The impact of dietary inclusion of silver nanoparticles on growth performance, intestinal morphology, caecal microflora, carcass traits and blood parameters of broiler chickens

Figures & data

Figure 1. Size and Shape of silver nano particles.

Table 1. Composition and chemical analysis of basal diets (% as fed basis).

Ingredient, %	Starter	Grower and Finisher
Corn	54.85	63
Corn gluten meal (60%)	4.5	4
Soybean meal, (48.5 %)	32	25
Sunflower oil	4.3	3.65
Limestone	1.5	1.5
Dicalcium phosphate	1.8	1.8
Salt	0.4	0.4
Vitamin - mineral Premix^a	0.3	0.3
DL-methionine	0.2	0.2
L- lysine	0.1	0.1
Antioxidants	0.05	0.05
Total	100	100
Chemical analysis
Dry matter (%)	90.10	89.93
Crude protein (%)	23	20
ME (Kcal/kg diet) ^b	3200	3200
Calcium (%)	1.08	1.06
Total phosphorus (%)	0.77	0.69
Non-phytae phosphorus (%)	0.44	0.43
Na (%)	0.17	0.17
Lysine (%)	1.24	1.05
DL-Methionine (%)	0.58	0.54
Methionine + cysteine (%)	1.06	0.88
Crude fibre (%)	2.95	2.92
Crude fat (%)	6.39	6.02

^a The vitamin and mineral premix provided per kg of diet: vitamin A, 4000000 IU; vitamin D3, 667000 IU; vitamin E 3334 mg; vitamin K3, 1167 mg; vitamin B1, 334 mg; vitamin B2, 1667 mg; vitamin B3, 3334 mg; B6, 500 mg; vitamin B12 33.4 mg, Folic acid, 334 mg; Biotin, 17 mg; Iron, 10; Copper, 2.167; Zinc, 18.334; Manganese 20.0; Iodine, 0.167; Cobalt, 0.034 and Selenium, 0.034.

^b Calculated based on NRC (1994) feed composition tables.

Table 2. Effect of different dietary treatments on body weight, feed intake and feed conversion ratio in broiler chickens.

Item	AgNPs levels (mg/kg diet)
	C	NS2.5	NS5	NS10	NS20	SEM^A	Sig.
Body weight (g)
Initial (day 8)	179.83	182.00	183.27	178.87	181.40	1.28	0.87
Day 28	1343.33	1398.33	1401.11	1368.89	1423.33	14.78	0.53
Final BW (day 42)	2500^c	2600^a	2580^ab	2560^ab	2535^bc	11.2	0.01
Feed consumption (g)
From day 8–28	1970	1945	1964.07	1958.33	1911.66	10.62	0.56
From day 28–42	2595.74	2624.07	2638.27	2622.41	2612.37	8.05	0.61
From day 8–42	4565.74	4569.07	4602.34	4580.74	4524.03	13.64	0.13
Weight gain (g)
From day 8–28	1163.5	1216.33	1217.84	1190.02	1241.93	14.62	0.55
From day 28–42	1156.67	1201.67	1179.56	1191.11	1111.67	18.57	0.63
From day 8–42	2320.167^c	2418^a	2397.4^ab	2381.13^ab	2353.6^bc	10.67	0.006
Feed conversion ratio
From day 8–28	1.69	1.60	1.61	1.65	1.54	0.02	0.17
From day 28–42	2.24	2.20	2.23	2.20	2.36	0.03	0.64
From day 8–42	1.97^a	1.89^b	1.91^b	1.92^b	1.91^b	0.02	0.01

^ASEM: pooled standard errors of means; Sig.: significant.

C (Control, basal diet); NS2.5, NS5, NS10, NS20 (Basal diet with 2.5, 5, 10, 20 ppm AgNPs), respectively.

^a,bMeans with different superscript letters in the same row are significantly different (p ≤ 0.05).

Table 3. Effect of different dietary treatments on carcass traits and relative organ weight and WHC in broiler chickens.

