Evaluation of Enterolobium ciclocarpum as dietary alternative to eliminate protozoa from the rumen

Evaluation of Enterolobium ciclocarpum as dietary alternative to eliminate protozoa from the rumen

Alberto Navas, Max Alberto Laredo, Aurora Cuesta, Héctor Anzola and Juan Carlos Leon

Elimination of ciliate protozoa from the rumen (defaunation) improves the protein to energy ratio in the nutrients absorbed by increasing the amount of bacterial and sometimes dietary amino acids available for absorption at the small intestine. The increased ratio of amino acids to volatile fatty acids in the nutrients absorbed leads to the better performance observed in defaunated animals.

There are however no commercial alternatives to obtain defaunated ruminants at the farm level. The present paper discusses the effect of supplementing sheep with different levels of tree legumes (Enterolobium ciclocarpum) on the concentration of ciliate protozoa in the rumen fluid.

The inclusion of E. ciclocarpum as supplement or basal diet in the diet of sheep, does not eliminate protozoa from the rumen, but causes a significant reduction of total protozoa numbers and a variation in the species of protozoa present in the rumen. Holotrichs are the most susceptible species to E. ciclocarpum.

Supplementation with E. ciclocarpum may be an alternative to increase the protein to energy ratio in the nutrients absorbed, by increasing the flow of dietary amino acids to the duodenum. At high levels of inclusion in the diet, it may also increase the flow of bacterial-N to the small intestine due to the lower number of protozoa in the rumen. However, the reduction in dry matter intake may affect animal performance when the proportion of E. ciclocarpum in the diet is high.

Elimination of ciliate protozoa from the rumen (defaunation) is an alternative to improve productivity of ruminants fed on fibre or fibre and soluble sugars (Bird 1991).

The presence or absence of protozoa in the rumen affects the amount and ratio of amino acids (of dietary and microbial origin) and volatile fatty acids (amount and proportion) in the nutrients absorbed. The rate and degree of fibre degradation in the rumen is altered by defaunation, but the direction and extent of its effect depends on the nature of the substrate (animal diet) for microbial growth (Kreuzer and Kirchgessner 1988), rumen volume and dilution rate (Demeyer 1988), and concentration of sulphur (Hegarty et al 1988) and ammonia (Perdok and Leng 1988) in the rumen fluid.

Independent of the diet offered, a consistent effect of elimination of protozoa is a larger availability of amino acids for absorption at the small intestine - amino acids of bacterial (Ushida et al 1988) and sometimes dietary origin (Michalowsky 1988). The increased ratio of amino acids to volatile fatty acids in the nutrients absorbed leads to the better performance observed in defaunated animals (Leng 1990).

There are however no commercial alternatives to defaunate cattle or sheep at the farm level. In this sense, tropical plants with high or medium content of saponin may be an alternative to be included at high levels for short periods of time in the diet of ruminants to eliminate protozoa from the rumen (Lu and Jorgensen 1987).

The present paper discusses the effect of supplementing sheep with different levels of Enterolobium ciclocarpum on the concentration of ciliate protozoa in the rumen fluid.

The experiments were carried out at ICA's (Instituto Colombiano Agropecuario) research centre TIBAITATA, which is located in Mosquera (Cundinamarca), at 2600 masl, 630 mm annual rainfall, 72% relative humidity and 13 °C air temperature.

Cross-bred sheep (30 kg live weight in average) were used for the experiments. Animals were fixed with permanent rumen cannula, 4 months before the commencement of the experiments.

EXPERIMENT 1: A complete randomised design with 4 animals per group was utilised. Sheep received hay of Pennisetum clandestinum and one of three levels of dry leaves of Enterolobium ciclocarpum (Control group: 0 g/d; Treatment 1: 100 g/d; Treatment 2: 300 g/d). E. ciclocarpum was offered at the morning meal before the P. clandestinum.

