Hematology and Histopathology of Broiler Chickens Supplemented with Tuna Black Flour

ABSTRACT

The present study evaluated the effect of the sex and dietary intake of tuna black flour (TBF)on the hematology and histopathology of broiler chickens. A total of 200 sexed broilers (Ross 380) were distributed according to a completely randomized design into four treatments (25 males and 25 females/experimental group).Different levels of inclusion of TBF (0, 1, 2, and 3%) were offered as a source of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), for 42 d. At the end of the experiment, blood samples and tissues were collected. The hematocrit value (Hemat), total protein (PROT-T), hemoglobin concentration (Hemog), erythrocytes concentration (Erythro) and total count and differential of leucocytes (Leuco): lymphocytes, heterophils, monocytes, eosinophils, and basophils were determined. Statistical analysis of the data was performed using the Analysis of Variance test, Duncan’s multiple range test, and descriptive analyses (p<0.05). Samples of heart, liver, and bursa of Fabricius were processed with the routine histological technique. The sex had no significant effect on the hematology. The feeding additional with 3% TBF had lower level of Hemat value and PROT-T. The Leuco had lower value with 1 and 2% of TBF. The Monocytes showed low value with 1 and 2% of TBF, and the eosinophils had high level with 1% of TBF. No cardiac lesions were detected in the broiler chickens in all treatments, normal deposition of fat in the epicardium, endocardium and pericardium were seen in all treatments.

Keywords:
Broiler; Hematology; Histopathology; ω-3 polyunsaturated fatty acids; Tuna black flour

INTRODUCTION

Cardiovascular disease (CVD) is one of the leading causes of death in men and women of all ethnic groups. Consuming saturated dietary fats and ω-6 polyunsaturated fatty acids (ω-6 PUFAs) increases the risk of CVD, whereas consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) may reduce CVD incidence (González & Leeson, 2000; Zuidhof et al., 2009). In recent years, ω-3 PUFAs have received considerable attention in both human and animal nutrition. Dietary supplementation with fish oil (FO), which is rich in ω-3 PUFAs, is reported to be nutritional (Pilevar et al., 2011; Al-Khalifa et al., 2012; Ganesan et al., 2014). Diet manipulation by incorporating FO or oilseeds in the hen diet is the usual way of producing ω-3 PUFAs modified chicken eggs (González & Leeson, 2000; Castro, 2002; Cherian & Hayat, 2009; Khatibjoo et al., 2011; Koppenol et al., 2014), but in many countries the consumption of marine products is very low; hence the benefit that could be derived from a diet rich in ω-3 PUFAs does not reach the majority of a population. Fatty acid (FA) content of the meat chicken can be modified through nutrition. Currently, linseed oil and marine products are used commercially to achieve this effect (Zuidhof et al., 2009; Morales et al., 2013b; Morales et al., 2013a; Ribeiro et al., 2013; Martínez et al., 2016). In Mexico, black tuna meat is not used for human consumption but is considered a by-product destined for animal consumption in the form of flour. The aim of the present study was to evaluate the effect of the sex and dietary intake of tuna black flour (TBF) on the hematology and histopathology of broiler chickens.

MATERIALS AND METHODS

Animals and experimental design

Institutional and national guidelines for the care and use of animals were followed and all experimental procedures were approved by the Committee of Colima University ethical review.Two hundred sexed broilers: (100) 1 d old male (Ross 308) and (100) 1 d old females (Ross 308), were obtained from a local commercial hatchery. The birds were housed in floor pens of identical size (1×2 m) using wood shaving as litter. The room was thermostatically controlled to produce an initial temperature of 32 °C on day 1 and reduced in by 0-5 °C per d, reaching 21 °C. The birds were distributed according to a completely randomized design into four treatments (25 males and 25 females/experimental group). All data were analyzed as a 2 x 4 factorial arrangement, taking into account the incorporation of sex and TBF levels as the main factors.

Determination of fatty acids in tuna black flour (TBF)

The FA and fatty acids to methyl esters (FAME) analyses were carried out on a Varian CP3800 gas chromatograph (JVA; Analytical Ltd., Dublin, Ireland) equipped with a flame ionization detector (FID). The column was a CP FFAP CB capillary column (30 m × 250 μm i.d., 0.32-μm phase thickness; Agilent Technologies Ireland Ltd., Little Island, Cork, Ireland). The concentration of the main fatty acids in TBF are shown in table 1.

