Evaluation of Enterococcus faecium NCIMB 11181 and Clostridium butyricum probiotic supplements in post-weaning rabbits reared under thermal stress conditions

Figures & data

Table 1. Ingredients and composition of the basal diet.

Components	[g/kg feed]	Nutrient levels	[g/kg feed]
Lucerne meal	340	Dry matter	910.5
Soybean meal	140	Crude protein (N × 6.25)	170.9
Barely grain	170	Ether extract	29.8
Wheat bran	220	Ash	86.3
Yellow corn	80	Neutral detergent fibre	344.9
Cane molasses	30	Acid detergent fibre	176.5
Dicalcium phosphate	10
Sodium chloride	5
DL-methionine	1
Premix^a	4
Total	1000

^aPremix contains per kg (minerals and vitamins mixture): vitamin A, 20,000 U; vitamin D₃, 15,000 U; vitamin E, 8.33 g; vitamin K, 0.33 g; vitamin B₁, 0.33; vitamin B₂, 1.0 g; vitamin B₆, 0.33 g; vitamin B₅, 8.33 g; vitamin B₁₂, 1.7 mg; pantothenic acid, 3.33 g; biotine, 33 mg; folic acid, 0.83 g; choline chloride, 200; manganese 80 g; zinc 60 g; iron 30 g; copper 4 g; iodine 0.5 g; selenium 0.1 g; and cobalt 0.1 g.

Table 2. Effect of dietary supplemental probiotics and colistin as growth promoters on growth performance of broiler rabbit exposed to heat stress conditions.

Parameter	Experimental groups					Pooled SEM	p-Value
	Control	Colistin	EF	CB	EF + CB
Body weight, g
d 35 (Initial)	688	712	685	698	689	7.33	.807
d 63	1246	1339	1371	1307	1380	24.21	.418
d 91 (Final)	1697^b	1782^ab	1898^a	1821^ab	1925 ^a	27.02	.042
Weghit gain, g
d 35–d 63	558	628	686	609	692	25.24	.438
d 63–d 91	452	443	528	515	546	16.63	.182
d 35–d 91 (Overall)	1010	1071	1214	1123	1237	28.92	.054
Relative growth rate, %
d 35–d 63	57.12	60.50	66.32	60.52	66.67	1.95	.485
d 63–d 91	30.99	28.69	32.54	32.87	33.06	1.11	.729
d 35–d 91 (Overall)	84.42	85.55	93.86	88.94	94.47	1.54	.117
Daily feed intake, g
d 35–d 63	73.65	68.14	73.36	71.00	67.57	1.22	.380
d 63–d 91	117.79	108.07	110.78	117.37	115.40	2.03	.491
d 35–d 91 (Overall)	95.72	88.10	92.07	94.19	91.49	1.30	.426
Feed: Gain
d 35–d 63	3.90	3.19	3.16	3.35	2.85	0.16	.333
d 63–d 91	7.33	6.94	5.98	6.93	6.05	0.26	.404
d 35–d 91 (Overall)	5.36^a	4.67^ab	4.28^b	4.76^ab	4.23^b	0.13	.028

SEM stands for standard error of the mean. ^a,bMeans in a row with different superscripts differ significantly.

Ef: control diet+ Enterococcus faecium NCIMB 11181; CB: control diet+ Clostridium butyricum; EF + CB: control diet+ Enterococcus faecium NCIMB 11181+ Clostridium butyricum.

Table 3. Effect of dietary supplemental probiotics and colistin as growth promoters on carcase traits (as % of pre-slaughter weight, SW) of broiler rabbit exposed to heat stress conditions.

Parameter	Experimental groups					Pooled SEM	p-Value
	Control	Colistin	EF	CB	EF + CB
Carcase yield, %	57.87	57.47	57.84	57.19	58.23	0.35	.931
Liver, g/kg SW	27.62	30.91	25.99	29.46	27.90	0.78	.355
Kidneys, g/kg SW	6.89	6.79	6.05	6.72	7.45	0.32	.792
Heart, g/kg SW	2.58	2.55	2.92	2.48	3.73	0.18	.160
Lungs, g/kg SW	6.65	7.62	6.65	6.95	7.63	0.33	.821
Spleen, g/kg SW	0.71	0.59	0.44	0.86	0.42	0.07	.242

SEM stands for standard error of the mean.

