Effect of multipurpose tree (MPT) supplements on ruminal ciliate protozoa

ANIMAL FEED SCIENCE AND TECHNOLOGY

ELSEVIER

Animal Feed Science Technology 67 (1997) 169-180

Effect of multipurpose tree (MPT) supplements on ruminal ciliate protozoa A.A. Odenyo, P.O. Osuji *, 0. Karanfil International Livestock Research Institute (IIN),

P.O. Box 5689, Aaiiis Ababa, Ethiopia

Accepted 16 September 1996

Abstract The effect of five multipurpose trees (MPTs), Acacia angustissima, Acacia saligna, Chamaecytisus palmensis, Leucaena pallida and Sesbania sesban on ciliate protozoa was investigated in rumen cannulated Ethiopian sheep. Both entodiniomorphs and holotrichs were counted. The protozoa counts from S. sesban supplemented diet were significantly (P < 0.04) higher than from other diets. Maize stover alone and maize stover supplemented with C. palmensis or L. pallidu did not have any significant (P > 0.05) effect on the numbers of ciliate protozoa. A. saligna supplemented diet reduced the numbers of protozoa from 1.60 X lo5 to 0.62 X lo5 cells ml-’ rumen fluid. Differences in ciliate numbers among other MPT supplements barely failed to reach significance (P < 0.06). Entodiniomorphs dominated (93.3%) the protozoa population in all diets, with Entodinium species being the most predominant (80.7%). None of the MPTs tested eliminated protozoa. Effect of eating S. sesban and placing it in the rumen on protozoa was tested. Protozoa numbers decreased in the sheep in which S. sesban was placed in the rumen while they remained high in the sheep that were allowed to eat the supplement. Relationships between protozoa1 numbers and in sacco fibre degradation and neutral detergent tibre (NDF) digestibility were also examined. Degradation rate (c) increased with increase in protozoa numbers. NDF digestibility was significantly (P < 0.01) lower in A. suligna supplemented diet than in others. No significant relationship was observed between ruminal pH and protozoa1 numbers with all diets. The animals on A. angustissima died after 9 and 21 days of the experiment. 0 1997 Elsevier Science B.V. Keywords: Multipurpose trees; Acacia angustissima; Acacia saligna; pallida; Sesbania sesban; Ciliate protozoa: Rumen pH

* Corresponding author. Tel.: (251) 1 33 82 90/(251)

Chamaecytisus palmensis; Leucaena

1 33 95 66; Fax: (251)

[email protected]. 0377-8401/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO377-8401(96)01118-2

1 33 87 55; E-mail:

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1. Introduction

Ciliate protozoa are associated with many functions in the rumen (Jouany et al., 1988). Presence or absence of ciliate protozoa in the rumen affects ruminal factors such as bacterial numbers and type, ammonia concentration, pH and rumen volume and dilution rate. These factors affect rate and extent of digestion in the rumen. Understanding the effect of any feedstuff on rumen microorganisms, such as protozoa, is useful in designing feeding strategies for farmers. Multipurpose trees (MPTs) have been identified as potential suitable leguminous supplements to basal roughages (Jones, 1979; Siaw et al., 1993; Richards et al., 1994). However, MPTs contain secondary plant compounds that may affect rumen microbes (Reed, 1986; D’Mello, 1992; Kumar, 1992). Preliminary studies at ILRI and the Rowett Research Institute @RI) with MPTs indicated that some MPTs, e.g. S. sesbun, may have defaunating activity (Newbold et al., 1994; El Hassan et al., 1995). If the effects of these substances are targeted specifically to rumen protozoa, a combination of high protein content and the defaunating activity of MPTs would greatly promote the growth of rumen bacteria under certain feeding regimes. Increased bacterial numbers in the rumen would result in greater microbial protein flow into the small intestine thus providing the host animal with more protein. Concomitantly, however, a decrease in rumen fibre digestion might be observed. In this study, changes in the population of ruminal ciliate protozoa during and after adaptation to roughage diets supplemented with various MPTs were monitored. Our hypothesis was that MPTs, when used as protein supplements, may alter the population of ruminal ciliate protozoa and consequently fermentation in the rumen.