Table 1
Concentration of fatty acids in tuna black flour.

Total lipids, %	8.32 ± 0.01
Total saturated fatty acids (SFA), mg/100 g	1814.68
Main SFA
Myristic (C14:0)	137.25 ± 0.72
Palmitic (C16:0)	1120.78 ± 35.12
Stearic (C18:0)	356.40 ± 4.58
Total monounsaturated fatty acids (MUFAs), mg/100 g	1063.41
Main MUFAs
Palmitoleic (C16:1)	213.85 ± 8.09
Oleic acid (C18:1, ω-9)	708.40 ± 15.85
Total polyunsaturated fatty acids (PUFAs), mg/100 g	1579.80
Main PUFAs
Linoleic (C18:2, ω-6)	118.83 ± 3.50
α Linolenic (C18:3, ω-3)	33.72 ± 1.86
γ Linolenic (C18:3, ω-6)	11.44 ± 3.28
Arachidonic (C20:4)	86.26 ± 3.28
Eicosapentaenoic (C20:5, ω-3)	258.88 ± 14.15
Docosahexaenoic (C22:6, ω-3)	1039.07 ± 47.79

• Data expressed as mean ± standard deviation.

Dietary treatments

Different levels of inclusion of TBF (0, 1, 2, and 3%) were offered as a source of ω-3 PUFAs in commercial sorghum-soybean diets, for 42 d. All diets were isonitrogenous and isocaloric. Diets were formulated in three phases: i) starter (1-14 d), ii) grower (15-35 d), and iii) finisher (36-42 d), the diets formulations are shown in table 2.

Table 2
Composition and nutrient content of starter, grower and finisher experimental diets (% inclusion of raw materials)..

Ingredient	Starter diets (1-4 d)				Grower diets (15-35 d)				Finisher diets (36-42 d)
	Tuna black flour (TBF, %)
	0	1	2	3	0	1	2	3	0	1	2	3
Sorghum	65.16	66.56	67.96	69.36	71.80	73.21	74.45	76.01	74.49	75.44	76.32	77.04
Soybean meal	28.03	26.26	24.48	22.70	21.54	19.77	18.06	16.21	18.55	16.94	15.41	14.03
Calcium phosphate	2.60	2.31	2.01	1.72	2.33	2.03	1.74	1.45	2.19	1.90	1.60	1.31
Calcium carbonate	0.76	0.71	0.66	0.62	0.71	0.66	0.62	0.57	0.73	0.68	0.63	0.58
Oil	1.97	1.57	1.18	0.78	1.70	1.31	1.00	0.52	2.23	2.23	2.23	2.23
Lysine	0.43	1.47	0.48	0.49	0.43	0.44	0.45	0.46	0.36	0.36	0.37	0.37
Methionine	0.41	0.39	0.39	0.39	0.41	0.40	0.40	0.40	0.32	0.32	0.32	0.32
Salt	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35
Vitamin premix1	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Mineral premix2	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Coccidiostate	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
Pigment	0.00	0.00	0.00	0.00	0.45	0.45	0.45	0.45	0.50	0.50	0.50	0.50
Calculated Analysis
CP %	20	20	20	20	18	18	18	18	17	17	18	18
ME kcal/kg	3000	3000	3000	3000	3050	3050	3050	3050	3070	3084	3121	3127
Calcium %	1.00	1.00	1.00	1.00	0.90	0.90	0.90	0.90	0.85	0.85	0.85	0.85
Phosphorus%	0.50	0.50	0.50	0.50	0.45	0.45	0.45	0.45	0.42	0.42	0.42	0.42
Methionine + cysteine %	1.00	1.00	1.00	1.00	0.90	0.90	0.90	0.90	0.83	0.83	0.83	0.83
Lysine %	1.40	1.40	1.40	1.40	1.20	1.20	1.20	1.20	1.06	1.06	1.06	1.06

• ¹
  Vitamin premix supplied the following per kilogram of complete feed: vitamin A 12 000 IU, vitamin D3 2 500 IU, vitamin E 30 IU, vitamin k3 2 mg, thiamine 2.25 mg, riboflavin 7.5 mg, pyridoxine 3.5 mg, cobalamin 0.02 mg, niacin 45 mg, D-pantothenic acid, 12.5 mg, biotin 0.125 mg and folic acid 1.5 mg; ²Mineral premix supplied the following per kilogram of complete feed: zinc 50 mg, copper 12 mg, iodine 0.3 mg, cobalt, 0.2 mg, iron 100 mg, selenium 0.1 mg, and manganese 110 mg.