Item	AgNPs levels (mg/kg diet)
	C	NS2.5	NS5	NS10	NS20	SEM^A	Sig.
Presloughter weight (g)	2515^ab	2594.00^a	2590.00^a	2573.33^ab	2500.00^b	13.90	0.05
Worm carcass weight (g)	1610^c	1788.33^a	1723.19^ab	1686.68^bc	1634.79^c	19.63	0.005
Dressing %	64.00^b	68.95^a	66.53^ab	65.54^b	65.39^b	0.53	0.01
Liver %	1.73^b	2.03^a	1.97^a	1.87^ab	1.72^b	0.04	0.006
Gizzard %	1.72^c	1.95^a	1.92^ab	1.83^b	1.69^c	0.03	0.001
Kidney %	0.61^a	0.67^a	0.63^a	0.64^a	0.51^b	0.02	0.014
Spleen %	0.13^b	0.18^a	0.17^a	0.15^ab	0.14^b	0.005	0.017
Proventriculus %	0.36	0.38	0.42	0.42	0.42	0.008	0.06
Heart %	0.46^b	0.53^a	0.49^ab	0.47^ab	0.42^b	0.13	0.05
Bursa %	0.13	0.15	0.14	0.14	0.15	0.003	0.06
Water-holding capacity (WHC)
Breast muscle, (WHC % )	70.55^a	73.42^a	71.41^a	65.58^b	63.87^b	0.82	0.003
Thigh muscle (WHC,% )	70.27^ab	73.71^a	70.95^a	70.10^ab	67.10^b	0.69	0.02

WHC: water-holding capacity.

^ASEM: pooled standard errors of means; Sig.: significant.

C (Control, basal diet); NS2.5, NS5, NS10, NS20 (Basal diet with 2.5, 5, 10, 20 ppm AgNPs), respectively.

^a,bMeans with different superscript letters in the same row are significantly different (p ≤ 0.05).

Table 4. The effect of dietary AgNPs level on residues of silver (mg/kg) in meat of broilers.

Item	AgNPs levels (mg/kg diet)
	C	NS2.5	NS5	NS10	NS20	SEM^A	Sig.
Breast muscle	0.01^b	0.04^b	0.06^b	0.07^ab	0.11^a	0.01	0.02
Thigh muscle	0.02^c	0.10^c	0.15^b	0.17^b	0.29^a	0.02	0.001

^a,bMeans with different superscript letters in the same row are significantly different (p ≤ 0.05).

^ASEM: pooled standard errors of means; Sig.: significant.

C (Control, basal diet); NS2.5, NS5, NS10, NS20 (Basal diet with 2.5, 5, 10, 20 ppm AgNPs), respectively.

Table 5. Numbers of dominant bacterial groups in the contents of caeca [log cfu/g] of broilers after treatment with increasing concentration of AgNano.

Item	AgNPs levels (mg/kg diet)
	C	NS2.5	NS5	NS10	NS20	SEM^A	Sig.
Day 28
Lactose negative bacteria	4.78	3.99	3.98	3.06	2.52	0.33	0.22
Lactose positive bacteria	6.41^a	6.31^a	5.35^ab	4.78^b	4.47^b	0.25	0.02
Enterococci	7.19^a	7.16^a	7.08^a	7.09^a	6.37^b	0.09	0.002
Lactobacilli	8.77	8.84	9.05	8.86	8.63	0.14	0.95
Day 42
Lactose negative bacteria	3.38	2.13	1.97	1.57	1.43	0.26	0.11
Lactose positive bacteria	4.98	4.62	4.61	4.46	4.21	0.14	0.61
Enterococci	6.19	5.16	5.91	5.65	6.09	0.18	0.45
Lactobacilli	9.29	9.31	9.61	9.83	9.37	0.13	0.72

^a,bMeans with different superscript letters in the same row are significantly different (p ≤ 0.05).

^ASEM: pooled standard errors of means; Sig.: significant

C (Control, basal diet); NS2.5, NS5, NS10, NS20 (Basal diet with 2.5, 5, 10, 20 ppm AgNPs), respectively.

Table 6. Effect of different dietary treatments on blood metabolites (mg/dL) in broiler chickens.

Item	AgNPs levels (mg/kg diet)
	C	NS2.5	NS5	NS10	NS20	SEM^A	Sig.
Total protein (mg/dlìL)	2.73^b	4^a	4^a	3.7^a	3.17^ab	0.18	0.03
Albumen (mg/dL)	1.43	1.77	1.93	1.86	1.73	0.07	0.14
Globulin (mg/dL)	1.3	2.32	2.07	1.84	1.43	0.14	0.09
Total Cholesterol (mg/dL)	52.00^b	75.00^a	74.67^a	69.00^ab	51.67^b	3.77	0.03
Triglycerides (mg/dL)	49	84.67	82.5	69.00	61.33	5.53	0.17
Urea (mg/dL)	11^c	13^bc	15^ab	16.5^a	14.5^ab	0.62	0.01
Creatinine (mg/dL)	0.30	0.27	0.30	0.55	0.35	0.03	0.05
IgG (mg/dL)	371.66	507	415.67	367.33	266.5	25	0.09
IgA (mg/dL)	60.33	69.66	65.33	52.5	45.5	3.54	0.25
Uric acid (mg/dL)	6.66	5.27	5.3	5.8	5.45	0.34	0.7
Calcium (mg/dL)	6.73	7.83	7.73	7.45	6.85	0.19	0.24
Phosphorus (mg/dL)	6.47^a	5.66^ab	4.7^b	4.60^b	4.60^b	0.27	0.05

