Effects of feeding goats with Leucaena leucocephala and Manihot esculenta leaves supplemented diets on rumen fermentation profiles, urinary purine derivatives and rumen microbial population

Figures & data

Table 1. Chemical composition of L. leucocephala and M. esculenta leaves supplemented diets (Mean ± SE).

Chemical composition, g kg DM^−1	T1	T2	T3	T4	T5	T6	T7	SEM	P
DM	93.03 ± 0.06^a	92.77 ± 0.06^ab	92.69 ± 0.05^ab	92.11 ± 0.03^c	92.25 ± 0.07^bc	92.54 ± 0.14^ab^c	92.63 ± 0.14^ab^c	0.08	*
Organic matter	84.44 ± 0.06^a	83.43 ± 0.07^b	82.66 ± 0.26^c	82.60 ± 0.06^c	82.03 ± 0.34^cd	81.41 ± 0.08^d	80.28 ± 0.12^e	0.32	**
Neutral detergent fibre	58.46 ± 2.15^a	52.01 ± 0.75^bc	49.79 ± 1.43^cd	46.12 ± 1.32^d	56.66 ± 1.75^ab	53.27 ± 1.42^bc	45.03 ± 1.84^d	1.16	**
Acid detergent fibre	31.89 ± 0.15^a	30.89 ± 1.34^ab	25.04 ± 0.74^c	21.41 ± 0.28^c	31.20 ± 0.22^ab	29.72 ± 0.13^b	24.46 ± 0.20^c	0.87	**
Acid detergent lignin	3.28 ± 0.24^c	3.84 ± 0.24^bc	5.01 ± 0.62^a	5.31 ± 0.07^a	5.22 ± 0.11^a	3.66 ± 0.17^bc	4.32 ± 0.38^ab	0.20	**
Crude protein	10.60 ± 0.09^e	14.70 ± 0.40^c	17.92 ± 0.47^b	21.78 ± 0.27^a	12.20 ± 0.37^d	14.24 ± 1.01^c	17.65 ± 0.11^b	0.82	**
Crude Fat	1.07 ± 0.06^e	0.94 ± 0.16^e	1.54 ± 0.02^d	1.89 ± .0003^bc	1.69 ± 0.002^cd	2.11 ± 0.05^b	2.80 ± 0.07^a	0.16	**

Notes: T1: Control, T2: 25% of L. leucocephala leaves, T3: 50% of L. leucocephala leaves, T4: 75% of L. leucocephala leaves, T5: 25% of M. esculenta leaves; T6: 50% of M. esculenta leaves; T7: 75% of M. esculenta leaves.

^abcValues on the same column with different superscript differ (P < .05). * = P < .05, ** = P < .01, NS = P > .05. SEM = standard error of means. DM = dry matter.

Table 2. Primers used for qPCR assay to target total bacteria, total protozoa, methanogens, R. albus, R. flavefaciens and F. succinogenes.

Target groups	Sequence 5′–3′	Annealing temperatures (°C)	References
Total bacteria	F: CGGCAACGAGCGCAACCC	55	Koike and Kobayashi (2001)
	R: CCATTGTAGCACGTGTGTAGCC
Total protozoa	F: CTTGCCCTCYAATCGTWCT	55	Sylvester et al. (2004)
	R: GCTTTCGWTGGTAGTGTATT
Methanogens	F: TTCGGTGGATCDCARAGRGC	58	Denman and McSweeney (2006)
	R: GBARGTCGWAWCCGTAGAATCC
R. albus	F: CCCTAAAAGCAGTCTTAGTTCG	55	Koike and Kobayashi (2001)
	R: CCTCCTTGCGGTTAGAACA
R. flavefaciens	F: TCTGGAAACGGATGGTA	60
	R: CCTTTAAGACAGGAGTTTACAA
F. succinogenes	F: GTTCGGAATTACTGGGCGTAAA	55	Lane (1991)
	R: CGCCTGCCCCTGAACTATC

Figure 1. Mean cumulative gas production of the treatment diets.

Table 3. Diets’ effects on in vitro ruminal pH, rumen ammonia-nitrogen, VFA concentration and IVDMD (Mean ± SE).

