The effects of dietary bee pollen on lipid peroxidation and fatty acids composition of Japanese quails (Coturnix coturnix japonica) meat under different stocking densities

ABSTRACT

The objective of this study was to investigate the effects of supplementation of bee pollen (1 g/kg) to the basal diet on lipid peroxidation and the fatty acids composition of Japanese quails under different stocking densities. Hundred and sixty Japanese quails were divided into 3 treatment groups each with four replicates consisting of 8 birds in control group and 16 birds in the other groups. Experimental groups were arranged as Control (160cm²/quail without supplementation), high stocking density (HSD) (80 cm²/quail without supplementation), and POL (80 cm²/quail and 1g/kg bee pollen). Plasma Malondialdehyde level in the HSD group was significantly higher than those of Control and POL groups (P < .01). The catalase activity in blood of Control and POL groups was significantly higher than those of the HSD group(P < .01). In the HSD group the total polyunsaturated fatty acid (PUFA) ratio was lowered in all tissues. Increased PUFA ratios were observed in breast muscle and kidney tissue (P < .001), leg muscle (P < .05) and liver (P < .01) tissue in pollen-supplemented quails compared with the other groups. The n–6 PUFA ratio of breast muscle, kidney tissue (P < .001) and liver tissue (P < .01) was higher in the POL group than the other groups. As a result, the present study suggests that bee pollen supplementation to 1 g/kg diet had potential protective activity on lipid peroxidation and tissue fatty acid composition of quails reared under HSD. In all groups, the bee pollen consumption increased PUFA levels in tissues, especially n-6 PUFA.

KEYWORDS:

• Quail
• stocking density
• lipid peroxidation
• fatty acids
• bee pollen

Introduction

Poultry meat is consumed because of possessing extremely low lipid content and high concentrations of polyunsaturated fatty acid (PUFA)) (Yan & Kim 2013. Fatty acid composition of the diet is a significant factor due to its affecting the fatty acid composition of the skeletal muscle of poultry that consume these diets (Dalkilic et al. 2009). Natural supplements have been widely used in the general population for health and well-being, and potential therapy on certain disease and conditions (Kennedy 2005; Wang et al. 2007). In this regard, bee pollen is widely used as a natural supplement, as it contains most of the essential elements needed for growth and developments in human beings and animals (Bell et al. 1983; Orzaez et al. 2002; Hascik et al. 2012). Bee pollen contains essential amino acids, proteins, unsaturated fatty acids, anthocyanins, ferulic acids, pantothenic acid, vitamins, minerals such as iron, manganese and zinc, and trace elements (Campos et al. 2003; Mutsauers et al. 2005). Also it contains significant amounts of flavonoids, carotenoids and phytosterols (Campos et al. 2003). Flavonoids are polyphenolic substances. Polyphenols have strong antioxidant capacity. They are capable of scavenging free radical due to metal chelation properties (Campos et al. 2003; Abdella et al. 2009; Teixeria et al. 2010). Stocking density has been defined as the number of birds being reared in a given housing area (Thaxton et al. 2006). It is an important environmental factor just like temperature, humidity, etc. (Faitarone et al. 2005; Celik et al. 2010). Stocking density may enhance oxidative destruction and causes MDA (Malondialdehyde) generation (Simsek et al. 2009). It has been reported in some studies (Seven et al. 2011; Khalil & El-Sheikh 2012) that pollen has antioxidant effects against lipid peroxidation caused by free radicals.

The aim of the present study was to determine the effects of bee pollen on lipid peroxidation and fatty acids composition of Japanese quails (Coturnix coturnix japonica) reared under different stocking densities.

Material and methods

Animals and sampling

One hundred and sixty 3-day-old Japanese quails (Coturnix coturnix japonica) were allocated to 3 treatment groups each with 4 replicates consisting of 8 animals in the control and 16 animals in the other treatment groups. All of birds were placed into cages with an internal dimension of 40×32 cm. While quails in control group were reared at 160 cm²/quail, both high stocking density (HSD) and bee pollen groups were reared at 80 cm²/quail. The quails were fed with starter diets for 21 days (Table 1). At 22nd day of experiment, experimental groups were randomly divided into three groups and fed with finisher diets for a period of 21 days. The pollen was obtained from a commercial firm in Turkey. The experimental groups were designed as no supplementing to finisher diet under optimum stocking density (Control-C); no supplementing to finisher diet under HSD; supplementing of 1g/kg bee pollen to finisher diet under HSD (POL). Corn and soybean meal-based feeds were formulated to be isonitrogenic and isoenergic according to the NRC (1994) recommendation. Pollen was homogeneously mixed carefully to the basal diet. The ingredients and chemical composition of the diets are presented in Table 1. Chemical composition of feed ingredients (dry matter, crude protein, aether extract and ash) as dried samples was analysed using AOAC (1995) procedures and crude fibre was determined by the methods of Crampton and Maynard (1970). The fatty acid compositions of diet and bee pollen were presented in Table 2 and Table 3, respectively.