^a,bMeans with different superscript letters in the same row are significantly different (p ≤ 0.05), ^ASEM: pooled standard errors of means; Sig.: significant.

C (Control, basal diet); NS2.5, NS5, NS10, NS20 (Basal diet with 2.5, 5, 10, 20 ppm AgNPs), respectively.

Figure 2. Representative micrographs for duodenum (A–E) and spleen (F-G) of broiler chickens treated with different levels AgNPs for 42 days, H&E stain. A) Duodenum from control group showing normal epithelium with no lesions. (B) Duodenum from 2.5 mg supplemented group showing normal structure of intestinal villi. (C) Duodenum from 5 mg group showing necrosis and sloughing of epithelium (arrow). (D) Duodenum from 10 mg showing epithelial metaplasia (arrow). (E) Duodenum from 20 mg group showing epithelial metaplasia (arrow). F) Spleen from control group showing normal red and white pulp with normal lymphoid follicle. (G) Spleen from 2.5 mg group showing normal red and white pulp with normal lymphoid follicle. (H) Spleen from 5 mg group showing congestion of the blood vessels, desquamation of endothelium (arrow) and perivascular infiltration. (I) Spleen from 10 mg group showing necrosis of the lymphoid follicle (arrow). (G) Spleen from 20 mg group showing necrosis of the lymphoid follicle (arrow).

Figure 3. Representative micrographs for liver (A–E) and kidney (F-J) of broiler chickens treated with different levels AgNPs for 42 days, H&E stain. A) Liver from control group showing normal hepatic architecture. (B) Liver from 2.5 mg group showing focal mononuclear infiltration (arrow). (C) Liver from 5 mg group showing congestion and vacuolar degeneration. (D) Liver from 10 mg group showing hepatocellular vacuolation (arrow). (E) Liver from 20 mg group showing hepatocellular vacuolation (arrow). F) Kidney from control group showing normal glomerulus and renal tubules. (G) Kidney from 2.5 mg group showing normal glomerulus and renal tubules. (H) Kidney from 5 mg group showing congestion of the glomerulus (arrow). (I) Kidney from 10 mg group showing severe congestion of the blood vessels (arrow). (J) Kidney from 20 mg group showing congestion of the glomerulus (arrow).

Figure 4. Representative micrographs of Transmission electron microscope of duodenum from broiler chickens treated with 20 mg AgNPs for 42 days showing nano particles of different size inside the nucleus (A) of the enterocyte.

Table 7. Summary of lesion score at 42 days in all studied groups.

Item	AgNPs levels (mg/kg diet)
	C	NS2.5	NS5	NS10	NS20
Duodenum lesions:
Necrosis & sloughing of epithelium	–	+	+	++	+++
Mononuclear infiltration	–	+	++	+++	+++
Spleen lesions:
Necrosis of Lymphoid follicle	–	–	+	++	++
Changes in bl.vs.	–	–	+	++	++
Liver Lesions:
Congestion	–	+	+	+++	+++
Focal accumulation of mononuclear cells	–	+	++	+++	+++
Hepatocellular vacuolation	–	+	+	+++	+++
Kidney Lesions:
Congestion of bl.vs	–	–	++	++	+++
Hemorrhages	–	–	–	+++	+++
Glomerular congestion	–	–	+	++	+++
Glomerular swelling	–	–	++	+++	+++
Mononuclear infiltration	–	+	++	+++	+++
Renal tubular degeneration	–	+	++	++	++

(- No lesions, + lesions present in 3–5 sections, ++ lesions present in 6–12 sections, +++ lesions present in 13–20 sections).

C (Control, basal diet); NS2.5, NS5, NS10, NS20 (Basal diet with 2.5, 5, 10, 20 ppm AgNPs), respectively.

NRC 1994. Nutrient requirements of poultry. 9th Rev ed. Washington, DC: National Academic Press.

 Google Scholar

Data availability statement

The corresponding author is committed to providing data that support the findings of the study upon request.