Effect of supplementation with a tree legume forage on rumen function
Alberto Navas-Camacho, Max A Laredo, Aurora Cuesta, Hector Anzola and Juan C Leon
Instituto Colombiano Agropecuario, AA 151123 El Dorado, Bogotá
Multi-purpose trees, as components of production systems in the tropics, play an important role in soil conservation and provision of shade, as well as being an important alternative as a source of protein-rich forage source. They are also more drought-resistant than grasses. The present experiment aimed at evaluating the effect on rumen function of supplementing sheep with three levels (0, 100 and 300g/d) of dry leaves of a tree legume (Enterolobium ciclocarpum), which is naturally consumed by ruminants when branches are accessible to them. The main results of the experiment were the following: It appears that dietary and maybe bacterial amino acid absorption at the small intestine was increased by supplementation with leaves of E. ciclocarpum, as indicated by the positive correlation between wool growth rate and intake of E. ciclocarpum. Supplementation with low levels (100 g/d; approximately 10% of total dry matter intake) of E. ciclocarpum, increased voluntary feed intake, digestible matter intake and nitrogen balance by increasing nitrogen digestibility. However, the high level of supplementation (300 g/d; approximately 34% DMI) reduced digestible dry matter intake, which, could be an important limitation to its inclusion in the diet at high levels. The population of rumen fungi was increased by supplementation with low and high levels of leaves of Enterolobium ciclocarpum. Rumen ciliate protozoa were increased by the inclusion of low levels in the diet (10% of DMI), but strongly decreased in sheep receiving high levels of supplementation (34% of DM). Rumen bacteria showed no response to supplementation. The presence of saponins in leaves of Enterolobium ciclocarpum may have contributed to the reduction in protozoa numbers in the rumen fluid, particularly Holotrich protozoa.
KEY WORDS: Ruminants, protozoa, fungi, legumes, trees, wool growth
Trees are playing an increasingly important role in agricultural production systems in the tropics. They have beneficial effects on soil fertility (by protecting soil from erosion and supplying nutrients by nitrogen fixation and incorporation of organic matter); they provide shade which helps to reduce heat stress of cattle in hot and humid areas; and they are an important alternative as forage source, due to their high production of edible, highly-acceptable biomass and drought resistance (Otsyina and McKell 1985; Preston and Murgueitio 1987).
Tree leaves have a high protein content (18-26% crude protein on average), and some of them have low rates of degradability in the rumen (Espinosa 1984). These characteristics, along with those mentioned above, make them an alternative source of by-pass protein to be evaluated as a supplement for ruminant production systems in the tropics.
The present experiment aimed at evaluating the effect on rumen function of supplementing sheep fed on pasture hay with leaves of a tree legume (i.e. Enterolobium ciclocarpum), which is naturally consumed by ruminants when branches are accessible to them.
Materials and methods
The experiment was carried out at ICA's (Instituto Colombiano Agropecuario) National Research Centre, Tibaitata, located at 14 km from Bogota at 2600 metres above sea level, with 13 °C average temperature and 83% relative humidity.
Twelve cross-bred castrated male sheep, averaging 27 kg live weight and 12 months old, fitted with permanent rumen cannulas were used. They were assigned to one of three diets (4 animals per diet) and housed in individual metabolic cages. The diets were:
Control: Hay of Pennisetum clandestinum ad
EC100: Pennisetum hay and 100 g/d of sun-dried leaves of Enterolobium ciclocarpum (E.c.).
EC300: Pennisetum hay and 300g/d of Enterolobium
All the animals were supplemented with urea (3g/d and 8g/d for animals without and with supplementation with Enterolobium) with the aim of ensuring 20 mgN-NH3/100 ml of rumen fluid. A mineral mixture (20 g/d) was also given.
The chemical composition of the diets is shown in Table 1.
|Table 1: Percentage of dry (DM), organic matter (OM), total (TP) and soluble protein (SP), and neutral detergent fibre (NDF) of Pennisetum clandestinum and Enterolobium ciclocarpum|
|(%)||(%)||(% of DM)||(% TP)||(% DM)|
Dry matter of feed was estimated by drying samples to constant weight in an air-forced oven at 65 °C for 48h. Organic matter was assessed by placing samples in a muffle furnace at 600 °C for 24h. Total protein (N x 6.25) was estimated by digestion with sulphuric acid, distillation and titration as described by Laredo and Cuesta (1985). Protein (N x 6.25) digested in HCl-Pepsin was used to estimate soluble protein (Laredo and Cuesta 1985). Cell wall content was estimated by using the neutral detergent fibre (NDF) procedure (Van Soest 1982).
