementation with L40 increased 29% VDMI (g/animal.day), while sheep fed L20 presented no difference (P < 0.05) to control and L60 diet. The average responses of apparent digestibility among leucaena diets were 384 (SE=10), 341 (SE=24), 288 (SE=53) and 365 (SE=24) g/Kg for total DM, CP, ADF and NDF, respectively, which were lower (P<0.05) than control diet (504, 630, 557, 459, respectively). It is supposed that the low digestibility found with supplementation was due to condensed tannin effect, high indigestible fibre contend and the additive action of both. Supplementation with L60 and L40 increased faecal nitrogen excretion (FN) and faecal acid detergent soluble nitrogen (P < 0.05). However, animals fed L20 reduced 64% urinary nitrogen (UN) loss related to control diet and presented similar FN than control (P > 0.05). L60 and control diet presented the highest N retention (P < 0.05). In general, the high level of leucaena supplementation (40 and 60%) should be exploited with higher digestible nutrients, in order to synchronize the “excess” protein in rumen and energy.

 

It is concluded that phenolic compounds, especially condensed tannin, present in leguminous may influence positively digestive parameter; however its effect depends on other factor such as presence of lignin, available energy and also its chemical characteristic.

Keywords: condensed tannin, leguminous, phenolic compounds, ruminant nutrition

Introduction 

The use of leguminous forages is an alternative of protein supplementation for animal fed with insufficient quality of diets. Leucaena leucocephala (Leucaena) is lifelong tropical legume that can be directly grazed or harvested and offered to animals as hay, silage or fresh (Mitidiere 1983). Leucaena contain phenolic compounds, which are considered anti-nutritional factor as it may reduce intake, digestibility and thus animal performance (D`Mello 1995; Waghorn 1994 et al). Nevertheless, some studies using tanniniferous forages reported increase in intake and no change in digestibility (Kaitho et al 1998a; Abdulrazak et al 2006), reduction in nematode infection (Niezen et al 1995) and in enteric methane emission (Waghorn et al 2002).

 

According to Barahona et al (1997), there are large differences in the condensed tannin structure especially among tropical legume species, which may explain differences in astringency (bound protein/ extractable CT) and in the effects on intake, digestion and nitrogen (N) utilization among forages of the same genus or even specie. The aim of this work was to evaluate the influence of graded levels of Leucaena leucocephala used as supplement feed for Santa Inês sheep diet on dry matter (DM) intake, apparent digestibility of nutrients and nitrogen balance.

 

Material and methods

Local and Leucaena origin

 

The in vivo experiment was conducted at the Centre for Nuclear Energy in Agriculture (CENA), University of São Paulo, Brazil. Leucaena was collected in the Research Institution of Animal Production located in Nova Odessa, São Paulo, Brazil. It was selected young stems with diameter smaller than 1 cm. Leucaena was harvested in three batches and was dried for 48 h and batches were grounded and mixed to obtain a uniform material. This material was conserved in bags, in a dry and dark place and it was sieved through 0.5 cm metal mesh before offering to the animals.

 

Animals, diets and feeding

 

Eight Santa Inês (BW 35 kg ± 2.6) male sheep, previously adapted to metabolic cages, were used in this study. Four diets were prepared based on Tifton 85 hybrid Bermuda grass (Cynodon dactylon Pers. × Cynodon nlemfuensis Vanderyst) and used for intake and digestibility trials. Diets L20, L40 and L60 consisted in three leucaena levels, 20, 40 and 60% of DM diet, respectively. Control diet was based on tifton hay plus protein concentrate, which consisted of 92 g soybean meal, 92 g citrus pulp and 3 g urea per animal per day (g/animal.day). The experiment was conducted in three periods of 21 days, allowing 2 days for animal rest. In each experimental period, two animals per treatment were allocated to individual pens for seven-day adaptation followed by seven-day intake trial. The intake trial carried out to estimate voluntary dry matter intake (VDMI) followed by digestibility trial during five days to estimate apparent digestibility of dry matter (DM), crude protein (CP), neutral detergent fibre (NDF), acid detergent fibre (ADF) and N balance. Animal were returned to the pens for two day rest before starting the next period. In both trials, animals had access to water and mineral lick ad libitum and the diets were offered at 8:30 am and 4 pm allowing refusals no higher than 20%.