Table 1. Ingredient composition and chemical composition of the experimental diets (g/kg).

Ingredients	Starter	Finisher
Maize	539.8	621.3
Soybean meal (%48)	266.3	–
Full fat soybean	124.8	276.5
Poultry meal	25	60
Soybean oil	9.4	14.3
Limestone	11.9	2.9
Bone meal	–	16.5
Dicalcium phosphate	11.9	–
Salt	3	2.1
DL-methionin	2.8	1.3
Vitamin–mineral premix^a	3.5	3.5
Lysine	1.6	1.6
Calculated content
ME, kcal/kg^c	3300	3030
Dry matter, %^b	89.40	90.50
Crude protein (CP), %^b	23.62	19.25
Aether extract, %^b	6.1	8.9
Ash, %^b	5.1	4.3
Crude cellulose, %^b	5.6	5.0
Calcium^c	10	10.2
Total phosphorus^c	5.5	5.6

^aVitamin–mineral premix provided per kilogram of diet: retinol, 4,5 mg; cholecalciferol 0.125 mg; Alpha-tocopherol, 100 mg; menadione, 4 mg; thiamine, 3 mg; riboflavin, 8 mg; niacin, 60 mg; pyridoxine, 5 mg; Ca-Dpantothenate, 18 mg; folic acid, 2 mg; D-biotin, 0.20 mg; Mn, 100 mg; Zn, 80 mg; Fe, 80 mg; Cu, 8 mg; Co, 8mg; Se, 0.3 mg; Iodine, 1 mg, Mo,1 mg; choline chloride, 500 mg.

^bAnalysed (AOAC, 1995).

^cBased on NRC (1994) feed composition tables.

Table 2. Fatty acid profiles of diet and feed additives (% fatty acids).

	Starter	Finisher	Pollen
SFA	22.58	21.94	33.88
MUFA	30.54	21.93	13.48
PUFA	46.36	55.81	52.38

Note: SFA, saturated fatty acid; MUFA, monounsaturated fatty acid

Table 3. The fatty acid composition of pollen (% fatty acids).

Lipid numbers	Name	(%)
C12:0	Lauric acid	1.00
C14:0	Myristic acid	2.86
C16:0	Palmitic acid	23.98
C18:0	Stearic acid	3.57
C22:0	Behenic acid	2.47
	ΣSFA	33.88
C161n7	Palmitoleic acid	1.28
C181n9	Oleic acid	8.42
C201n9	Eeicosenoic acid	0.89
C221n9	Erucic acid	2.89
	ΣMUFA	13.48
C182n6c	Linoleic acid	18.93
C182n6t	Linoleic acid	0.32
C183n6	Gamma linolenic acid	1.09
C183n3	Alfa-linolenic acid	31.53
C202n6	Eicosadienoic acid	0.16
C205n3	Eicosapentaenoic acid	0.35
	ΣPUFA	52.38

All groups were kept under the same environmental conditions. Photoperiods of 23 h/day during 3–42 days of periods were maintained. The chicks were raised in a temperature-controlled room at 36°C for the first week. Thereafter, the temperature was reduced by 3 degrees each week to a minimum of 22±2°C. Average relative humidity was determined as 65%. Feed and water were given ad libitum. On 42nd day of the study 6 quails from each group with their body weight near the group average were weighed and slaughtered. The blood, breast, leg muscle, liver and kidney samples were collected. Blood samples were taken into tubes containing anticoagulant (2% sodium oxalate). The samples were centrifuged at 200 g for 5 min at +4°C; then the plasma was removed immediately and stored at −20°C until analysed. The tissue samples were stored at −20°C until analyses. They were thawed at +4°C and homogenized just before analysis.

MDA concentration in plasma, the end product of lipid peroxidation was measured according to the method of Satoh (1978). MDA concentration in plasma was expressed as nmol/ml. Catalase (CAT) activity was estimated by measuring the breakdown of H₂O₂ at 240 nm according to the method of Aebi, (1984). CAT activity in blood was expressed as kat/hHb.

Extraction of lipids from the tissue (kidney, liver, breast and leg) samples, experimental diets (starter and finisher) and pollen samples were performed according to the method by Hara and Radin (1978). According to this method, 1 g of liver, kidney and muscle (leg and breast) tissue and diet pollen specimens were broken down in a Micra–D.8 homogeniser for 1 min with 10 ml hexane isopropanol. The tissue homogenate was centrifuged at 4500 rpm for 10 min to seperate the tissue pellet. Fatty acid methyl esters were prepared according to the methylation method by Christie (1992). Fatty acids in the lipid extract were converted to methyl esters by 2% sulphuric acid (v/v) in methanol. Fatty acids were analysed in SHIMADZU GC 17 ver. 3 gas chromatography. A 25 cm-long MACHERY-NAGEL (Germany) capillary colon with a 0.25 µm internal diameter and a PERMABOND 25 µ film thickness was used in analyses.