Samples of rumen fluid were taken just before and 4h after feeding via the rumen cannula by using a 50 ml syringe and a 50 cm-long plastic probe attached to the syringe. Rumen fluid pH was determined using a Millivolt Meter 611 potentiometer. Concentration of ammonia nitrogen in the rumen fluid was estimated by distillation and titration as described by Laredo and Cuesta (1985). The dilution curve of chromium in the rumen (determined by atomic absorption spectrophotometry) after a single injection (0.5 ml/kg live weight) of a CrEDTA solution (2.77 mg Cr/ml solution) via cannula was used to estimate rumen volume and dilution rate of rumen fluid as described by Binnerts et al (1968). Measurements of rumen dry matter and protein in sacco degradability of the pennisetum hay and the dried enterlobium leaves were carried out following the technique described by Orskov et al (1980).
Estimation of protozoa population in the rumen fluid was by diluting 8 ml of rumen fluid with 16 ml of a formal saline solution (1 part of formol 37% and 9 parts of saline 0.9% solution) and counting protozoa under light-microscopy (10x) using a Neubauer chamber. To assess bacterial population, samples of rumen fluid were diluted 1:3 in formal saline solution and again diluted to 103 in formal saline solution. Crystal violet (20 ml) was added to 200 ml of this solution and the stained bacteria were read under light-microscopy (40x) using a Neubauer Chamber. Rumen fungi population was estimated by incubating for 24h in the rumen strips of pennisetum hay put into nylon bags. After taking out the bags from the rumen they were carefully washed with tap water, and stained for 2 min with lactophenol cotton blue; sporangia were counted under light-microscopy (20x).
A 7-day nitrogen balance and dry matter and protein (N x 6.25) digestibility in the whole tract was conducted. Feed refusals, faeces and urine were collected and weighed daily and a 10% sample was kept at -20 °C until they were analyzed. Measure of wool growth rate was carried out as described by Wodzika and Bigham (1968).
Results and discussion
Protozoa numbers in the rumen fluid:
Sheep supplemented with 100 g/d of enterolobium (representing approximately 10% of the DM intake) showed a higher concentration of total rumen protozoa (22% and 10% higher before and 4h after feeding respectively) than sheep receiving no supplementation (P<0.01, Table 2). However, higher levels of supplementation (300g/d, 34% of DM intake) decreased the concentration by 40 and 59% before and after feeding respectively. Marin (1990) also found an increase of 21% in the total concentration of protozoa in cattle supplemented with 13% of the diet dry matter as compared with those receiving no supplementation.
|Table 2: Concentration of Entodiniomorphs (ENT), Holotrichas (HOL) and total protozoa (TOT) in the rumen fluid of sheep fed on Pennisetum clandestinum and levels of Enterolobium ciclocarpum. Values are before (BF) and 4h after feeding (AF).|
|Plus 100 g/d of EC||1.48||0.019||1.50||1.33||0.024||1.35|
|Plus 300g/d of EC||0.66||0.007||0.67||0.48||0.004||0.48|
Unlike rumen bacteria, protozoa use protein of low solubility as a nitrogen source for cell synthesis (Michalowski 1989). Protein of Enterolobium had a low degradability in the rumen (less than 50% after 48h of rumen incubation, Table 11) and its tannin content (3% condensed tannin in DM), apparently brought about the reduction in protein degradability of the whole diet (Leinmüller et al 1991). It is possible therefore that the greater availability of slowly degradable protein in the diet of the sheep supplemented with Enterolobium (10% of the diet DM) may have been associated with the increase of protozoa numbers on this diet.
The high level of inclusion of Enterolobium decreased the protozoa numbers in the rumen fluid (Table 2), which may be related to the toxic effect of the saponins (13% in the DM) on protozoa (Lu and Jorgensen 1987). At the high level of supplementation, the saponin could have outweighed the tannin effect on protozoa. Rumen infusion of 2% and 4% alfalfa saponin decreased the protozoa population by 37 and 47% as compared to sheep receiving no saponin (Lu and Jorgensen 1987). Saponin concentration in the present experiment was 1.2 and 4% of the diet DM for the animals receiving 100 and 300 g/d enterolobium, respectively.
Holotrich were the protozoa most susceptible to the Enterolobium (Table 2). They were reduced by 73% and 90% (before feeding) as the level of Enterolobium in the diet increased from 0 to 10 and 34% of the DM intake (P<0.01, Table 2). Conversely, the low level of Enterolobium increased the Entodiniomorph population by 26% (samples before feeding) and 13% (after feeding, P<0.01). However, the high level of supplementation strongly decreased the population (40 and 59% before and after feeding respectively) as compared to those sheep receiving no Enterolobium. Navas-Camacho et al (1992), also found that in sheep fed E.c. (100% DM) for 5 days, Holotrich were apparently eliminated from the rumen, while Entodiniomorph were strongly reduced (to 103/ml).