Urea was offered to get a minimum of 200 mg N-NH₃ /L of rumen fluid (3 g/d to control group and 8 g/d for T1 and T2). Fresh and clean water was offered ad libitum and 20g/d of a mineral mix was supplemented.

EXPERIMENT 2: A complete randomised design with 2 animals per group was utilised. The control group received 800g/d of hay of Pennisetum clandestinum; the experimental animals received 800 g/d of dry leaves of Enterolobium ciclocarpum for a 8-day period. After this period animals were placed to graze on P. clandestinum.

All animals were subjected the day 1 of the experiment to a 24h fasting period. Clean and fresh water was available ad libitum during the whole experiment. Mineral mix (20 g/d) was offered to all animals.

PROTOZOA NUMBERS IN THE RUMEN FLUID: Rumen fluid samples were taken via cannula, by using a 50 ml syringe connected to a plastic tube (50 cm-long) with a plastic cone with 2 mm-diameter holes in its distal extreme. Samples of rumen fluid (50 ml) were taken just before feeding and 4h post-feeding, and 4 ml diluted in 16 ml of a 1:9 formol saline solution (1 part of formol 37% and 9 parts of saline 0.9% solution). The remaining sample (46 ml) was used for pH, and ammonia concentration determination. Protozoa numbers was counted in a Neubauer chamber under 100x.

RUMEN FLUID pH: Rumen fluid pH was estimated immediately after sample was taken by using an Orion Research Digital potentiometer.

RUMEN N-NH₃ CONCENTRATION: Ammonia-N concentration was estimated by using the micro-Kjeldalh procedure.

IN SACCO DIGESTIBILITY: Dry matter (DM) and protein degradability were estimated in sacco following the procedure described by Orskov et al (1980).

E. ciclocarpum is highly accepted by sheep. There was no rejection of leaves during the experimental period (10d) of the second experiment, in which, leaves were offered as the only feed. During the first experiment, leaves of E. ciclocarpum (100 and 300 g/d) were eaten in approximately 15 minutes during 4 months when offered in the morning meal.

Low levels of inclusion of E. ciclocarpum (10% DM) increased dry matter intake (DMI) by 13%. However, sheep fed higher levels in the diet showed similar DMI to those receiving no supplementation (P<0.01; Table 1), indicating a lower intake of P. clandestinum by sheep fed the legume (858 vs 594 g/d in the Control and T2 groups respectively).

Table 1. Dry matter intake of Pennisetum clandestinum (P.c.), total diet (P. clandestinum + Enterolobium ciclocarpum, E.c.) and digestible dry matter in sheep fed P. clandestinum and three level of E. ciclocarpum.

	P.c.	P.c. + E.c.	Digestible D.M.
DIET	(g DM/d)	(g DM/d)	(g DM/d)

P. clandestinum	858	858	471
P.c.+ E.c. (100g/d)	874	966	552
P.c.+ E.c. (300g/d)	594	869	427
P	0.01	0.01
EEM	0.012	0.012

The effect of supplementation on DM intake was apparently accounted for by its effect on DM rumen degradability. Low levels of inclusion tended to increase dry matter digestibility of P. clandestinum (10% higher), while higher levels of E. ciclocarpum in the diet decreased it by 6% (P=0.13; see Table 2). Similar results were reported by Goetsch and Owens (1985) who found that 608 mg saponin (sarsaponin) increased organic matter digestibility in the rumen of cattle by 12% (from 32% in the control group to 44% in the cattle receiving saponin).

EFFECT OF FASTING ON PROTOZOA NUMBERS: Fasting (24h) reduced protozoa numbers in the rumen fluid by 60% (see Table 3). It is likely that protozoa without substrate has remained attached to rumen epithelium (Abe et al 1981) and therefore protozoa free in the rumen fluid had been found decreased. However, after feeding, protozoa numbers in the rumen fluid did not increase (see Table 4), suggesting there was in fact a reduction of protozoal biomass as a result of fasting.