Analytical testing

The diets were fed in mash form, mixed in house, and were analyzed for gross energy by bomb calorimetry (Robbins & Firman, 2006). The crude protein (CP) was measured to the method 968.06 of Association of Official Analytical Chemists (AOAC, 2002) using the Nitrogen/Protein Determinator (FP-428; LECO Instruments Ltd., Mississauga, Ontario, Canada). Phosphorus and calcium content were measured to the method 985.01 (AOAC, 2002) using the Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). Methionine, cysteine, and lysine were measured by High-Performance Liquid Chromatography (HPLC).

Blood collection and determination of hematology parameters

All birds were sacrificed by exsanguination and necropsied immediately. Blood samples were collected from the jugular vein using 6 mL vacuum tubes with Ethylene Diamine Tetra Acetic Acid (EDTA) as anti-coagulant (BD Vacutainer 367863; Becton-Dickinson Co., Franklin Lakes, United States). The serum was separated by centrifuging at 1 500 x g for 15 min using a portable centrifuge (Porta-Spin C828; UNICO, Dayton, United States). The Hemat value was counted using standard Wintrobe method and haemocytometer. The PROT-T was measured by Biuret using UV/Vis spectrophotometer (Biochemistry Analyzer ES-218; KONTRO Lab., Guidonia, Italy). The Hemog concentration was analyzed by the HemoTest. The Erythro value was counted using Improved Neubauer, and total count and differential of Leuco were analyzed by blood smear and Giemsa dye.

Tissue processing and histological analysis

The portion of the heart, liver and bursa tissues were fixed in 10% neutral buffered formalin, they were trimmed, processed, (Pathology Laboratory of Colima University, Mexico), sectioned at approximately 5 μm, mounted on a glass slide, stained with hematoxylin and eosin. The evaluation of hematoxylin and eosin stained sections was conducted by a pathologist blinded to the treatments. The histological structures of the heart, liver, and bursa of Fabricius were observed using a light microscope under low (×10) and high (×40) magnification. Photographs were taken for better illustration of the results.

Statistical analysis

The comparison between groups was assessed by Analysis of Variance using (PROC ANOVA, SAS, System, v. 8.2, Cary, NC). Differences among treatments were examined by Duncan’s multiple range test. Significance (p-value) was evaluated at 0.05.

RESULTS

The effects of the sex and dietary intake of TBF on the hematology of broiler, are shown in table 3 and 4. The sex had no significant effect (p>0.05) on the Hemat value, PROT-T, Hemog, Erythro and total count and differential of Leuco. Dietary intake of TBF had no significant effect (p>0.05) on the Hemog, Erythro, lymphocytes, heterophils, and basophils.

Table 3
Effects of the sex and dietary intake of tuna black flour (TBF) on the hematology of broiler chickens, n= 200 sexed broilers (Ross 380).