Lu and Jorgensen (1987) suggested than Entodiniomorphs were the protozoa most susceptible to saponins. The difference between their and our results may be associated with a different biological effect of a different kind of saponin (Bondi et al 1973), or with the presence of other secondary metabolites in Enterolobium.
FUNGAL AND BACTERIAL POPULATION: Rumen fungi (numbers of sporangia grown on blades of Pennisetum hay) increased by 2 and 31% as the level of Enterolobium in the diet increased from 0 to 10 and 34% of DM intake (P<0.1, Table 3). Akin and Windham (1989) reported a positive effect of alfalfa on the population of rumen fungi, which suggest the presence of growth factors in, or the rumen environment created by, both herbaceous and tree legumes.
|Table 3: Number of sporangia cultivated for 24h in blades of leaves of pennisetum hay in the rumen of sheep fed on Pennisetum hay and Enterolobium|
|Diet||Sporangia numbers (#/mm2)|
|Plus 100 g/d EC||81|
|Table 4: Bacterial population in the rumen fluid of sheep fed Pennisetum clandestinum and Enterolobium ciclocarpum|
|Diet||Before Feeding||After Feeding|
|Plus 100 g/d EC||15.8||11.7|
|Plus 300g/d EC||12.9||11.1|
The reduction of predation of zoospores by protozoa (Orpin 1975), may be the explanation of the increased rate of colonisation of the pennisetum hay. However, the number of sporangia did not correlate with protozoa numbers in the rumen fluid (neither before feeding: r=0.26, P>0.1; nor after feeding: r=0.42, P>0.1).
The level of Enterolobium in the diet had no effect on bacteria numbers in the rumen fluid (Table 4).
Rumen degradation and total digestibility
The data are in Table 5.
|Table 5: Dry matter in sacco degradability of Pennisetum clandestinum and Enterolobium ciclocarpum alone or as mixtures in sheep fed on P. clandestinum and E. ciclocarpum. Values are the variables of the equation Y=a+b(1-e-ct) (Orskov et al 1980).|
|Diet||Sample in the bag||n||a||b||c||a+b|
*M1: Mixture 1 = 0.9g Pennisetum + 0.1g EC
*M2: Mixture 2 = 0.7g Pennisetum + 0.3g EC
The low level of supplementation with Enterolobium increased the rumen degradability (fractions a+b) of both Pennisetum hay (from 67 to 74%; P=0.13) and total diet (from 67 to 70%; P<0.01). However, the high level of inclusion of Enterolobium in the diet decreased rumen dry matter degradability. Dry matter degradability of Pennisetum was decreased by 6% and that of the total diet by 10%. Solubility (fraction 'a' of the exponential equation), potential degradability of the non-soluble fraction (fraction 'b') and rate of degradation (fraction 'c') were nevertheless not affected (P>0.30) by Enterolobium supplementation. The degree of rumen degradation of Enterolobium (a+b) was not affected by its level of inclusion in the diet (P>0.5), but the low level of supplementation increased the rate of rumen degradation (P<0.05).
Several studies (Gordon and Phillips 1989; Akin et al 1989; Soetanto 1985) have indicated that a greater fungal population is associated with a greater degradability of the fibrous fraction of the diet. Rumen fungi produce fibrolytic enzymes (Gordon and Phillips 1989), but also contribute to increase fibre degradation by decreasing fibre resistance to size reduction (Akin et al 1989) and increasing sites available for bacterial action (Bauchop 1989).
In the present experiment, despite the fact that fungal colonisation on Pennisetum was greater in sheep receiving the high level of supplementation with Enterolobium (Table 3), dry matter degradability was found to be decreased in these animals, as compared to sheep receiving no supplementation (from 67 to 61%).
The presence of tannins in the Enterolobium could have not only reduced protein degradability, because of the formation of linkages with the protein, but also may have formed linkages with the fibrous fraction of the diet (Leinmüller et al 1991) and therefore affected its rumen degradation. Despite the increase in fungal population and a similar bacterial density in the rumen fluid, the high level of inclusion of Enterolobium appears to have reduced their activity in the rumen.