 

Sampling procedure

 

The ingredients of the diets (tifton hay, leucaena and concentrate) were weekly sampled and pooled as a sample per period, which was frozen for chemical determination. Refusals and faeces were individual and daily weighed before morning feeding and a pool sample per period and per animal was also frozen for later analysis. Urine was collected in plastic bucket containing 100 mL of sulphuric acid 10 % and protected with two layers of thin cotton tissue. Total urine volume and pH were daily measured and the volume was completed to 3 L with tap water. After homogenising, 20 mL of this solution were sampled and pooled as a sample per period and per animal, which was kept frozen for later chemical determination.

 

Chemical determination

 

The collected samples of feed offered, refusal and faeces were thawed at room temperature, dried in a forced-air oven at 40^o C maximum and grounded to 1 mm for chemical determination and to 0.5 mm (60 Mesh) for phenolic compounds analysis.

 

Determination of dry matter (DM; AOAC 2005: method 934.01), ash (AOAC 2005: 942.05), crude protein (CP; AOAC 2005: 954.01), acid detergent fibre (ADF) and lignin (ADL); (AOAC 2005: method 973.18) and neutral detergent fibre (NDF) (AOAC 2005: method 2002.04 without amylase). Acid detergent insoluble protein (ADIP) refers to the protein content in ADF. Acid detergent soluble protein (ADSP) was estimated by subtracting ADIP from total protein (CP-ADIP).

 

Phenol compounds were extracted using acetone 70 % in ultra-sound bath, according methodology described in Makkar (2000). Determination of total tannin (TT) was carried on adding to the extract 100 mg PVPP (polyvinylpoliypyrrolidone). The total phenols (TP) and total tannin were determined applying Folin 2N and 1.25 mL sodium carbonate solution on an aliquot of the extract. The concentration of total phenols and total tannins were expressed as tannic acid equivalent, g/kg DM.  Condensed tannin (CT) was determined using Butanol-HCl and the concentration was expressed in leucocyanidin equivalent as A 550nm x 78.26 x dilution factor) / % DM (Porter et al 1986).

 

Total digestible nutrients (TDN) in the forages were estimated according to the equation:

75.1 + {(6.25 x log %N) – 0.75 x [-4.32 + (0.92 x % ADF)]} (Du Toit 1998; Longo et al 2000).

The TDN in the concentrate was estimated by nutrition tables found in the NRC (1985).

 

Statistics analysis

 

All variables were subjected to analysis of variance (ANOVA) in a randomized block design with three blocks (period), four diets and eight animals, using the general linear model procedures (proc glm) of the statistical package of SAS (2001). Means were compared by Student`t test at 0.05 error limit and linear and quadratic regressions were applied to analyze the influence of the three leucaena levels.

 

Results 

Chemical composition

 

Chemical composition of ingredients and diets is presented in Table 1.

No statistical analysis was performed on these data, but it was observed that DM, ADF and TDN presented narrow range among diets and CP, ADSP, ADL and phenolic compounds had higher differences among diets. Energy content in the diets for finishing sheep was low according to NRC sheep (1985). Diet L60 presented the highest values of CP, ADSP, ADL, ADIP and phenolic compounds (TP, TT and CT) and the lowest NDF/CP, ADF/CP, cellulose and hemicelluloses. Control diet showed close ADSP, ADF, NDF and hemicelluloses values to L40 but higher cellulose. Protein and energy relation, CP/TDN and ADSP/TDN, was similar between L20 and control, however ADSP/TDN was slighter lower in L20 (0.11 and 0.13, respectively). L60 presented the highest CP/TDN, ADSP/TDN and g CP/Kg DMD but the lowest relation between fibre (NDF, ADF) to protein. The relation between g CP/Kg digestible DM was high for diet L60 and L40, (337 and 270, respectively), which may promote high protein escape in these diets. Chemical determination of the plants showed that in average 19 % of total protein was bound to ADL (ADIN/N) in leucaena diets while only 8 % protein was unavailable in the control diets.

 

Intake and digestibility

 

The results obtained regarding to intake and digestibility are presented on Table 2.

It was not observed rejection of leucaena and also no symptoms of leucaena toxicity. Animals fed L40 showed 29% increase of VDMI (999 g/animal.day) related to control (776 g/animal.day). Although, there was no difference (P > 0.05) between L20 and L60 to the control, VDMI of L60 (865 g/animal.day) was numerically 11% higher than control. Among leucaena diets, the increasing amount of leucaena was associated with a quadratic effect of VDMI (P < 0.05).

 

Supplementation increased CP intake but not soluble protein (ADSP) intake, which showed similar values (P < 0.05) between L60 and L40 (100 and 81 g/animal.day, respectively). L40 presented also similar CP and ADSP intake than control diet. Fibre intake (ADF and NDF) were similar among the leucaena diets (P > 0.05), however comparing to the control diet, L40 showed higher ADF and NDF intake (P < 0.05).