2. Materials and methods 2.1. Chemical anulysis of feeds Dry matter (DM), organic matter (OM) and nitrogen were analysed following the procedures of the Association of Official Analytical Chemists (Hehich, 1990). Neutral detergent fibre (NDF) was analysed according to Van Soest and Robertson (1985). 2.2. Animals and diets Eighteen rumen cannulated Ethiopian sheep (average weight 29.7 SD f 2.18 kg) were randomly allotted to one of six treatments, i.e. three animals per treatment. The animals were previously fed grass hay ad libitum and wheat bran. Maize stover was the basal diet and was supplemented with: Acacia angustissima, Acacia saligna, Chamaecytisus palmensis, Leucaena pallida and Sesbania sesban. Each animal was offered 1000 g day-’ total feed. The supplements were offered at the level of 30% as fed (300 g) of total feed. Water and mineral licks were available ad libitum. The chemical composition of the diets is presented in Table 1.

A.A. Odenyo et al./Animal Table 1 Chemical

composition

of the feedstuffs

Feedstuff

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171

used in the experiment

Chemical component DM”

N

OM (g kg-’ DM)

E;-

I DM)

(g kg-

Acacia angustissima 15 132 b Acacia saligna

873.9 902.6

912.6 893.4

314.9 457.5

33.2 22.5

Chamaecytisus palmensis Leucaena pallida 14189 b Sesbania sesban 10865 b Maize stover

865.6 881.6 916.8 940.2

938.9 886.3 891.3 889.5

335.5 273.0 244.4 671.6

33.4 48.4 41.4 06.3

’DM)

a DM, dry matter (on as-fed basis). b ILCA accession numbers.

2.3. Protozoa counts

Rumen fluid was collected before the morning feed. The samples were taken after 0, 7, 14, 21, 28, 35, 42, 49, 56 and 63 days of supplementation. Rumen fluid was strained through cheese cloth and 10 ml of rumen fluid was transferred into a sample bottle containing 40 ml of formal-saline solution (8.1 g of NaCl: 100 ml formalin (37% formaldehyde); 900 ml distilled water) to give a 1:5 dilution. Further dilutions (1:20) were made as needed to give 30 to 50 cells per field. Prior to examination, 10 ml of the diluted samples were pipetted into a small test tube and two drops of iodine (10 g iodine; 100 ml 95% ethanol) were added after which the samples were mixed. Four drops of the stained samples were put in the groove of the counting chamber. Both entodiniomorphs and holotrichs were counted using an objective lens with a 40 X magnification. Four fields were counted and the counts were repeated twice for accuracy. Using the chamber specifications (volume = 1000 mm3, depth = 0.2 mm, area = 16 mm’>, calculations were made as follows: Volume No. of protozoam- ’ rumen fluid =

Depth X Area

X Dilution X Average count

2.4, ESfect of eating S. sesban or placing it in the rumen on protozoa Four animals were supplemented with 300 g of S. sesban. Two animals were allowed to eat the supplement, while the remaining two were supplemented by placing the supplement in the rumen through the cannulae. The animals were fed S. sesban for 14 days after which the samples were taken three times per day and pooled on a daily basis. The samples were taken for a period of 1 week. The protozoa were counted. 2.5. Nylon bag degradability Nylon bag incubations (Orskov and McDonald, 1979) were used to examine the relationship between protozoa1 numbers and in sacco degradation of fibre. Native grass