Rumen values of pH (Table 6) and concentration of ammonia in the rumen fluid (Table 7) were not associated with the lower rumen dry matter degradability found in sheep receiving the high level of supplementation with Enterolobium. Bacterial cellulolytic activity is increased at neutral pH (Russell and Dandbrowski 1980), and rumen pH was increased as the level of Enterolobium in the diet increased.
|Table 6: Rumen fluid pH of sheep fed Pennisetum clandestinum and Enterolobium ciclocarpum|
|Diet||n||Rumen fluid pH|
|Before feeding||4h after feeding|
In all animals rumen ammonia-N concentration was at, or above, the level (20 mg N-NH3/ml) (Table 7), found to maximise rumen fibre degradation and feed intake in ruminants fed on forages (Perdok and Leng 1989).
Similarly, dilution rate was not apparently related to the differences found in dry matter degradability among treatments (Table 8). The level of inclusion of E.c. tended to reduce dilution rate (and therefore increase the retention time of solids), which should have increased (instead to decrease) the degree of dry matter degradation in the rumen.
|Table 7: Concentration of ammonia-N in the rumen fluid of sheep fed Pennisetum clandestinum and Enterolobium ciclocarpum. Values are for samples taken before feeding (BF) and 4h after feeding (AF).|
|Diet||Ammonia-N in rumen fluid (mg N/100 ml)|
|Pre - experimental||Experimental|
|Table 8: Rumen volume and dilution rate of rumen fluid of sheep fed on Pennisetum clandestinum and Enterolobium ciclocarpum|
|Diet||Rumen volume||Dilution rate|
Dry matter digestibility in the whole digestive tract showed the same pattern as that of rumen degradability (Table 9). Supplementation with Enterolobium apparently did not affect the partial coefficients of dry matter degradation for the rumen and intestine, as was found by Lu and Jorgensen (1987) in response to saponin infusion in the rumen: the infusion increased the coefficient for intestinal degradation.
|Table 9: Dietary dry matter digestibility in the whole digestive tract in sheep fed Pennisetum clandestinum and Enterolobium ciclocarpum. Trial was carried out during the 5th and 12th experimental week.|
|Diet||Dry matter digestibility (%)|
|n||5th week||12th week|
Rate of wool growth depends essentially on the amounts of amino acids (particularly sulphur amino acids) absorbed from the small intestine (Ferguson et al 1967). Therefore, wool growth rate is an indirect, simple and economic measurement of the relative amount of amino acids available and absorbed at the small intestine (Leng et al 1984).
The amount of amino acid absorbed was apparently increased as the level of Enterolobium in the diet increased, as is indicated by the greater rate of wool growth in supplemented sheep (Table 10). This increment in amino acid availability probably was of dietary and maybe also of microbial origin.
|Table 10: Wool growth rate in sheep fed on Pennisetum clandestinum and Enterolobium ciclocarpum|
|Diet||Wool growth rate|
|n||(g/10cm2 of patch)|
As was mentioned above, the protein of Enterolobium had a low rumen degradability, and its inclusion in the diet may have reduced also the degradability of the total dietary protein because of its tannin content (Leinmüller et al 1991; Table 11). The protein degradability of the Pennisetum was not affected by the low level of Enterolobium but it was decreased from 82% to 69% in sheep supplemented with the high level of Enterolobium (34% of dietary DM). This reduction in the rate of degradation of the protein in the rumen should have had the effect of increasing the amount of by-pass protein available to the sheep given the diet containing Enterolobium.
|Table 11: Protein in sacco degradability of Pennisetum clandestinum and/or Enterolobium ciclocarpum in sheep fed on P. clandestinum and E. ciclocarpum. Values are the variables of the equation Y=a + b(1-ect) (Orskov et al 1980)|
|Diet||Sample in the bag||a||b||c||a+b|
*M1: Mixture 1 = 0.9 g Pennisetum + 0.1 g EC
*M2: Mixture 2 = 0.7 g Pennisetum + 0.3 g EC
It is now well known that the elimination of protozoa from the rumen increases the efficiency of bacterial synthesis in the rumen and its flow rate to the small intestine (see Preston and Leng 1989). It is possible therefore that the reduction in the protozoa population in sheep receiving 300 g/d of Enterolobium brought about an increase in bacterial synthesis in the rumen (Diaz et al 1992) and therefore greater availability of bacterial protein at the small intestine.