 

Control diet showed higher (P < 0.05) digestibility of DM, CP, ADF and NDF (g/Kg) than leucaena diets, except NDF digestibility of L20 that was significantly similar to control (392 and 459, respectively). Animals fed control diet had 504 g/Kg digested, while animal supplemented with leucaena presented in average 384 g/Kg (± 8.6). Among leucaena diets, it was not detect differences (P > 0.05) in the digestibility of DM, CP, ADF and NDF, however, numerically, diet L40 present the lowest value of DM and CP digestibility (372 and 320 g/Kg, respectively) and L60 showed the lowest ADF digestibility (251 g/Kg DM). This promoted linear and quadratic effect (P < 0.05) for DM, CP, ADF and NDF digestibility among the leucaena diets.

 

Nitrogen balance: faecal and urinary nitrogen losses

 

Animals supplemented with L60 and L40 had higher (P < 0.05) faecal nitrogen excretion (FN) and faecal acid detergent soluble nitrogen (ADSN) than control and L20 (Table 3).

L20 presented the lowest (P < 0.05) urinary N (UN), followed by L40, L60 and control (1.8, 3.6, 4.2 and 5.0 g N/d, respectively). In addition, nitrogen (N) retention in L20 was lower (P < 0.05) than control and similar (P > 0.05) to L40. There was no difference (P > 0.05) in UN and N retention between L60 and control diets. Despite the high N excretion (UN and FN), L60 presented similar N retention than control. Nevertheless, N retention data presented high coefficient of variance (53%) (data not showed), which suggested that careful interpretation should be taken on this variable.

 

Discussion 

Despite the higher content of indigestible protein in L60 followed by L40 and L20, the relation between indigestible protein to the total crude protein (ADIP/CP) had no difference (P > 0.05) among leucaena diets (averaged 19 ± 0.82). This happened because L60 presented not only high CP but also high soluble crude protein, which regulated this relation. On the other hand, control diet presented lower ADIP/CP (8.2) than leucaena diets, which gave undesirable advantages in terms of digestibility. However, these results were expected as the tanniniferous leguminous presents not only high protein content but also high lignin. Besides, condensed tannin can bind molecules in the plant cell, depending on the dry treatment applied. In fact, when tanniniferous plants are offered fresh, they present better results in digestibility than when sun-dried process is applied (Hammond 1995). Leucaena forage used in this study was sun dried, which may explain the high ADIP (39 g/kg DM) found in the plant.

 

VDMI was positively affected by leucaena content in the diets in spite of phenolic compounds. According to Forbes (2000), voluntary intake is controlled by physiological, metabolic and physical aspects of the feed. In a study with sheep fed Lotus pedunculatus, it was observed that animals preferred leaves instead of stem although there was higher tannin content in the leaves (Waghorn et al 1994). Control and L20 presented similar but lower VDMI than L60 and L40. Control and L20 contained similar amount of tifton hay (1012 and 960 g/animal.day, respectively), which enriched these diets with longer fibre and slowed the escape of feed residues. According to Forbes (2000), rumen fill is the dominant factor in the intake of forages and any factor that may decrease the residence time will increase voluntary intake. It seems that control and L20 diets reached the rumen capacity by activating the stretch receptors in the rumen wall and consequently reducing intake. In the control diet, the high digestibility and the replacement of leucaena for more digestible concentrate were not enough to stimulate VDMI and compensate the negative physical action of tifton. Nevertheless, the presence of CT in L20 may have promoted higher protein escape due to tannin binding at the rumen. This hypothesis is sustained by the lower urinary nitrogen in L20 than control (Table 3), which suggested reduction of protein degradation in L20. Consequently, the passage rate in L20 increased, contributing to keep VDMI at the level of control diet (52 vs. 54 Kg BW^0.75, respectively). It is supposed that if the leguminous used in this study had no tannin or other complexing agent, L20 would presented lower VDMI than control.

 

The amount of condensed tannin in the leucaena used in this study was lower than reported by Karda et al (2001) and Abdulrazak et al (2006), however, digestibility was highly reduced, which suggested that other factors besides CT may have influenced digestibility. The low digestibility reached in leucaena diets were probably due to (i) high concentration of ADL in leucaena diets, (ii) influence of CT and (iii) additive action of both, ADL and CT. Besides, a high protein to carbohydrate ratio in the diet could lead to a relatively low microbial protein to VFA ratio in the end-products of fermentative (Leng 1993), which means low digestibility.