Ef: control diet+ Enterococcus faecium NCIMB 11181; CB: control diet+ Clostridium butyricum; EF + CB: control diet+ Enterococcus faecium NCIMB 11181+ Clostridium butyricum.

Table 4. Effect of dietary supplemental probiotics and colistin as growth promoters on hematological parameters of broiler rabbit exposed to heat stress conditions.

Parameter	Experimental groups					Pooled SEM	p-Value
	Control	Colistin	EF	CB	EF + CB
Erythrogram
RBCs, 10^6×µL	4.73	4.60	5.33	4.78	4.99	0.09	.063
Hb, g/dL	10.93^b	11.30^b	12.43^a	11.33^b	12.30^a	0.18	.011
Hct, %	34.47	37.50	39.10	36.73	36.23	0.60	.156
MCV, fL	72.82^b	81.72^a	74.16^ab	76.85^ab	76.52^ab	1.02	.038
MCH, pg/dL	23.17	24.60	23.65	23.70	24.89	0.22	.062
MCHC, g/dL	31.91	30.17	31.90	30.84	32.55	0.33	.147
Platelets, 10^3/µL	363.25	290.75	311.75	347.75	324.75	19.41	.819
Leukogram
WBCs, 10^3/µL	5.75	4.94	5.81	5.78	4.63	0.23	.367
Lymphocyte, %	64.75	60.69	64.38	65.58	61.99	1.39	.819
Monocytes, %	2.00	2.00	2.25	2.25	2.25	0.08	.736
Neutrophils, %	25.58	35.98	31.29	29.42	33.35	1.57	.293
Eosinophils, %	2.25	1.25	2.00	2.00	2.00	0.14	.234

SEM stands for standard error of the mean. ^a,bMeans in a row with different superscripts differ significantly.

Ef: control diet+ Enterococcus faecium NCIMB 11181; CB: control diet+ Clostridium butyricum; EF + CB: control diet+ Enterococcus faecium NCIMB 11181+ Clostridium butyricum; RBCs: red blood cells; Hct: the haematocrit; Hb: haemoglobin; MCV: mean corpuscular volume; MCHC: mean corpuscular haemoglobin concentration; WBCs: white blood cells.

Table 5. Effect of dietary supplemental probiotics and colistin as growth promoters on protein and lipid profiles and immunological indices of broiler rabbit exposed to heat stress conditions.

Parameter	Experimental groups					Pooled SEM	p-Value
	Control	Colistin	EF	CB	EF + CB
Protein profile
TP, g/dL	6.00^b	5.27^b	8.04^a	7.53^a	7.91^a	0.28	<.001
ALB, g/dL	2.22	2.91	2.55	2.36	3.00	0.12	.136
GLOB, g/dL	3.78^c	2.36^b	5.49^a	5.18^a	4.91^a	0.29	<.001
A/G, %	0.61^b	1.89^a	0.46^b	0.47^b	0.61^b	0.13	<.001
Lipid profile
TC, mg/dL	238.00^a	195.25^ab	166.00^b	195.25^ab	178.00^b	5.42	.039
TG, mg/dL	283.25^a	200.25^b	188.00^bc	174.75^bc	122.75^c	14.60	.002
HDL, mg/dL	35.25^d	39.25^cd	46.75^b	44.25^bc	55.00^a	1.81	<.001
LDL, mg/dL	147.35	91.20	109.80	97.95	115.85	8.84	.318
VLDL, mg/dL	54.28^a	40.08^b	37.60^bc	34.93^bc	24.53^c	2.78	.003
Immunological indices
C3, mg/dL	118.25^b	130.00^ab	141.25^a	130.00^ab	139.75^a	2.43	.005
Lysozyme, U/ml	10.75^c	14.25^bc	17.75^ab	17.75^ab	20.75^a	1.00	.004

SEM stands for standard error of the mean. ^a,bMeans in a row with different superscripts differ significantly.

