Livestock Research for Rural Development 23 (1) 2011 Notes to Authors LRRD Newsletter

Citation of this paper

Comparative rumen microbial population in sheep fed Dicantium annulatum grass supplemented with Leucaena leucocephala and Hardwickia binata tree leaves

Sultan Singh and S S Kundu*

Plant Animal Relationship Division, Indian Grassland and Fodder Research Institute, Jhansi-284003, India
* National Dairy Research Institute, Karnal, India


In a switch over experiment 4 male adult Muzzafarnagari sheep (mean wt. 26.93 1.74 kg)  were fed Dicanthium annulatum-Leucaena leucocephala (DA-LL) and Dicanthium annulatum- Hardwickia binata (DA-HB) in 75:25 ratios, respectively in  2 separate feeding trials to assess the effect of tree leaves supplementation on protozoa population and their generic distribution, bacteria population and fungus counts. Sheep were maintained on each diet for more than 3 months and rumen liquor was collected twice at 0 and 4 h post feeding from each animal to ascertain the rumen microbes.


Mean protozoa counts were 5.34 and 3.95 x105/ml in rumen of sheep supplemented with LL and HB, respectively. Entodiniomophs were the most dominant protozoa species in rumen liquor of sheep on DA-LL (90.82) and DA-HB diets (87.59 %).  Total viable bacteria and cellulolytic bacteria were significantly (P<0.05) higher in rumen of sheep fed DA-LL (16.05 and 21.46) than DA-HB diet (8.53 x 108/ml and 9.18 x 107/ml). The activity of amylolytic and proteolytic bacteria was 8.84 and 7.70 on DA-LL and 5.04 x 108/ml and 4.45 x 107/ml on DA-HB diet respectively. Mean sporangia counts were comparable in sheep rumen liquor on both diets, while total fungi as thallus forming unit (TFU) were (P<0.05) higher in sheep fed DA-LL (12.87) than DA- HB (6.32 x104/ml).  On both diets bacterial population was more at 0 h post feeding, while fungi was more at 4 than 0 h of feeding.


Sheep rumen liquor had higher activities of protozoa, bacteria and fungi on DA-LL diet. 

Key words: bacteria, dicanthium aanulatum, fungi, protozoa, sheep, tree leaves


The diet of the small ruminants and camels consistently contains tree leaves and shrubs more than other livestock diets (Devendera 1989). In many parts of the globe browse species leaves form a natural part of the diet of many ruminant species and have been used as source of forages for livestock (Aganga 2003).  At the time when grazing provides animals only dried mature grass which is poor in nutritive value and palatability and often inadequate, browse plants provides green forage as supplement to poor quality crop residues (grass, straw and stovers)  (Toutain 1980.) Tree foliage usually rich in protein and minerals are used as dry season supplement to poor quality natural pasture, grazing land and /or poor quality crop residues (Singh 2004; Mtui et al 2008; Aganga and Mesho 2008).  Presence of lignin and other anti-nutritional components mainly the tannins, saponins and phenolics hampers their use as animal feed. Anti microbial properties of plant secondary metabolites have been reviewed by Wallace (2004). Studies on effect of supplementation with tropical trees like Leucaena leucocephala and Hadwickia binata on rumen microbial population seems to be obscure. The present study evaluates the effect of these tree leaves supplementation to Dicanthium annulatum grass diet on rumen microbe’s activity in sheep.


Materials and methods 

Animals and diets


In two separate feeding trials 4 adult male Muzzafarnagari sheep with mean body weight 25.622.13 kg and 28.251.7 kg were fed Dicanthium  annulatum-Leucaena leucocephala (DA-LL) and Dicanthium  annulatum-Hardwickia binata (DA-HB), respectively. Sheep in both trials were offered grass and tree leaves in 75: 25 ratios, respectively (% DM basis). The animals on each dietary regimen were maintained more than 90 days. Animals were offered half of the total grass and tree leaves once at 9 a.m. and remaining half at 12.00 noon. The animals had   free access to clean drinking water and were maintained under hygienic conditions. The chemical composition of DA with LL and HB has been reported earlier (Singh and Kundu 2010).  After more than 1 month of feeding rumen liquor samples were collected twice at monthly interval from animals at 0 and 4 h post-prandial to estimate protozoa count, their generic distribution and fungus sporangia numbers. Microbial population as bacterial numbers (total viable bacteria, amylolytic bacteria, cellulolytic, proteolytic bacteria) and total fungal population were ascertained in freshly drawn rumen liquor.


