Effect of supplementing the Dicanthium annulatum hay diet with tree leaves on microbial activity in the rumen of goats

Four adult goats (mean body weight 29.0±2.65 kg) were fed Dicanthium annulatum Leucaena leucocephala (DA-LL) and Dicanthium annulatum Hardwickia binata (DA-HB) in 50:50 ratio in switch-over experiment to assess the effect of tree leaves supplementation on rumen concentration of protozoa, their generic distribution, bacteria and fungi. Animals were maintained on each diet for more than 3 months and rumen liquor was collected at 0 and 4 h post feeding. Mean protozoa counts were 5.98 and 5.03×105/ml in goats supplemented with LL and HB, respectively. Entodiniomopha were the most dominant (88.0%) rumen protozoa on both diets. Total viable, amylolytic and cellulolytic bacteria population was higher (P<0.05) in rumen of goats fed DA-LL (23.74, 11.22 and 29.66) than DA-HB diet (10.52 × 108/ml, 6.31 × 108/ml and 11.02 × 107/ml), while the concentration of proteolytic bacteria was 8.64 and 6.23 × 107/ml in rumen of goats fed earlier and later diets, respectively. Mean sporangia counts were comparable in rumen liquor of goats fed both types of diets (0.31 vs 0.33 × 105/ml), while total fungi was higher (P<0.05) in goats fed DA-LL (9.62) than DA-HB (5.04 × 104/ml). On both diets bacteria population was higher at 0 h post feeding, while population of fungi was higher at 4 than 0 h of feeding. Results revealed that goats had higher activities of bacteria and fungi on LL supplemented diet.


INTRODUCTION
Browse species (tree and shrubs foliage) with grasses constitute a major component of pasture and grazing systems and provide a major part of cellulosic biomass to ruminants particularly to goats and sheep.Tree foliages usually rich SINGH S. ET AL.
in protein and minerals are used as dry season supplement to poor quality natural pasture, grazing land and /or poor quality crop residues (Devendra, 1981;Kibon and Ørskov, 1993).Presence of lignin and other anti-nutritional components mainly the tannins and phenolics hampers their use as animal feed.Tannins and phenolics had bacteriostatic and bactericidal effect on rumen microbes (Henis et al., 1964;Martin and Akin, 1988), fungi (Akin and Rigsby, 1987) and protozoa (Akin, 1982).Work with Leucaena leucocephala and Hadwickia binata tropical trees on rumen microbial population (bacteria, protozoa and fungi) seems to be obscure.The present work was carried out to assess relative effect of these tree leaves supplementation to Dicanthium annulatum grass hay on rumen microbial activity in goats.

Animal feeding and rumen sampling
Four adult local male goats (mean body weight 29.0±2.65 kg) were maintained on Dicanthium annulatum (DA) -Leucaena leucocephala (LL) and Dicanthium annulatum (DA) -Hardwickia binata (HB) diets (50:50 ratios on % DM basis) for more than 3 months on each diet in switch-over experiment.Animals were offered half grass (conserved as hay) and tree leaves (collected from whole plant canopy and wilted for 24 h before feeding) together once at 9 a.m. and remaining half at 12.00 noon.The animals maintained hygienically had free access to clean drinking water.The chemical composition of grass (DA) and tree leaves (LL and HB) is presented in Table 1.After more than 1 month of feeding rumen liquor samples were collected at 1 month interval for 2 consecutive days from animals at 0 and 4 h post parandial to estimate protozoa counts, their generic distribution and fungal sporangia numbers.Total viable bacteria, amylolytic, cellulolytic and proteolytic bacteria, and total fungal population were ascertained too in fresh rumen liquor samples collected 1 month interval twice for individual animal on each diet.