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hay (Sululta hay) was ground through a 2-mm sieve. Approximately 2.5 g of sample was placed in nylon bags (6 cm X 12 cm) with a pore size of 41 pm. The bags were individually labelled, tied with nylon string and then were further tied to a plastic weight to keep the bags in place in the rumen. The bags were then incubated for 3, 6, 12, 24, 48, 72, 96 and 120 h in the rumen of animals given the six dietary treatments. After incubation, all bags were rinsed under tap water then washed in a five-cycle washing machine (Tefal Altematic, Finland) for 6 min per cycle. The bags were then dried in an oven at 60°C for 48 h, and dry matter (DM) disappearance calculated. All samples were incubated in triplicate. The disappearance of DM was described by the exponential model (Orskov and McDonald, 1979). 2.4. Digestibility

and N balance procedures

After 63 days on MPT supplements, the animals were moved to metabolism crates for a 2-day adaptation period. Dietary treatments remained the same. Total feed intake, faeces and urine outputs were recorded. About 10% (fresh weight) of total faeces collected was frozen. At the end of the trial, all frozen samples for each animal were thawed and mixed. About 1 kg was weighed, dried at 60°C for 48 h then analysed for DM, organic matter (OM), nitrogen (N) and NDF for digestibility estimates. Urine (pH below 3) was collected into containers with 100 ml (10%) hydrochloric acid. A 100~ml aliquot from each animal was stored at 5°C and used for N analysis. 2.7. Ruminal pH Protozoa are sensitive to pH changes towards acidity and cannot survive outside the range between 5.5-8.0 (Hungate, 1966). Effect of MPTs on ruminal pH and the relationship between pH and the numbers of ciliate protozoa was therefore investigated. The pH of the rumen fluid from all animals was measured immediately following collection with a digital pH meter (Kent, model 7045/46, Stonehouse, Gloucestershire, UK). 2.8. Statistical analysis The data were subjected to an analysis of variance procedure for a completely randomized design using the general linear model (GLM) available in SAS @AS, 1987). The protozoal numbers counted at each time point and the pH values were investigated using a repeated measures analysis of variance @AS, 1987). The analysis used the split plot approach with the Greenhouse-Geiser approximate (conservative) significance tests (Littell et al., 1992).

3. Results 3.1. Protozoa1 counts The results of protozoal counts are presented in Table 2. Rumen fluid from animals supplemented with S. sesban had significantly (P < 0.04) higher numbers of protozoa while sheep supplemented with A. saligna had the lowest protozoal counts. The

173

A.A. Odenyo et ai./Animal Feed Science Technology 67 (1997) 169-180 Table 2 Mean protozoa1 numbers in the rumen of sheep fed maize stover supplemented

with various multipurpose

trees

(MPTs) Supplements and protozoa species A. angustissima Entodiniomorphs Holotrichs A. saligna Entodiniomorphs Holotrichs C. palmensis Entodiniomorphs Holotrichs L. pallida Entodiniomorphs Holotrichs S. sesban Entodiniomorphs Holotrichs Maize stover Entodiniomorphs Holotrichs Statistical significance

Protozoa1 numbers in rumen fluid (X lo5 ml14

7

1.14 0.04

0.66 0.05

0.65 0.03

0.89 0.08

1.52 0.08

0.72 0.04

0.53 0.05

0.62 0.05

0.88 0.04

0.71 0.03

0.78 0.06

0.66 0.03

0.57 0.04

0.58 0.03

0.93 0.07

2.03 0.16

1.37 0.15

0.92 0.09

1.22 0.08

1.37 0.09

1.42 0.13

1.52 0.21

1.20 0.06

1.28 0.10

1.14 0.07

1.23 0.09

1.54 0.11

1.27 0.15

1.52 0.13

1.13 0.11

1.05 0.12

1.28 0.19

0.96 0.10

1.30 0.13

1.26 0.07

1.58 0.12

2.57 0.22

2.58 0.22

2.82 0.36

2.22 0.19

2.42 0.22

2.45 0.36

2.33 0.12

2.65 0.18

1.43 0.02

1.35 0.05

1.39 0.06

1.10 0.03

1.47 0.06

0.81 0.04

0.79 0.03

1.88 0.04

1.03 0.05

1.08 0.04

NS

NS

NS

2l

‘) at sampling day

o

**

28

35

42

49

56

63

NA NA

NA NA

NA NA

NA NA

NA NA

NA NA

*1/

**

**

t *

NS

**

’

A Statistical significance test was based on total protozoa (entodiniomorphs NS, not significant; * P < 0.05; * ’ P < 0.01; * * * P < 0.001. NA, not available since animals died after 9 and 21 days.