 

L60 presented the lowest NDF/CP, which gave advantages on intake as it delayed the rumen fill. However, these advantages were limited by the excess of protein related to energy supply resulting in similar VDMI as L40. According to Poppi and McLennan (1995), forages that exceed 210 g CP/Kg digestible organic matter (DOM), high protein losses may be expected. Diet L60 and L40 had 337 and 270 g CP/Kg DOM, respectively, suggesting higher protein escape in those diets. Nevertheless, the higher relation between protein and energy (CP/TDN and ADSP/TDN) in L60 than L40 (Table 1) may have disturbed rumen environment with no synchronicity between protein degradation and energy available, which may had negatively influenced intake and digestibility.

 

Indigestible nutrients, such as ADL contained in ADF fraction, are inversely related to digestibility. Leguminous present higher concentration of ADL due to the erect stems and thus, if the proportion of leaves is high when fed leguminous, lower ADL is offered to the animals (Forbes 1995). In this study, despite of shivering the leucaena, a lot of pieces of stem were included in the leucaena diet, giving disadvantages to these supplements on digestibility.

 

High FN was found in L60 and L40 but not in L20, although all leucaena diets presented similar CP digestibility. The high g CP/Kg DOM and CT concentration found in L40 and L60 could be one of the causes for increasing protein escape. The high faecal N found in the supplemented animals is also mentioned in other studies using Leucaena leucocephala (Kaitho et al 1998b, Karda et al 2001). However, different results were found like, Bengaly et al (2007), which results presented no difference in apparent digestibility, faecal N increased while urinary N excretion was not affected neither N retention.

 

Different authors, stand that the protein released from the tannin-protein complex depends on not only from the abomasums pH, but also on the chemical characteristics of tannin molecule (Kaitho et al 1998a, Barahona et al 1997, McNeill et al 1998). Consequently, could be possible that tannin-protein complex was or was not released in the abomasums, which would differently affect digestibility and nitrogen losses in faeces. In addition, CT could also attach to endogenous or microbial protein, which would enhance the soluble N excreted (McNeill et al 1998).

 

The amount of soluble nitrogen in faeces (FADSN) was higher when fed L40 and L60, probably due to the increase of dietary CP intake, microbial N or endogenous protein attached to CT. However, this study was not able to confirm this information, as we have not worked with protein marker. Although the results suggested an influence of CT, more studies evaluating true digestibility and endogenous nitrogen losses should be performance for better understandings.

 

Regarding to CT, it is well known that CT binds protein and other macromolecules in rumen, which may reduce availability of the nutrients to microorganisms degradation (Barry and Duncan 1984, Makkar et al 1988, D´Mello 1995, McNeill et al 1998, McAllister et al 2005). The advantage in this process is that excessive ammonia production in the rumen could be reduced, decreasing urinary N losses. As a result, nitrogen retention may increase in ruminants fed tannin-rich plants (Kaitho et al 1998b) or remain without effect (Carulla et al 2005; Bengaly et al 2007).

 

Based on the results of faecal and urinary nitrogen losses, it was expected that L20 presented higher or at least similar N retention as control diet, however, it showed lower value. It is possible that the low nitrogen intake and the high nitrogen bound to fibre (ADIP) affected negatively nitrogen retention in L20. Urinary and faecal nitrogen losses in L40 were higher than in L20 but similar to L60, however the N retention was similar for  L40 and L20 and was higher in L60. It is hypothesized that the higher amount of protein in the rumen fed L60 and L40 was compensated by the high amount of CT, which controlled protein degradation and nitrogen retention. N retention showed high coefficient of variance (CV = 53%), probably due to problems in the calculating daily N retention, which result in a highly unstable data. In addition, fibre determination in tannin rich plant could be distorted due to insoluble complexes that can appear in the residue (Makkar et al 1995), which would overestimate nitrogen loss in faeces. Thus, N retention was carefully taken into interpretation of nitrogen balance among diets.

 

Conclusions 

• Leucaena leucocephala had good acceptability by Santa Inês sheep and voluntary intake was positively affected. However, digestibility of dry matter, crude protein and fibre was reduced.

• Increased level of Leucaena leucocephala increased intake of crude protein and soluble protein. However, the level of energy in the diet should also increase in order to synchronize protein/energy and avoid protein escape from the rumen.

• Supplementation of 20 % Leucaena offered the best result as was the most compatible with the basal diet offered in this study. However, recommendation of inclusion level of tanniniferous forage in ruminant diet depends not only on the quantity of phenolic compound but on its chemical characteristics and on the energy content of the base diet.

• It is suggest that a performance study be processed before introducing tanniniferous forage in the feed system.

 

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