However, although the literature is consistent in indicating that
defaunation results in a higher amino acid availability at the small intestine, the effect of reducing, rather than eliminating, the ciliate population is not as clear. Meyers et al (1986) found that a seven-fold increase in the concentration of ciliate protozoa in sheep had no effect on the efficiency of bacterial synthesis in the rumen or the flow rate of microbial protein to the small intestine. Navas-Camacho (1991), on the other hand, found no interaction between the effect of defaunation on wool growth rate, live weight gain or feed conversion ratio, and the protozoa numbers in sheep fed on diets having three ratios of fibre to sucrose. Sheep with the highest level of sucrose in the diet (45% of the DM), had 3 times as many rumen protozoa in the rumen as sheep receiving the lowest level of sucrose (15% of DM). There was a tendency (P>0.15) for nitrogen balance to increase in sheep fed 10% Enterolobium, but the high level of inclusion of Enterolobium tended to decrease nitrogen retention (Table 12).
|Table 12: Nitrogen intake (NI), nitrogen excreted in faeces (NEF), nitrogen excreted in urine (NEU) and nitrogen retention (NR) in sheep fed on Pennisetum clandestinum and Enterolobium ciclocarpum. The trials were carried out during the 5th and 12th experimental week.|
|Pennisetum (Week 5)|
|Pennisetum (Week 12)|
Increased faecal excretion of nitrogen accounted for the differences in nitrogen retention (Table 12). Nitrogen excretion in faeces increased with the high level of inclusion of Enterolobium in the diet, which could be associated with the level of tannin (McBrayer et al 1983; Hill et al 1987). Hill et al (1987) found that the increase in the concentration of tannin in the diet of young steers from 1.2 to 5.8% of DM, decreased nitrogen retention due to a greater faecal excretion of nitrogen.
There was no difference among treatments in the proportion of nitrogen excreted in urine.
Effect of the level of supplementation with enterolobium ciclocarpum on voluntary feed intake and live weight gain.
Leaves of E.c. were highly accepted by sheep. This plant is normally browsed in the field when animals (sheep, goats or cattle) have access to the lower branches of this tree. Sheep that had no previous contact with E.c. leaves shown also a good acceptability to it when offered as the only diet for 5 days (Navas-Camacho et al 1992).
The low level of supplementation with Enterolobium increased the intake of the basal diet (Pennisetum) by 2% and total dry matter intake by 13% (P<0.01; Table 13), which was not affected by animal liveweight (Table 13). The increase in dry matter intake could have been brought about by the positive effect of low levels of inclusion of Enterolobium on dry matter digestibility (Table 5). Digestible dry matter intake was 17% higher than that of those sheep receiving no Enterolobium. Sheep supplemented with the high level of Enterolobium had a lower dry matter digestibility, lower (31%) intake of the basal forage (Pennisetum) and lower (10%) digestible dry matter intake.
|Table 13: Dry matter (DMI) and digestible dry matter intake (DDMI) of sheep fed on Pennisetum clandestinum and Enterolobium ciclocarpum|
The negative effect of high levels of inclusion of this kind of tree legume on digestible matter intake represents an important limitation for its use as a supplement in forage diets. However, the positive effect of a low level make it an interesting alternative as a supplement to grazing or fibrous-residues fed to ruminants in the tropics.
Daily live weight gain was rather low for all treatments, and although it tended to be higher for the supplementation treatments, there was considerable variation within treatments as shown by the high variance for this parameter (Table 14).
|Table 14: Liveweight gain (LWG) of sheep fed on Pennisetum clandestinum and Enterolobium ciclocarpum|
|Diet||Liveweight gain (g/d)|
The experimental animals were under constant stress because of their permanence in metabolic cages and being subjected to continuous manipulation because of the sample taking. One must also question the validity of feed intake and growth data obtained with animals with rumen fistulas. Nevertheless, the data tend to substantiate the rumen and wool growth studies in indicating a positive response on animal production through supplementation with low levels of Enterolobium (10% of the diet DM).
Supplementation with low levels (10% of dietary DM) of Enterolobium ciclocarpum, increases voluntary feed intake, digestible matter intake and nitrogen balance by increasing nitrogen digestibility. High level of supplementation with Enterolobium ciclocarpum (34% of dietary DM) reduced digestible dry matter intake, which could be an important limitation to its inclusion in the diet at this level.
The rumen fungal population was increased by supplementation with both low and high levels of leaves of Enterolobium ciclocarpum. Rumen protozoa were increased by low levels of inclusion of Enterolobium in the diet, but strongly decreased by high levels of supplementation. Rumen bacteria showed no response to Enterolobium supplementation.
The presence of saponins in the leaves of Enterolobium ciclocarpum may have contributed to the reduced protozoa numbers in the rumen fluid, particularly the Holotrich protozoa.
The experimental findings are interpreted as indicating an increase in dietary and maybe bacterial amino acid availability and absorption at the small intestine in response to supplementation with leaves of Enterolobium ciclocarpum.
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(Received 1 July 1993)