+ holotrichs)

for each MF’T

population of protozoa remained almost the same in sheep supplemented with C. palmensis, L. pallida and those on maize stover alone. The population of entodiniomorphs was higher (P < 0.04) in sheep supplemented with S. sesban than in sheep fed any other supplement. In sheep supplemented with A. saligna, the population of protozoa decreased from 1.60 X lo5 ml-’ (Day 0) to 0.62 X lo5 ml-’ of rumen fluid by Day 21 then only slightly increased but still remained below initial numbers. Entodiniomorphs were most abundant (93.3%) and species of Enrodinium were the most predominant (80.7%) in all diets. The protozoa numbers in this study could have been slightly underestimated due to filtration of feed particles before enumeration. Some protozoa might have been sequestered on feed particles and therefore filtered out. Sheep allowed to eat S. sesban were compared with those in which the supplement was put in the rumen. The numbers of ciliate protozoa remained higher in sheep that were allowed to eat the supplement and decreased in those sheep which were fed through the cannulae (Fig. 1). 3.2. Nylon bag degradability Table 3 shows the results of the nylon bag degradability study. Dry matter (DM) disappearance at 48 h was significantly different (P < 0.01) among MPTs. The extent of

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0

1

Feed Science Technology 67 (1997) 169-180

2

3

4

6

5

I

samplingPeriod (Days) Fig. 1. Effect of eating S. sesban 10865 (A ) or placing it into the rumen (0) on the ruminal ciliate protozoal numbers in Ethiopian highland sheep.

degradation, intercept (a), potentially degradable component (b), potential degradability (PD), rate of degradation (c) and lag time (TL (h)) were affected by the type of supplement. The rate of degradation (C (h- ’>>was highest in sheep supplemented with S. sesban and lowest in sheep supplemented with A. saligm. 3.3. Digestibility and N balance The digestibility and N balance results are presented in Table 4. Dry matter digestibility was significantly different (P < 0.01) among supplements. Digestibility of

Table 3 Nylon bag dry matter disappearance (mg g-’ ) of Sululta hay incubated in the rumen of Ethiopian highland sheep fed maize stover supplemented with various multipurpose tree (MIT) leaves MPl supplement

Incubation time (h) 12

24

Acacia angustissima 15132 Acacia saligna Chamaecytisus palmensis L.eucaenapallida 14189 Sesbania sesban 10865

199.4 162.9 204.3 183.6 210.1

254.6 184.3 243.3 278.5 314.2

Maize stover

164.1 218.5

SE(n=9) Statistical significance

459.9 327.8 458.4 504.2 502.7 461.6

12.0 32.2 29.8 *** * ’ NS

SE, standard error of the mean. NS, not significant; P > 0.05; ??

a

48

?? ??

P < 0.01;

??

72

96

469.5 443.4 548.8 567.0 592.2 524.5

561.5 107.9 502.1 113.7 597.3 104.0 595.9 92.9 608.1 96.4 571.9 88.5

29.8 35.8 * NS

* * P < 0.001.

4.99

*

b

PD

c(h-‘)

n.(h)

561.6 1163.5 651.1 614.6 595.0 597.4

669.6 1277.3 755.1 707.5 691.4 685.9

0.0165 0.0041 0.0162 0.0191 0.0218 0.0165

- 1.688 -4.271 - 1.120 0.073 - 0.237 0.4306

0.002

0.795

61.42 ??

*

??

.