Parameters	T1	T2	T3	T4	T5	T6	T7	SEM	P
pH	6.60 ± 0.12	6.77 ± 0.03	6.70 ± 0.10	6.60 ± 0.10	6.73 ± 0.07	6.77 ± 0.03	6.60 ± 0.12	0.03	NS
NH₃-N^1, ppm	0.68 ± 0.25	1.32 ± 0.75	0.70 ± 0.06	1.28 ± 0.78	0.79 ± 0.09	0.96 ± 0.21	0.95 ± 0.27	0.14	NS
VFA, mol/100mol
Acetate	63.03 ± 1.00	62.25 ± 1.13	64.05 ± 0.54	62.42 ± 0.59	62.51 ± 0.78	63.33 ± 0.74	62.63 ± 1.33	0.33	NS
Propionate	19.15 ± 0.39^ab	20.15 ± 0.47^a	19.08 ± 0.31^ab	19.91 ± 0.24^a	18.65 ± 0.27^b	18.42 ± 0.3^b	19.12 ± 0.54^ab	0.15	*
Butyrate	17.82 ± 0.69	17.60 ± 0.86	16.87 ± 0.45	17.68 ± 0.47	18.85 ± 0.65	18.25 ± 0.71	18.25 ± 1.08	0.27	NS
A:P^2	3.31 ± 0.12^ab	3.11 ± 0.12^b	3.37 ± 0.08^ab	63.14 ± 0.05^ab	3.36 ± 0.08^ab	3.44 ± 0.08^a	3.31 ± 0.15^ab	0.04	NS
TVFA^3, mmol/L	69.10 ± 2.74	71.66 ± 3.18	72.47 ± 2.11	71.54 ± 1.38	67.97 ± 1.79	73.56 ± 2.02	74.00 ± 3.41	0.93	NS
IVDMD^4	48.28 ± 1.93^a	18.98 ± 3.55^b	24.93 ± 5.98^b	30.66 ± 5.09^ab	23.71 ± 1.85^b	22.09 ± 4.19^b	48.28 ± 1.93^b	0.70	*

Notes: T1: Control, T2: 25% of L. leucocephala leaves, T3: 50% of L. leucocephala leaves, T4: 75% of L. leucocephala leaves, T5: 25% of M. esculenta leaves; T6: 50% of M. esculenta leaves; T7: 75% of M. esculenta leaves.

^abValues on the same column with different superscript differ (P < .05). * = P < .05, NS = P > .05. SEM = standard error of means.

¹NH₃-N = Ammonia-nitrogen, ²A:P = Acetate to propionate ratio, ³TVFA = Total volatile fatty acid, ⁴IVDMD = In vitro dry matter digestibility.

Table 4. Diets effects on in vivo ruminal pH, rumen NH₃-N and VFA concentration of the goats (Mean ± SE).

Parameters	T1	T2	T3	T4	T5	T6	T7	SEM	P
pH	6.83 ± 0.13^b	7.00 ± 0.15^ab	7.13 ± 0.03^ab	7.27 ± 0.13^a	6.93 ± 0.09^ab	7.07 ± 0.18^ab	7.07 ± 0.67^ab	0.05	NS
NH₃-N^1, ppm	1.39 ± 0.05^c	1.57 ± 0.09^abc	1.51 ± 0.04^bc	1.72 ± 0.05^ab	1.76 ± 0.06^a	1.66 ± 0.10^ab	1.64 ± 0.06^ab	0.03	**
VFA, mol/100 mol
Acetate	75.13 ± 1.39^ab	77.17 ± 1.04^a	71.79 ± 0.92^b	71.59 ± 1.06^b	73.88 ± 1.54^ab	75.10 ± 1.95^ab	73.78 ± 1.58^ab	0.56	NS
Propionate	17.11 ± 0.88^ab	15.52 ± 0.53^b	18.49 ± 0.81^a	17.68 ± 0.95^ab	17.63 ± 0.94^ab	18.37 ± 1.15^a	19.25 ± 0.73^a	0.34	*
Butyrate	7.75 ± 0.57^bc	7.31 ± 0.65^bc	9.72 ± 0.51^ab	10.72 ± 0.51^a	8.49 ± 0.75^abc	6.53 ± 1.39^c	6.96 ± 1.06^c	0.34	**
A : P ^2	4.55 ± 0.31^ab	5.06 ± 0.21^a	3.93 ± 0.22^b	4.18 ± 0.25^b	4.35 ± 0.31^ab	4.23 ± 0.34^ab	3.92 ± 0.21^b	0.11	*
TVFA^3, mmol/L	80.39 ± 4.08^ab	84.83 ± 2.44^a	72.80 ± 1.96^bc	68.35 ± 3.40^c	74.60 ± 2.80^bc	76.17 ± 3.87^abc	71.29 ± 2.51^bc	1.31	**

Notes: T1: Control, T2: 25% of L.leucocephala leaves, T3: 50% of L.leucocephala leaves, T4: 75% of L.leucocephala leaves, T5: 25% of M. esculenta leaves; T6: 50% of M. esculenta leaves; T7: 75% of M. esculenta leaves.

^abcValues on the same column with different superscript differ (P < .05). * = P < .05, ** = P < .01, NS = P > .05. SEM = standard error of mean.