Table 2. Dry matter in sacco degradability of P. clandestinum in sheep receiving P. clandestinum and three levels of leaves of E. ciclocarpum. Values are the coefficients of the equation: y= a+b(1-e ^ct); (Orskov et al 1980)

TREATMENT	n	a	b	c	a+b

P.cl.	20	18.6	48.2	0.06	66.78
P.cl. + E.c.(100g/d)	20	22.6	52.1	0.05	73.67
P.cl. + E.c.(300g/d)	20	17.6	44.9	0.07	62.54
EEM		2.10	2.06	0.005	1.65
P		0.66	0.53	0.30	0.13

Table 3. Effect of 24h fasting on protozoa numbers in rumen fluid of sheep fed on Pennisetum clandestinum

PROTOZOA NUMBERS		PROTOZOA NUMBERS-After
(Ax10⁵)	n	24h fasting (Ax10⁵)	P

4.14	8	1.65	0.05

EFFECT OF DIETARY LEVEL OF E. ciclocarpum ON PROTOZOA NUMBERS: Low levels (10% DM) of inclusion of dry leaves of E. ciclocarpum in the diet of sheep increased protozoa numbers in the rumen by 29% (see Table 5). However, sheep receiving higher proportions of E. ciclocarpum in the diet (ca. 34%) showed a large reduction of protozoa in the rumen fluid (43% as compare to protozoa numbers in the rumen fluid of sheep receiving no E. ciclocarpum).

Lu and Jorgensen (1987) reported a reduction of 34 and 47% of protozoa numbers in sheep infused intraruminally with 2 and 4% DM alfalfa saponin respectively. In the present experiment sheep receiving 1% saponin showed conversely a higher concentration of protozoa in the rumen. However, saponin at 4% DM had a strong detrimental effect on rumen protozoa as reported by Lu and Jorgensen (1987).

Table 4. Entodiniomorph, Holotrich and Total protozoa numbers in the rumen fluid of sheep receiving once daily Pennisetum clandestinum or Enterolobium ciclocarpum. All animals were placed to graze on Pennnisetum clandestinum since day 8 of the experiment.Samples were taken just before feeding.

	Entodiniomorph		Holotrich		TOTAL
	(A x 10⁵)		(A x 10⁵)		(A x 10⁵)
Experimenta
day	E.c.	P.c.	E.c.	P.c.	E.c.	P.c.

1	3.1	3.2	0.07	0.14	3.5	3.6
2	1.0	1.5	0.03	0.06	1.5	1.9
3	0.06	1.2	0.0	0.02	0.09	1.3
4	0.008	1.3	0.0	0.03	0.08	1.5
5	0.002	0.7	0.0	0.06	0.02	0.81
7	0.002	0.9	0.0	0.04	0.003	1.02
8	0.003	0.9	0.0	0.03	0.003	0.98
16	0.5	0.5	0.0	0.02	0.45	0.63
23	1.2	0.5	0.0	0.08	1.2	0.63
25	1.5	0.8	0.003	0.04	1.5	0.85

Table 5. Protozoa numbers in the rumen fluid of sheep fed once daily on Pennisetum clandestinum and three levels of Enterolobium ciclocarpum (E.c.)

	BEFORE FEEDING	4h AFTER FEEDING
DIET	(A x 10⁵)	(A x 10⁵)

P. clandestinum	1.17	1.21
P.c.+ E.c. (100g/d)	1.50	1.35
P.c.+ E.c. (300g/d)	0.67	0.48
EEM	0.036	0.033
P	0.01	0.01

The effect of leaves of E. ciclocarpum on protozoa numbers may be associated with a combined effect of saponin and the low soluble protein content of E. ciclocarpum. Protozoa numbers in vitro were reported to be increased as the solubility of the protein in the substrate decreased (Michalowski 1988). Similarly, Navas (1991) reported that supplementation to sheep with cotton seed meal (50% by-pass protein) increased protozoa numbers in the rumen fluid.