Factor	Hemat1	PROT-T2	Hemog3	Erythro4	Leuco5
Sex
Female	32.27±3.56a	3.95±0.51a	11.07±3.25a	1.98±0.41a	1.78±0.61a
Male	32.22±2.64a	3.90±0.51a	11.34±3.03a	2.11±0.46a	1.81±0.32a
p 6	0.95	0.67	0.77	0.40	0.59
EEM7	1.77	0.71	1.77	0.66	0.38
Tuna black flour (TBF)
0%	33.10±1.96a,b	4.12± 0.04a	11.33±3.04a	2.06±0.60a	2.14±0.54a
1%	33.80±3.32a	4.04±0.63a,b	11.65±3.63a	2.21±0.50a	1.64±0.39b
2%	32.05±2.72a,b	3.90±0.04a,b	10.85±3.04a	1.93±0.30a	1.47±0.29b
3%	30.04±2.86b	3.64± 0.11b	10.98±3.11a	2.00±0.11a	2.00±0.29a
p 6	0.01	0.03	0.93	0.55	0.00
EEM7	1.69	0.62	1.79	0.66	0.63
Interaction
Female *0%TBF	32.30±1.78a,b	4.22±0.25a	12.72±3.95a	2.00±0.22a	2.27±0.71a
Male *0%TBF	34.00±1.87a	4.02±0.08a	9.95±0.70a	2.26±0.82a	2.01±0.32a,b
Female *1%TBF	35.40±3.57a	4.04±0.50a	12.69±4.31a	2.29±0.54a	1.45±0.35a,b
Male *1%TBF	32.20±2.38a,b	4.04±0.49a	10.61±2.90a	2.14±0.50a	1.82±0.37a,b
Female * 2%TBF	33.30±2.13a,b	4.04±0.25a	9.72±0.69a	1.82±0.37a	1.36±0.86b
Male *2%TBF	30.80±2.86a,b	3.76±0.46a	11.97±4.15a	2.05±0.17a	1.57±0.39a,b
Female * 3%TBF	28.18±2.33a,b	3.50±0.33a	9.13±1.47a	1.98±0.19a	2.43±0.77a
Male *3%TBF	31.90±3.04b	3.78±0.52a	12.83±3.33a	2.01±0.80a	1.83±0.47a,b
p 6	0.01	0.35	0.05	0.55	0.06
EEM7	1.60	0.62	1.74	0.67	0.61

• ¹
  Hematocrit value (%); ²total protein (g/dL); ³hemoglobin concentration (g/dL); ⁴erythrocytes concentration (x10¹²/L); ⁵leucocytes value (x10⁹/L); ⁶significance; ⁷standard error of measurements; data expressed as mean ± standard deviation; significant differences were obtained between groups indicated with different letters.

Table 4
Effects of the sex and dietary intake of tuna black flour (TBF) on the leucocytes of broiler chickens, n = 200 sexed broilers (Ross 380).

Factor	Lymphocytes	Heterophils	Monocytes	Eosinophils	Basophils
Sex
Female	64.25 ± 16.75a	26.80 ± 14.60a	2.70 ± 2.02a	3.05 ± 2.06a	3.25 ± 2.29a
Male	54.75 ± 17.66a	35.70 ± 14.58a	3.60 ± 2.56a	2.60 ± 2.21a	2.85 ± 2.43a
p 1	0.07	0.05	0.08	0.86	0.58
EEM2	4.14	3.81	1.51	1.45	0.52
Tuna black flour (TBF)
0%	52.10 ± 9.88a	36.80 ± 8.41ª	4.30 ± 2.26a	2.90 ± 0.73b	3.90 ± 1.10a
1%	53.30 ± 15.41a	33.00 ± 14.46ª	5.10 ± 1.96a	4.80 ± 2.14a	3.80 ± 1.87ª
2%	69.90 ± 10.08a	24.60 ± 10.04ª	1.30 ± 1.05b	2.30 ± 0.61b	2.80 ± 3.35ª
3%	62.70 ± 26.06a	30.60 ± 22.79ª	1.90 ± 1.44b	2.00 ± 0.52b	1.70 ± 2.05ª
p 1	0.06	0.30	0.00	0.00	0.13
EEM2	4.08	3.87	1.31	0.53	0.71
Interaction
Female *0%TBF	53.40 ± 2.40a	35.40 ± 3.84a	4.60 ± 2.40a,b	2.80 ± 0.83a	3.80 ± 1.09a
Male *0%TBF	50.80 ± 14.48a	38.20 ± 11.81a	4.00 ± 2.34a,b,c	3.00 ± 0.70a	4.00 ± 1.22a
Female *1%TBF	65.20 ± 6.72a	21.80 ± 5.31a	3.60 ± 0.89a,b,c	4.80 ± 1.92a	4.60 ± 2.07a
Male *1%TBF	41.40 ± 11.63a	44.20 ± 11.34a	6.60 ± 1.51a	4.80 ± 2.58a	3.00 ± 1.41a
Female * 2%TBF	72.80 ± 9.73a	20.00 ± 6.36a	1.20 ± 0.44c	2.60 ± 1.34a	3.40 ± 3.13a
Male *2%TBF	67.00 ± 10.63a	29.20 ± 11.56a	1.40 ± 1.51b,c	2.00 ± 1.50a	2.20 ± 3.83a
Female * 3%TBF	65.60 ± 30.76a	30.00 ± 27.12a	1.40 ± 1.51b,c	2.00 ± 2.91a	1.20 ± 1.30a
Male *3%TBF	59.80 ± 23.70a	31.20 ± 20.80a	2.40 ± 1.34b,c	2.00 ± 1.87a	2.20 ± 2.68a
p 1	0.46	0.35	0.06	0.96	0.56
EEM2	4.03	3.79	1.27	0.79	1.02

