Experimental ochratoxicosis A in Nile tilapia and its amelioration by some feed additives

Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.

Figures & data

able 1 Outline of the experimental design.

Group	Diet
1	Basal diet + 0% (OTA)^* (negative control)
2	Basal diet + 500 ppb OTA (positive control)
3	Basal diet + 500 ppb OTA + 0.1 g/kg Garlen Extra4^**
4	Basal diet + 500 ppb OTA + 30 g/kg Nigella sativa^***
5	Basal diet + 500 ppb OTA + 0.1 g/kg Garlen Extra4 + 30 g/kg Nigella sativa
6	Basal diet + 500 ppb OTA + 0.5 g/kg Fero Bind Pro^****(Mycotoxins binder)

Ochratoxin A.

* Garlen Extra₄: Commercial feed additive, contain Garlic extract (25%), blend of volatile oil (10%).A product from Media http://Vet.%20Co. for Imp. And Exp. Kafr El-Sheikh. Egypt. Manufactured by: Hefei Royal Eagle Imp. & Exp. Co. Ltd. China.

** Nigella sativa: Fresh prepared seeds powder. The proximate analysis of Nigella sativa dry biomass as feed base provided: 92.93% dry matter, 7.07% moisture, 19.04% crude protein, 21.4% crude fat, 5.3% Ash, 47.19% total carbohydrate, 0.27% Ca, 0.51% total phosphorus, 4316.11 Metabolizable Energy (Metabolizable Energy was calculated according to [55].

*** Fero Bind Pro: Commercial feed supplement acting as mycotoxins binder consisted of yeast (cell wall extract) vitamin E & Se, enzymes, butylated hydroxyl toluene (BHT), bentonite and clay minerals, Betaine and herbal extract (Quillaya saponaria).

able 2 Ingredients Proximate analysis of the experimental different dietary treatments.

Ingredients	Treatments	Basal diet	2	3	4	5	6
Ochratoxin A		–	+	+	+	+	+
Garlen Extra4		–	–	0.010	–	0.010	–
Nigella sativa		–	–	–	3.0	3.0	–
Fero Bind Pro		–	–	–	–	–	0.050
Yellow corn (7.5%)		34	34	36.29	34.9	34.89	33.95
Soybean meal (48%)		39	39	39	38.7	38.7	39
Fish Meal (Menhaden, 64%)		10	10	10	10	10	10
Wheat bran (16.4%)		5	5	2.1	2	2	5
Corn glutine (60%)		5	5	5.5	5	5	5
Soybean oil		5	5	5.100	4.4	4.4	5
Vitamins & mineral mixture^*		0.3	0.3	0.3	0.3	0.3	0.3
Dicalcium phosphate		1.7	1.7	1.7	1.7	1.7	1.7
Total (kg)		100	100	100	100	100	100
Values between parentheses are determined crude protein content (N×6.25)
Proximate analysis
Dry matter (DM)		90.12	90.12	90.12	90.17	90.17	90.13
Moisture		9.88	9.88	9.88	9.83	9.83	9.87
Crude protein (CP)		31.54	31.54	31.54	31.54	31.54	31.54
Ether extract (EE)		7.755	7.755	7.83	7.715	7.71	7.753
Crude fiber (CF)		2.75	2.75	2.52	2.73	2.73	2.75
Nitrogen free extract (NFE^**)		41.275	41.275	41.54	41.385	41.39	41.247
Ash		6.80	6.80	6.69	6.80	6.80	6.84
Methionine		0.60	0.60	0.60	0.60	0.60	0.60
Lysine		1.84	1.84	1.84	1.84	1.84	1.84
Calcium		1.01	1.01	1.01	1.02	1.02	1.02
Total phosphorus		1.03	1.03	1.01	1.01	1.01	1.03
Digestable Energy (DE kcal /kg)		3090.76	3090.76	3090.33	3094.74	3094.52	3089.66