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Table 4 Mean dry matter (DM), organic matter (OM), nitrogen (N), neutral digestibility (D) and nitrogen balance (NJ3) (g day-‘) for Ethiopian supplemented with various MPTs Supplement

SE(n=9) Statistical

OM

N

NDF

365.47 548.80 509.85 562.79 514.49

381.91 566.77 541.56 601.79 544.05

108.73 511.64 322.64 741.11 113.27

220.16 495.38 410.92 521.64 538.84

-0.04 2.11 2.11 8.54 - 0.42

32.73 **

32.62 NS

30.13 NS

41.86 NS

0.587 ***

DM

Acacia saligna Chumaecyfisus palmensis Leucaena pallida Sesbania sesban Maize stover

significance

SE, standard error of the mean. NS, not significant: * P < 0.05;

detergent fibre (NDF) (g kg-l ) highland sheep fed maize stover NB(gday-‘)

* * P < 0.01: * * * P < 0.001.

supplemented diets was much lower (P < 0.02) than the unsupplemented diets. There were no significant differences (P > 0.05) in NDF digestibility among diets. Nitrogen balance was significantly different (P < 0.01) among diets. Sheep supplemented with S. sesbun had the highest positive (8.53 g day-‘) N balance, while those supplemented with A. saligna and those fed maize stover alone both had negative N balances (-0.03 and -0.42 g day-‘, respectively).

A. sdigna

3.4. Ruminal pH Ruminal pH was not significantly (P > 0.05) affected by supplementation with MPTs. The pH values in all diets, with the exception of the A. s&gnu supplemented diet, ranged between 6.6 and 7.0. This was within the optimum pH for fibre digestion. There was no significant relationship between the ruminal pH and the numbers of protozoa.

4. Discussion There is increasing interest in rumen protozoa because of the concern that increased numbers in the rumen may reduce the bacterial biomass leading to a negative effect on the efficiency of feed utilization by ruminants under certain feeding regimes. Feedstuffs that decrease or keep the concentration of rumen protozoa at a low level should, therefore, increase the efficiency of feed utilization. Our results showed that none of the MPTs fed as supplement had defaunating activity. The S. sesban supplemented diet maintained higher numbers of ruminal ciliate protozoa (1.3 X 105-2.8 X 10’ ml-’ rumen fluid) (Table 2). This is contrary to results of El Hassan et al. (1995) and Newbold et al. (1994), which suggested that S. sesban defaunates sheep. The results in this study, however, agree with those of Leng et al. (19911, that S. sesban has no anti-protozoa activities.