Leaves protein of E. ciclocarpum have a low rumen in sacco degradability (lower than 50% after 48h of rumen incubation; see Table 6). Therefore, supplementation with a low-soluble protein, associated with a low level of saponin, may explain the increase in protozoa numbers in the rumen of sheep receiving 100 g daily E. ciclocarpum. On the other hand, the detrimental effect on protozoa numbers of a higher level of E. ciclocarpum. in the diet, may be related to the higher (3 times higher) dietary saponin concentration in sheep receiving 34% DM E. ciclocarpum.

Sheep receiving leaves of E. ciclocarpum as their only diet for 8 days (second experiment), showed, after 3-5 days of being in treatment, a much more markedly reduction in protozoa numbers (from 10⁵to 10³) than those receiving 34% DM of it (Table 4). However it was not possible to eliminate protozoa from the rumen.

Table 6: Protein in sacco degradability of leaves of Enterolobium ciclocarpum (E.c.) in sheep fed on Pennisetum clandestinum (P.c.) and three levels of E. ciclocarpum. Values are the coefficient of the following equation as described by Orskov (1980): Yt = a + b(1-e-ct)

DIET	a	b	c	a + b

P.c. + E.c. (100g/d)	22.58	30.07	0.048	52.65
P.c. + E.c. (300g/d)	19.58	53.47	0.053	40.42

Lu and Jorgensen (1987) suggested that Entodinium may be the most susceptible species of rumen protozoa to saponin. In the present experiment holotrichas were nevertheless the most susceptible group of protozoa, meanwhile Entodinium (<50u) tended to increase in proportion (see Table 7). Moreover, it appears that holotricha were eliminated from the rumen in sheep receiving E. ciclocarpum as the only diet (Table 4). Holotricha were observed again only after 15 days grazing on Pennisetum clandestinum which could have been because of re-contamination from the other animals these group was place to graze with after the treatment.

Entodiniomorphid showed a faster trend to increase in numbers since sheep were placed to graze again (see Table 4).

The different results found in the present experiment to those reported by Lu and Jorgensen (1987), may be related to the different biological activity of different kind of saponin present in alfalfa and E. ciclocarpum (Bondi et al 1973).

Rumen pH was not apparently associated with the effect of diet on protozoa numbers and variation in the proportions of different species of protozoa in the rumen (see Table 8).

Table 7: Proportion (%) of entodiniomorph anf holotrich protozoa in the total protozoa number in the rumen fluid of sheep fed once daily on Pennisetum clandestinum and three levels of Enterolobium ciclocarpum (E.c.). Values of samples taken before feeding and 4h after feeding.

	Sampling	Entod.	Entod.	Entod.	Holotr.
DIET	time	(<50u)	(50-100u)	(>100u)

P. clandestinum	Before	73.0	6.6	6.2	14.4
"	After	83.6	6.3	7.4	2.7
P.c.+E.c.(100g/d)	Before	86.9	5.9	6.4	0.8
"	After	87.2	6.7	5.7	0.4
P.c.+E.c.(300g/d)	Before	86.2	6.8	6.9	0.2
"	After	86.7	6.5	6.7	0.08

The inclusion of E. ciclocarpum as supplement or as the basal diet does not eliminate protozoa from the rumen, but causes a significant reduction of total protozoa numbers and a variation in the species of protozoa present in the rumen. Holotrichas are the most susceptible species to E. ciclocarpum.

Fruits of plants with high saponin content (i.e. Sapindus saponaria), may be included in the diet along with leaves of E. ciclocarpum to increase the saponin concentration in the rumen for a short period of time (i.e. 3-5 days) and evaluate their combined effect on rumen protozoa.

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Table 8. Rumen pH of sheep fed on Pennisetum clandestinum or leaves of E. ciclocarpum (values 4h after feeding).

P. clandestinum	E. ciclocarpum	P

6.66	6.75	0.56