The used vitamins & mineral mixture* (Multivita Co.) composed of vitamin A 12000000 IU, vitamin D3 2200000 IU, vitamin E 10000 mg, vitamin K3 2000 mg, vitamin B1 1000 mg, vitamin B2 5000 mg, vitamin B6 1500 mg, vitamin B12 10 mg, Niacin 30000 mg, Biotin 50 mg, Folic acid 1000 mg, Pantothenic acid 10000 mg, Iron 30000 mg, Manganese 60000 mg, Copper 4000 mg, Zinc 50000 mg, Iodine 1000 mg, Cobalt 100 mg, Selenium 100 mg, calcium carbonate (CaCO₃) carrier to 3000 g.**NFE = Nitrogen free extract and calculated by difference {100 − (moisture% + CP% + EE% + CF% + Ash%). Carbohydrates = NFE + CF.

ig. 1a Experimentally OTA intoxicated O. niloticus showing enlarged pale liver with necrotic foci, enlarged gall bladder, congested enlarged spleen and congested gills.

ig. 1b Experimentally OTA intoxicated O. niloticus showing enlarged pale liver, enlarged gall bladder, congested enlarged spleen, congested gills and kidneys.

able 3 Effect of different treatments on growth performance and survivability of Oreochromis niloticus at the end of the experiment.

Group	Survival rate	Growth parameters
		Initial body weight	Final body weight	Body weight gain	F.C.R^*	RGR^**
1	100%	60.69 ± 0.09a	98.55 ± 0.1a	37.86 ± 0.04a	1.7 ± 0.07c	47.55
2	53%	60 ± 3.05a	70.46 ± 0.1f	10.46 ± 0.1e	3 ± 0.1a	16.04
3	93%	56.75 ± 0.15a	76.79 ± 0.03d	20.04 ± 0.14d	2.8 ± 0.05ab	30.01
4	53%	55.67 ± 0.21a	77.16 ± 0.04c	22.49 ± 0.03c	2.5 ± 0.02b	33.86
5	100%	53.67 ± 1.4a	77.86 ± 0.01b	24.51 ± 0.07b	2.4 ± 0.02b	37.26
6	93%	55 ± 1.15a	75.23 ± 0.04e	20.23 ± 0.12d	2.4 ± 0.2b	31.07

Data represented as Mean ± SE, FCR^*: Feed Conversion Ratio. RGR^**: Relative Growth Rate. Means within the same column of different letters are significantly different at (P < .05).

able 4 Results of differential leukocyte count among different groups at the end of the experiment.

Group	N	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
1	7	31 ± 2.8a	58 ± 10.3 a	4 ± 0.57 a	4 ± 1.15 a	3 ± 0.57 a
2	7	17 ± 0.57c	72 ± 4.04 a	5 ± 0.57 a	3 ± 0.57 a	3 ± 1.15 a
3	7	22 ± 0.57 bc	68.3 ± 1.6 a	5 ± 0.57 a	3 ± 0.57 a	2 ± 0.57 a
4	7	27 ± 1.7 ab	63 ± 2.8 a	4.6 ± 0.33 a	2.6 ± 0.33 a	1 ± 0 a
5	7	30 ± 2.3 a	61 ± 2.3 a	4 ± 1.15 a	4 ± 0.57 a	2 ± 0.57 a
6	7	24 ± 4.04 abc	63 ± 2.3 a	6 ± 1.15 a	3 ± 0.57 a	4 ± 1.7 a

Data represented as Mean ± SE. Means within the same column of different letters are significantly different at (P < .05).

able 5 Results of serum total protein, albumin, globulin and albumin/globulin ratio among different groups at the end of the experiment.

Group	N	Total protein (g/dL)	Albumin (g/dL)	Globulin (g/dL)	A/G ratio
1	7	5.60 ± 0.29a	2.11 ± 0.63 a	3.500 ± 0.2 ab	0.58 ± 0.15
2	7	3.38 ± 0.03b	1.21 ± 0.06b	2.17 ± 0.01b	0.55 ± 0.03
3	7	3.730 ± 0.6bc	1.23 ± 0.07b	2.500 ± 0.17 ab	0.49 ± 0.03
4	7	4.4 ± 0.4abc	1.35 ± 0.03 ab	3.023 ± 0.07 ab	0.46 ± 0.003
5	7	5.130 ± 0.5ab	1.36 ± 0.03 ab	3.77 ± 0.076 a	0.38 ± 0.008
6	7	3.500 ± 0.c	1.26 ± 0.06 ab	2.24 ± 0.18b	0.62 ± 0.05

Data represented as Mean ± SE. Means within the same column of different letters are significantly different at (P < .05).

able 6 Effect of different treatments on serum AST, ALT, urea and creatinine of Oreochromis niloticus at the end of the experiment.