Analytical techniques


Moir (1951) method was followed for enumerating the total protozoa in formalized rumen liquor. Generic identification of protozoa was done as per morphological features compiled by Kudo (1960), Hungate (1966) and Ogimote and Imai (1981). Pre- reduced anaerobic dilution fluid was prepared as per Bryant and Burkey (1953) for culturing bacteria. For starch hydrolysing bacteria estimation 0.1 ml iodine-potassium iodide solution (1:2) in the tubes was added.  For enumeration of cellulolytic bacteria Whatman No. 1 filter paper strip was used and media was prepared as per Mann (1968). For estimation of proteolytic bacteria and anaerobic fungi media composition given by Abou-Akkada and Blackburn (1963) and method of Theodorou and Trinci (1988), respectively were followed. Most probable number of bacteria and fungi were computed using Mc Crady table based on the positive tubes. Data on protozoa count, bacterial numbers and fungus population was subjected to one way analysis using general linear model of SPSS version 12 and Duncan’s multiple range test was applied to test the significance as per Snedecor and Cochran (1968).


Results and discussion 

Protozoa counts


Mean total protozoa numbers were (P<0.05) higher in rumen liquor of sheep fed DA-LL (5.34) than DA-HB (3.95 x105/ml) based diets (Table 1).

Table 1.  Protozoa concentration in sheep rumen fed DA grass-tree leaves diets

Protozoa number



Total protozoa x 105/ml    0h



                                        4 h






Entodiniomorphs             0h



                                        4 h






Holotrichs                        0h



                                        4 h






a,b means within a row bearing different superscripts differ significantly( p<0.05)

Entodiniomorphs were the most dominant (90 %) protozoa in the rumen liquor of sheep on both the tree leaves supplemented diets. Average numbers of Entodiniomorhs and Holotrichs were 4.85 and 0.50 on DA- LL vis--vis 3.46 and 0.48 x105/ml on HB supplemented diet. Fenn and Leng recorded protozoa population in the range of 3.7-5.6 x105/ml in sheep on bentonite and without bentonite supplemented diets which are within the range of present observations. Protozoa populations in sheep rumen liquor on different straw-concentrate diets (Prasad and Pradhan 1990) also substantiate the present results.  Isotricha and Entodinium were the most predominant species and constitute 50 and 80% of the Holotrichs and Entodiniomorphs population, respectively (Figure 1).


H: Holotrichs, I: Isotricha, D: Dasytricha, C: Charon, B: Blepharoprosthium, E: Entodiniomorphs, En: Entodinium, Di: Diplodinium, O: Ostracodinium, Os: Ohryoscolex, Ep: Epidinium, Dip: Diploplstron, M: Metadinium, Eu: Eudiplodinium, PP: Polyplastron, Er: Eremoplatron, U: Unidentified

Figure 1.  Generic distribution of protozoa in rumen of sheep fed DA grass-tree leaves