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However, the results from this study compared with those of El Hassan et al. (1995) and Newbold et al. (1994) suggest that S. sesban may have substance(s) toxic to protozoa found in Scottish sheep but not Ethiopian sheep. Ethiopian sheep graze in the wild and therefore may be expected to be more exposed to different browse plants thus evolving protozoa strains resistant to many toxic chemicals inherent in these plants. It is also possible that the Ethiopian sheep harbour protozoa populations different from those found in Scottish sheep. This is yet to be investigated. The experiment on the effect of eating or placing S. sesban into the rumen showed that, in the sheep allowed to eat S. sesban, the protozoa numbers remained high while in those animals where the supplement was placed in the rumen via a cannula, the numbers decreased substantially. However, defaunation did not occur. These results suggested that physiological activities associated with eating, including saliva secretion in the mouth, may play a role in making poisonous substance(s) found in S. sesban less toxic to ciliate protozoa. Acacia saligna supplemented diets reduced the population of ciliate protozoa. Whether this reduction was due to toxins in A. saligna or to insufficient nutrients is not yet clear. A. saligna has generated a lot of interest as a potential feed supplement because of its attributes, i.e. good adaptation to semi-arid climate, use as a quick method for reafforestation and very high biomass production (Gutteridge, 1994). However, A. saligna contains high soluble phenolics and low nitrogen (Table 1; Woodward and Reed, 1989). The nitrogen balance results (Table 4) showed that feeding A. saligna resulted in a negative N balance. These nutritional limitations, and not toxicity directed specifically to the protozoa, might have accounted for the low protozoa numbers. If the general nutritional deficiency argument is correct, then both bacterial and fungal populations might also be affected. Previous ILRI studies (ILCA, 1986) showed that sheep supplemented with A. saligna lost weight. Our results showed very low (5.4 g day-‘) weight gain. These results may suggest that A. saligna, even with its positive adaptive features and high biomass, has limitations as a protein supplement for ruminants. Protozoa1 numbers were also reduced in sheep supplemented with A. angustissima. However, the sheep fed this supplement became sick and died after 9 and 21 days of the experiment suggesting the presence of very potent toxin(s) in this plant. It is not clear whether the toxicity of A. angustissima is a plant to microbe, or a plant to animal interaction in the rumen or both. Detailed work on A. angustissima toxicity is being carried out in our laboratory. Degradation parameters (a, b, c and a + b) were estimated (Orskov and McDonald, 1979) and these were related to protozoa1 populations using a step wise regression procedure available in SAS (1987). R2 values were generally highest when c alone came into the model. R2 improved markedly when c and b were in the model and only marginally when c, b and a formed the model. These would suggest that c and b degradation parameters had the greatest effect on the protozoa population. The higher the rate of degradation and the potentially degradable fraction the higher the protozoa population. The converse, i.e. the effect of protozoa1 numbers on parameter c, was further investigated using the regression procedure (SAS, 1987). The result showed that the regression of c on the protozoa population in the middle of the incubation period

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was significant (P < 0.010, R* = 0.417). The rate of degradation, c, increased as the protozoa population (x) increased. The regression equation was c = 0.0080 (SE 0.003) + 0.0055 (SE 0.002)X. The intercept and slope were significant (P < 0.03 for the intercept and 0.02 for the slope). The result, therefore, would suggest that reduction in protozoa numbers does not result in increased rate of fibre degradation. This conclusion does not agree with the Leng (1982) suggestion that defaunation enhances libre degradation. The degradation data also suggested that supplementation with S. sesban enhanced degradation. A number of factors may have contributed to better fibre degradation in sheep supplemented with S. sesban. Sesbania sesbun has more nitrogen and soluble carbohydrates (Table 1) and a higher concentration of macrominerals (Bonsi et al., 1995) as compared with other MPT supplements used. These nutrients are important for the growth of rumen microorganisms including the protozoa. Higher numbers of protozoa also may have contributed to improved fibre digestion. Most of the ciliates in S. sesbun supplemented diet were entodiniomorphs. Large entodiniomorphs have been shown to have high a-L-arabinofuranosidase and PD-xylosidase activities (Williams et al., 1984) which are important in fibre degradation. Furthermore, Entodinium spp. were the most predominant; these small entodinia have been shown to improve in vitro cellulose digestion (Ushida et al., 1987). Also, protozoa provided a stable environment for fibre degradation by supplying sufficient ammonia required for the growth of fibrolytic bacteria. The relationship between protozoa1 numbers and degradation of cellulosic materials has been examined by several workers. The conclusions have, however, been equivocal. Defaunation has been said to enhance fibre digestion (Leng, 1982). High cellulolytic activity has been reported in the rumen of defaunated sheep fed a low quality straw based diet (Soetanto et al., 1985; Romulo et al., 1986). Such high cellulolytic activity was attributed to an increased fungal population consequent upon defaunation. This suggested that defaunation could enhance tibre digestion through an increased fungal population. However, in the present trial fibre degradation was not improved in the rumen of sheep supplemented with A. suligna which had the lowest numbers of protozoa. On the other hand, many studies have emphasized the contribution of protozoa in fibre digestion. Several reports (Ushida and Jouany, 1990; De Smet et al., 1992) indicated that fibre digestion is depressed when animals are defaunated. Defaunation reduced the apparent digestibility of ADF of ammonia treated straw in the rumen from 0.60 to 0.58 and in the total tract from 0.66 to 0.62 (Ushida and Jouany, 1990). Ushida et al. (1991) showed that cellulolytic bacteria are not affected by the presence of protozoa in a cellulosic diet. Additionally, Kurihara et al. (1978) suggested that protozoa may actually stimulate the growth of cellulolytic bacteria. Cellulolytic activity of bacteria in the rumen increased after inoculation with ciliate protozoa on a cellulosic diet (Jouany and Senaud, 1979) but the cellulolytic flora was only reduced by protozoa when the diet contained 30-50% starch (Jouany, 1989). Bird et al. (1990) concluded that very low protozoa density in the rumen is as detrimental as high density. In most developing countries, ruminants are fed mainly poor quality roughages, native pastures and crop residues. These feeds are very high in fibre and low in protein. If defaunation enhances fibre digestion, producers in these countries could benefit from