¹NH₃-N = Ammonia-nitrogen, ²A:P = Acetate to propionate ratio, ³TVFA = Total volatile fatty acid.

Table 5. Effect of L. leucocephala and M. esculenta leaves supplemented diets on urinary PD of goats (Mean ± SE).

PD (μmol/kg BW⁰˙⁷⁵d^−1)	T1	T2	T3	T4	T5	T6	T7	SEM	P
Allantoin	196.97 ± 22.26^ab	227.15 ± 33.26^a	141.81 ± 16.34^bc	152.47 ± 17.16^bc	136.96 ± 5.72^bc	226.35 ± 14.99^a	94.50 ± 2.66^bc	9.26	**
Uric acid	14.82 ± 3.79^b	18.02 ± 2.17^ab	17.69 ± 4.29^ab	18.27 ± 3.74^ab	17.55 ± 1.26^ab	25.95 ± 4.07^a	15.76 ± 0.23^ab	1.19	NS
Hypoxanthine	10.17 ± 0.25^ab	12.44 ± 2.12^a	10.26 ± 0.74^ab	12.83 ± 1.32^a	9.75 ± 2.36^ab	10.69 ± 2.47^ab	6.11 ± 0.75^b	0.71	NS
Xanthine	11.19 ± 0.23^bc	16.23 ± 0.95^a	12.06 ± 1.30^b	15.30 ± 0.60^a	8.75 ± 0.33^c	10.20 ± 0.99^bc	9.23 ± 0.45^c	0.49	**
Total PD^1	223.17 ± 24.12^ab	271.83 ± 31.63^a	177.89 ± 20.92^bc	196.83 ± 17.51^b	173.00 ± 13.59^bc	273.18 ± 21.23^a	125.60 ± 3.17^c	10.09	**

Notes: T1: Control, T2: 25% of L. leucocephala leaves, T3: 50% of L. leucocephala leaves, T4: 75% of L. leucocephala leaves, T5: 25% of M. esculenta leaves; T6: 50% of M. esculenta leaves, T7: 75% of M. esculenta leaves.

^abcValues on the same column with different superscript differ (P < .05). ** = P < .01, NS = P > .05. SEM = standard error of means. BW = body weight.

¹Total PD = Total purine derivatives.

Table 6. Effect of L. leucocephala and M. esculenta leaves supplemented diets on rumen microbial population in goats (Mean ± SE).

Microbial population, Log10 copy no. per ml of DNA extract	T1	T2	T3	T4	T5	T6	T7	SEM	P
Total bacteria	10.75 ± 0.14	10.97 ± 0.38	10.96 ± 0.43	10.26 ± 0.28	11.02 ± 0.51	11.11 ± 0.44	11.35 ± 0.61	0.16	NS
Methanogens	5.82 ± 0.03	5.98 ± 0.08	5.75 ± 0.14	5.86 ± 0.36	5.58 ± 0.11	5.59 ± 0.10	5.78 ± 0.26	0.07	NS
Total protozoa	7.21 ± 0.19^c	7.53 ± 0.08^abc	7.70 ± 0.06^ab	7.86 ± 0.12^a	7.27 ± 0.20^c	7.81 ± 0.05^ab	7.41 ± 0.13^bc	0.06	**
R. albus	8.26 ± 0.20^a	7.52 ± 0.13^b	6.80 ± 0.31^b	6.87 ± 0.44^b	7.49 ± 0.20^b	7.25 ± 0.07^b	7.29 ± 0.12^b	0.11	**
R. flavefacien	11.31 ± 0.07^c	11.44 ± 0.11^bc	11.58 ± 0.02^abc	11.66 ± 0.08^abc	11.87 ± 0.16^ab	11.84 ± 0.16^ab	11.99 ± 0.27^a	0.07	NS
F. succinogenes	7.66 ± 0.16^a	6.85 ± 0.20^bc	7.33 ± 0.13^ab	6.88 ± 0.26^bc	6.97 ± 0.06^bc	7.19 ± 0.09^bc	6.79 ± 0.04^c	0.07	**

Notes: T1: Control, T2: 25% of L .leucocephala leaves, T3: 50% of L. leucocephala leaves, T4: 75% of L. leucocephala leaves, T5: 25% of M. esculenta leaves; T6: 50% of M. esculenta leaves; T7: 75% of M. esculenta leaves.

^abcValues on the same column with different superscript differ (P < .05). ** = P < .01, NS = P > .05. SEM = standard error of means.

Koike S, Kobayashi Y. 2001. Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FEMS Microbiol. Lett. 204(2):361–366. doi: 10.1111/j.1574-6968.2001.tb10911.x

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