Mughetti et al (2007) also reported highest occurrence of Entodiniomorphs species (64.0 %) of total protozoa in rumen of sheep fed diet of different rumen degradability which substantiates the present findings. Occurrence of Blepharoprosthium and Ophryoscolex species was lowest in rumen liquor of sheep on both DA-LL (0.15 and 0.27) and DA-HB (0.6 and 0.85 %) diets, respectively. These findings are partially in agreement of the observations of earlier workers (Selim et al 1996; Gonzalez et al 2007) who observed no occurrence of Ophryoscolex species in cattle and sheep, however, (Mughetti et al 2007) found Ophryoscolex in range of 5.51 to 7.17 % in sheep rumen. Variability in protozoal counts in rumen of sheep supplemented with different multipurpose tree leaves has been reported (Odenyo et al 1997). These workers further reported that Entodiniomorphs are most dominant (93.3%) on the all the tree leaves supplemented diets. This substantiates the present results where Entodiniomorphs constitute 88 % of total protozoa numbers.  With respect to feeding hour protozoa numbers did not follow similar pattern in rumen of sheep on both diets.


Bacterial activity


Sheep rumen liquor had significantly (P<0.05) higher population of total viable bacteria on DA diet supplemented with LL (16.05) than HB (8.53 x 108/ml) supplemented diet Table 2).

Table 2.  Bacterial activity in goat rumen fed DA grass-tree leaves diets

Bacteria population



Total viable (x 108/ml)        0h



                                           4 h






Amylolytic (x 108/ml)         0h









Cellulolytic (x107/ml)        0h









Proteolytic (x107/ml)         0h









a,b means within a row bearing different superscripts differ significantly ( p<0.05)

Similarly the mean population of amylolytic, celluloltic and proteolytic bacteria were significantly (P<0.05) higher in rumen liquor of sheep on DA-LL (8.84, 21.46 and 7.70) than DA- HB based diet (5.04 x 108/ml, 9.18 x 107/ml and 4.45 x 107/ml), respectively which are consistent with observations of Prasad and Pradhan (1990) recorded in sheep on different straw-concentrate diets. On both diets sheep rumen liquor had higher bacterial numbers at 0 than 4 h post feeding. The lower bacteria population at 4 h post feeding may be dilution factor due to feed intake.  Effect of tree leaves supplementation on bacterial activity of ruminants has been reported earlier (Wang et al 2000; Singh et al 2007).


The higher bacterial activities in goats rumen on LL supplemented diet may be attributed to its higher CP and better degradability (Singh 2004) resulting in optimum metabolites concentration for conducive rumen environment for multiplication and proliferation of bacteria.  The higher bacterial activity in sheep rumen liquor on DA-LL diet may partly be attributed to higher mineral contents (S, P and Mg) and more protein in LL (Norton 1994).


Fungal population


Sheep fed DA grass supplemented with LL and HB had identical number of fungal sporangia counts (0.39 and 0.41 x105/ml Table 3) in its rumen liquor.

Table 3.  Fugal sporangia counts and fungus activity in sheep rumen fed DA grass-tree leaves

Fungus activity



Sporangia count (x105/ml)   0h



                                            4 h






Fungus as TFU (x 104/ml)   0h









a,b means within a row bearing different superscripts differ significantly (p<0.05)

TFU:  Thallus forming unit

However, fungus activity (TFU) was significantly (P<0.05) higher in rumen liquor of sheep fed LL (12.87) vis--vis HB (6.32 x104/ml) supplemented diet. Fungal activity as TFU was relatively more at 4 than 0 h feeding (before feeding).  This observation is supported by Orpin (1977) who reported an increase in zoospore numbers after feeding due to presence of component (s) of the diet that stimulate the release of zoospores from the sporangia. Alike protozoa and bacteria, rumen fungal population is also affected by physical and chemical makeup of diet. Higher the dietary fiber contents more the rumen fungal population (Grenet et al 1989). The reason for higher fungal activity on LL supplemented diet is not known. Higher activities of fungus on DA-LL diet in goats has been reported by Singh et al (2007). Higher mineral contents particularly of sulphur in LL (Norton 1994) might be possible cause of increased fungal activity as S is one of the most limiting factors for fungal growth.




Authors are thankful to Indian Council of Agricultural Research for funding this work under Young Scientist Scheme. Facilities provided by Director IGFRI are dully acknowledged.



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Received 25 September 2010; Accepted 22 October 2010; Published 5 January 2011

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