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defaunation. However, only very few in vivo studies on the effect of defaunation on rumen fibre digestion have been reported. It is thus clear that the role of protozoa in ruminant digestion is still under debate (Veira, 1986; Hobson and Jouany, 1988). It appears that defaunation benefits animals on high energy and low protein diets (Bird and Leng, 1978, 1985).

5. Conclusions This study has demonstrated that Acacia angustissima, Acacia saligna, ChumaeL.eucaena pallida and Sesbania sesban have no defaunating activity in Ethiopian sheep. It also showed that A. angustissima was toxic to sheep. It was also evident that an increase in protozoa numbers enhanced fibre degradation and that defaunation may not be beneficial under certain regimes. cytisus palmensis,

Acknowledgements The authors are grateful to ILRI staff at the Debre Zeit Research Station and the biometricians for their technical assistance.

References Bird, S.H. and Leng, R.A., 1978. The effects of defaunation of the rumen on the growth of cattle on low-protein high energy diets. Br. J. Nutr., 40: 163-167. Bird, S.H. and Leng, R.A., 1985. Productivity responses to eliminating protozoa from the rumen of sheep. Rev. Rural Sci., 6: 109-l 17. Bird, S.H., Nolan, J.V. and Leng, R.A., 1990. Nutritional significance of rumen protozoa. Paper presented at VIIth International Symposium on Ruminant Physiology (1989), Satellite Symposium on Regulation of Microbial Metabolism in the Rumen Ecosystem. Hakone, Japan. Bonsi, M.L.K., Osuji, P.O. and Tuah, A.K., 1995. Effect of supplementing teff straw with different levels of leucaena or sesbania leaves on degradabilities of teff straw, sesbania, leucaena, tagasaste and vemonia and on certain rumen and blood metabolites in Ethiopian Menz Sheep. Anim. Feed. Sci. Technol., 52: 101-129. De &net, S., Demeyer, D.I. and van Nevel, C.J., 1992. Effect of defaunation and hay: concentrate ration on fermentation, tibre digestion and passage in the rumen of sheep. Anim. Feed Sci. Technol., 37: 333-344. D’Mello, J.F.P., 1992. Chemical constraints to the use of tropical legumes in animal nutrition. Anim. Feed Sci. Technol., 38: 237-261. El Hassan, SM., Lahlou-Kassi, A., Newbold, C.J. and Wallace, R.J., 1995. Antimicrobial factors in African multipurpose trees. In: R.J. Wallace and A. Lahlou-Kassi (Editors), Rumen Ecology Research Planning, Proc. Workshop. ILRJ, Addis Ababa, Ethiopia, pp. 43-54. Gutteridge, R.C., 1994. Other species of multipurpose forage tree legumes. In: R.C. Gutteridge and H.M. Shelton (Editors), Forage Tme Legume in Tropical Agriculture. CAB International. Helrich, K. (Editor), 1990. Official Methods of Analysis of the Association of Official Analytical Chemists. 82 PP.

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