Analysis
Grass and tree leaves samples were analysed for proximate and cell wall polysaccharide according to AOAC (1980) and Goering and Van Soest (1970).Method described by Moir (1951) was followed for counting the total protozoa in formaldehyde-conserved rumen liquor.For generic identifi cation, morphological features of Kudo (1960), Hungate (1966) and Ogimoto and Imai (1981) were used.For culturing bacteria pre-reduced anaerobic dilution fl uid was prepared as described by Bryant and Burkey (1953).For starch hydrolysing bacteria estimation 0.1 ml iodine-potassium iodide solution (1:2) in the tubes was added.For culturing cellulolytic bacteria Whatman No. 1 fi lter paper strip was used and media was prepared as described by Mann (1968).For enumerating proteolytic bacteria and anaerobic fungi composition of media described by Abou- Akkada and Blackburn (1963) and method of Theodorou and Trinci (1988), respectively, were followed.Most probable number of proteolytic bacteria was computed using McCrady table based on the positive tubes.
Data were analysed according to Snedecor and Cochran (1968).

RESULTS AND DISCUSSION
The results revealed that goats supplemented with LL tended to have higher numbers of protozoa (5.98) than those from HB supplementation (5.03×10 5 /ml; Table 2).Entodiniomorpha were the most dominant (88%) protozoa in the rumen liquor of goats on both diets.Mean counts of Entodiniomorha and Holotricha were 5.26 and 0.70 on DA-LL vs 4.48 and 0.52×10 5 /ml on HB supplemented diet.Percent generic distribution of protozoa revealed that Isotricha and Entodinium were predominant species and constituted 50 and 80% of the Holotricha and Entodiniomorpha population, respectively (Figure 1).Blepharoprosthium and Ophryoscolex occurrence was lowest in rumen liquor of goats both on LL (0.4 and 0.4%) and HB (0.5 and 1.1%) supplemented diets, respectively.Variability in protozoa counts in rumen of sheep supplemented with different multipurpose tree leaves has been reported by Odenyo et al. (1997).They further reported that Entodiniomorpha are most dominant (93.3%) on all the tree leaves-supplemented diets being in accordance with the present results where Entodiniomorpha constitute 88% of total protozoa numbers.Mean total viable bacteria concentration was signifi cantly (P<0.05)higher in rumen liquor of goats supplemented with LL (23.74) than with HB leaves (10.52×10 8 / ml; Table 3).Similarly the mean activities of amylolytic, cellulolytic and proteolytic bacteria were signifi cantly (P<0.05)higher in rumen of goats fed 29.66 and 8.64) than DA-HB based diet (6.31×10 8 /ml, 11.02×10 7 /ml and 6.23×10 7 /ml, respectively).The higher bacterial activities in goats rumen on LL supplemented diet may be attributed to its higher CP content and degradability (Singh, 2004) resulting in optimum metabolites concentration for rumen environment conducive for multiplication and proliferation of bacteria.
Rumen liquor from goats fed diet supplemented with LL and HB had similar fungal sporangia counts (0.31 and 0.33 × 105/ml; Table 4).However, fungus activity (TFU) was signifi cantly (P<0.05)higher in rumen liquor of goats fed LL (9.62) vs HB (5.04×104/ml) supplemented diet.The reason for higher fungal activity on LL supplemented diet is not known.However, higher mineral contents particularly of S in LL might be responsible for increased fungal activity in the rumen of goats.

CONCLUSIONS
The results presented here indicate that activity of rumen microorganisms and particularly that of bacteria and fungi is higher in rumen of goats on Leucaena lancocephala supplemented diet and thus goats can be supplemented with up to 50% of total diet (dry matter basis) with Leucaena lancocephala for higher rumen microbial activity.

Table . 1
Chemical composition of grass-tree leaves, % DM basis Chemical constituents

Table 2 .
Protozoa concentration in goat rumen fed grass-tree leaves diet

Table 3 .
Bacterial activity in goat rumen fed grass-tree leaves diet

Table 4 .
Fungal sporangia counts and fungus activity in goat rumen fed grass-tree leaves diet