Group	Parameters
	AST (uL/mL)	ALT (uL/mL)	Urea (mg/dL)	Creatinine (mg/dL)
1	23 ± 2.3b	4 ± 1b	6.3 ± 2.3b	0.35 ± 0.07c
2	58.5 ± 20.4a	10 ± 2 a	11.3 ± 1.8a	0.88 ± 0.07a
3	49.5 ± 1.4 ab	8 ± 4ab	6.8 ± 1.4b	0.47 ± 0.09bc
4	46 ± 2.3 ab	8 ± 1 ab	6.85 ± 1.05b	0.44 ± 0.07bc
5	31 ± 0.57ab	7.9 ± 0.12 ab	6.55 ± 0.75b	0.43 ± 0.08bc
6	57 ± 5.7a	8 ± 3 ab	8.95 ± 0.45ab	0.58 ± 0.14b

Data represented as Mean ± SE. Means within the same column of different letters are significantly different at (P < .05).

ig. 2a Negative control group, kidney showing normal histological structure. H&E. ×200.

ig. 2b Negative control group, liver showing normal lobular architecture with normal vacuolation of fat and glycogen. H&E. ×200.

ig. 3a Ochratoxin positive control group, kidney showing marked vacuolar degeneration of the renal tubular epithelium. H&E. ×200.

ig. 3b Ochratoxin positive control group, kidney showing focal interstitial mononuclear cell infiltrations predominantly macrophages and lymphocytes. H&E. ×200.

ig. 3c Ochratoxin positive control group, kidney showing wide dilatation of renal blood vessels. H&E. ×200.

ig. 3d Ochratoxin positive control group, kidney showing massive tubular necrosis. H&E. ×200.

ig. 3e Ochratoxin positive control group, kidney showing focal vacuolation and necrosis of the renal tubules and cystic tubular dilatation. H&E. ×200.

ig. 3f Ochratoxin positive control group, kidney showing cystic tubular dilatation, focal mononuclear cells infiltrations, increase of glomerular space and shrinkage of the glomerular tufts. H&E. ×200.

ig. 4a Ochratoxin positive control group, liver showing dilatation of hepatic blood vessels and vacuolation of the hepatocytes. H&E. ×200.

ig. 4b Ochratoxin positive control group, liver showing hydropic degeneration of the hepatocytes, rarefaction, increased eosinophilia of the cytoplasm and pyknosis of the nuclei. H&E. ×200.

ig.4c Ochratoxin positive control group, liver showing necrosis of the hepatocytes. H&E. ×200.

ig. 4d Ochratoxin positive control group, liver showing area of necrosis of the hepatocytes infiltrated with mononuclear cells. H&E. ×200.

ig. 5a Garlen Extra4 treated group (Group 3), kidney showing vacuolation of the renal tubular epithelium as well as mononuclear cell infiltrations. H&E. ×200.

ig. 5b Garlen Extra4 treated group (Group 3), liver showing hydropic degeneration of the hepatocytes. H&E. ×200.

ig. 6a Group 4 (Nigella sativa treated group), kidney showing marked vacuolation of renal tubular epithelium and dilatation of renal blood vessels. H&E. ×200.

ig. 6b Group 4 (Nigella sativa treated group), liver showing congestion and vacuolation of the hepatocytes. H&E. ×200.

ig. 7a Group 5 (Garlen Extra4 and Nigella sativa treated group), kidney showing mild vacuolation of the renal tubular epithelium. H&E. ×200.

ig. 7b Group 5 (Garlen Extra4 and Nigella sativa treated group), liver showing hydropic degeneration and congestion. H&E. ×200.

ig. 8a Group 6 (Fero Bind Pro treated group), kidney showing marked tubular vacuolation and focal necrosis of renal tubular epithelium. H&E. ×200.

ig. 8b Group 6 (Fero Bind Pro treated group), liver showing marked dilatation of hepatic blood vessels and vacuolation of the hepatocytes. H&E. ×200.