• ¹
  Significance; ²standard error of measurements; data expressed as mean ± standard deviation; significant differences were obtained between groups indicated with different letters.

The feeding with thea dditional3% TBF had lower level of Hemat value and PROT-T (p<0.05). The Leuco had lower concentration with 1 and 2% of TBF. The Monocytes showed low value with 1 and 2% of TBF, and the eosinophils had high level with 1% of TBF. The interaction between sex and TBF affected Hemat with the increased dietary intake of TBF level (table 3). The Leuco concentration increased in female with 1 and 2% of TBF and male with 0, 1, 2 and 3% of TBF. Thus, the sex with the increased dietary intake of TBF had lower value of monocytes. No significant gross abnormalities were noted in any of the birds examined. No cardiac lesions were detected in the broiler chickens in all treatments, normal deposition of fat in the epicardium, endocardium and pericardium were seen in all treatments. Liver with multifocal hepatitis in all broiler chickens and the bursa of Fabricius was not affected by the dietary intake of TBF (figure 1).

Figure 1
Effect of the dietary intake of tuna black flour (TBF) on the histopathology of heart, liver and bursa of Fabricius of broiler chickens.

DISCUSSION

The diets that contain ω-6 PUFAs, can promote many diseases, including CVD (Simopoulos, 2016). Conjugated linoleic acid (CLA) has been reported to reduce CVD(Shen et al., 2018).To enhance human consumption of CLA, several enriched foods, mostly animal products, are being produced either by adding ω-3 PUFAs directly during the finisher phase of rearing or by modifying animal diets. The ω-3 PUFAs contribute to increase the number of calories for broiler chickens. However, it has been shown that the inclusion of over 6% FO in poultry diets, can exert some negative effects, such as a compromised oxidative balance in live animals and a higher oxidative susceptibility of the derived meat (González et al., 2013; Leskovec et al., 2018).In the present study up to 3% of the dietary intake of TBF was supplied, as a source of ω-3 PUFAs. Blood biochemical parameters may provide useful information for the evaluation of the health status of broiler chickens and reflect many metabolic alterations of organs and tissues for example heart, liver and bursa of Fabricius (Kudair & Al-Hussary, 2010). Serum proteins are mainly synthesized in the liver, and among other functions, participate in cell coagulation, and the body defense against foreign agents (Melillo, 2013). According to Harr (2002), PROT-T values of broiler chickens tend to be lower than those of mammals, ranging from 2.5 to 4.5g/dL. The values found for PROT-T (table 2), are consistent with the reference values. The 3% of the dietary intake of TBF had lower Hemat value and PROT-T, this situation assumes a low level in globulin (GLOB) rejecting a process of hepatic inflammation. High levels of ω-3 PUFAs affect the immune function in broiler chickens by reducing lymphocyte proliferation, mainly monocytes (Wang et al., 2011). The monocytes transform into macrophages and are actively involved in phagocytosis. Therefore, a reduction of monocytes (table 3) also reduces phagocytosis (Al-Khalifa et al., 2012).

The tight link between hepatic metabolic, dietary intake of methionine in all isonitrogenous and isocaloric diets of the experiment, regulation of adiponectin receptor2 (AdipoR2), fattyacyl-coenzyme Aoxidase1(Acox1), and carnitine palmitoyltransferase 1 (Cpt1) seems to explain the absence of cardiac lesions in broiler chickens. Considering the above, we maintain that it is reasonable to add 3% of the TBF dietary intake to commercial chicken feed, making it a significant source of ω-3 PUFAs for consumers of chicken.

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Publication Dates

• Publication in this collection
  20 Dec 2019
• Date of issue
  2019

History

• Received
  14 July 2019
• Accepted
  08 Sept 2019