Ef: control diet+ Enterococcus faecium NCIMB 11181; CB: control diet+ Clostridium butyricum; EF + CB: control diet+ Enterococcus faecium NCIMB 11181+ Clostridium butyricum; TP: total protein; ALB: albumin; GLOB: globulin; A/G: albumin/globulin ratio; TC: total cholesterol; TG: total triglycerides; HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low-density lipoprotein; C3: complement component 3.

Table 6. Effect of dietary supplemental probiotics and colistin as growth promoters on caecal volatile fatty acids (VFA) of broiler rabbit exposed to heat stress conditions.

Parameter	Experimental groups					Pooled SEM	p-Value
	Control	Colistin	EF	CB	EF + CB
Caecal VFA concentration (mmol/L)
Total	102.88^b	113.44^ab	126.73^a	120.96^ab	128.85^a	2.98	.018
Acetate	80.01	87.97	98.42	91.62	94.96	2.28	.083
Propionate	6.64^b	7.22^b	9.15^a	8.97^a	10.42^a	0.37	<.001
Butyrate	15.21^b	16.87^b	18.00^b	19.09^ab	22.39^a	0.48	.029
Valerate	1.02	1.38	1.16	1.28	1.08	0.06	.233
Caecal VFA proportions (%)
Acetate	77.77	77.55	77.66	75.74	73.70	0.78	.774
Propionate	6.45^b	6.36^b	7.22^b	7.42^ab	8.09^a	0.25	.017
Butyrate	14.78	14.87	14.20	15.78	17.38	0.68	.708
Valerate	0.99	1.22	0.92	1.06	0.84	0.05	.054

SEM stands for standard error of the mean. ^a,bMeans in a row with different superscripts differ significantly.

Ef: control diet+ Enterococcus faecium NCIMB 11181; CB: control diet+ Clostridium butyricum; EF + CB: control diet+ Enterococcus faecium NCIMB 11181+ Clostridium butyricum.

Table 7. Effect of dietary supplemental probiotics and colistin on histomorphometric measurements in the intestine of broiler rabbit exposed to heat stress conditions.

Parameter	Experimental groups					Pooled SEM	p-Value
	Control	Colistin	EF	CB	EF + CB
Histomorphometry measurements
Villus length (µm)	222.20^d	304.80^c	332.60^bc	367.20^b	589.80^a	26.30	<.001
Villus width (µm)	191.00^a	160.80^b	143.60^b	135.00^b	107.80^c	6.60	<.001
Crypt depth (µm)	88.00^a	68.40^b	48.60^c	49.00^c	35.00^c	4.49	<.001
Submucosa (µm)	153.00^a	99.00^b	86.60^bc	88.40^bc	69.40^c	6.93	<.001
Muscle (µm)	69.40^c	79.40^bc	90.40^b	85.40^b	106.20^a	2.92	<.001
Serosa (µm)	66.60^ab	77.00^a	45.60^bc	35.00^c	32.00^c	4.78	.001

SEM stands for standard error of the mean. ^a,bMeans in a row with different superscripts differ significantly. The

Ef: control diet+ Enterococcus faecium NCIMB 11181; CB: control diet+ Clostridium butyricum; EF + CB: control diet+ Enterococcus faecium NCIMB 11181+ Clostridium butyricum.

Figure 1. Representative photomicrographs of duodenum of growing rabbit reared under summer conditions of different experimental groups. (A–C) control group fed non-supplemented diets, (D–F) colistin group, (G–I) Enterococcus faecium (EF) NCIMB 11181 group, (J–L) Clostridium butyricum (CB) group, (M–O) EF + CB group. Villi: V; Necrotic masses: N; Intestinal glands: G; Muscular layer: M; Serosa: red arrow; Crypt: C; Inflammatory cell infiltrations: I; Epithelium: E; Erosions: S; Stroma: ST; Intraepithelial lymphocytosis: L; Hyperplasia: HV; Congestion: black arrow; Goblet cell: green arrow (1st, 2nd, 3rd column of photos stained with H&E at 40×, 100×, and 400×, respectively).