Statistical analysis

After normality test, Shapiro-Wilk, the data were subjected to analysis of variance, significant differences were further subjected to Duncan's multiple range test (SPSS 2006). The results were considered as significant when P values were lower than 0.05.

Results and discussion

The PUFA ratio of pollen was higher than those of monounsaturated fatty acid (MUFA) and saturated fatty acid (SFA) (Table 3). Dietary bee pollen supplementation showed positive effects on stress, reducing lipid peroxidation due to antioxidant substances in its structure (Campos et al. 2003) and increasing the PUFA level in some tissues due to high PUFA content in its structure (Table 3). The dietary PUFA supplementation can exert benefits on antioxidative status, and alter the animal performance and health (Chen et al. 2012). These results were considered to be due to the antioxidative capacity (Eraslan et al. 2009) and suitable fatty acids profile of bee pollen (Chen et al. 2012).

Poultry may be need antioxidant effective dietary supplements or enzymes under stress while they only need to creals-soybean based diets under normal conditions (Tatli Seven et al. 2008; Kianfar et al. 2013; Makhdum et al. 2013). Microclimate conditions around the birds deteriorate when the number of birds per unit of space increases. They move less and exhibit decreased walking ability, resulting in locomotion problems and difficulty accessing feeders and drinkers. The birds have to spend more time standing than resting, which results in social anarchy for resting birds. All of these problems cause physical and physiological stress to the birds (Agarwal et al. 2003; Seven et al. 2014b). The use of different management practices, equipment and several dietary alternatives has been recommended to alleviate such an environmental stress (Seven et al. 2014b).

The increasing MDA levels in plasma are an indicator of oxidative stress. We determined that the MDA levels in plasma were significantly higher in the HSD group when compared to the Control and POL groups (P < .01) (Table 4). In the present study, HSD decreased blood CAT (P < .01). POL supplementation was significantly decreased MDA, while increased CAT (Table 4). Living organisms are able to adapt to oxidative stress by inducing the synthesis of antioxidant enzymes and damage removal/repair enzymes (Tatli Seven et al. 2009). In the present study, increasing of plasma MDA level and decreasing of blood antioxidant enzymes activities (such as CAT) in the HSD group may be evidence of oxidative stress that caused of HSD (Tatli Seven et al. 2009; Eraslan et al. 2009; Seven et al. 2014a). POL supplementation partially improves oxidative stress caused by HSD. The present study results were in agreement with results of a study related to effect on MDA activity of POL supplementation (Eraslan et al. 2009). Bee products contain numerous phenolic compounds (Khalil & El-Sheikh 2012; Feas et al. 2012). As a bee product pollen is a good source of healty compounds such as phenolics. Bee pollen might be suggested for useful properties in the prevention of disease in which free radicals occur (Feas et al. 2012). Besides, it has been reported that bee pollen has metal chelation properties that react with free radicals (Abdella et al. 2009). Their function is to hunt down free radicals and neutralize them. In so doing, they not only prevent free radicals from causing damage but also repair any damages (Tatlı Seven et al. 2012). In a recent study, Yildiz et al. (2013) clearly showed that chesnut bee pollen had protective effects against carbon tetrachloride-induced hepatic damage in rats. Chestnut bee pollen showed this effect by protecting the hepatocytes from the oxidative stress and promoted the healing of the liver damage induced by carbon tetrachloride toxicity. They analysed antioxidant properties of chesnut bee pollen before the treatment and determined that the chestnut bee pollen possessed many phenolic compounds, which are the factors of high antioxidant properties. It was reported that bee pollen supplementation decreases markers of oxidative stress, enhancing the antioxidant system of animals under stress (Khalil & El-Sheikh 2012).

Table 4. Effects of bee pollen on MDA (µg/ml homojenat) and CAT (kat/hHb) activities.

	Control	Stocking density (HSD)	Pollen (POL)	P
MDA	2.66 ± 0.38^c	4.43 ± 0.56^a	3.38 ± 0.35^b	**
CAT	4.12 ± 0.68^a	2.60 ± 0.52^b	3.46 ± 0.54^a	**

^a,b,cMean values with different superscripts within a row differ significantly.

**P < .01.

Lipids mobilize from adipose tissue for increasing energy needs during stress, especially unsaturated fatty acids (Mumma et al. 2006). The total PUFA ratio of all tissues was found to be lower in the HSD group (Table 5). Especially PUFA ratios of breast and kidney tissue were lower than those in other groups (P < .001). Lipid peroxidation causes reduction of PUFA in the phospholipid fraction of the tissues (Morrissey et al. 1994). Results of Mumma et al. (2006) are consistent with our results, as given in Table 5. In the present study, the total SFA ratio of leg muscle was higher in the HSD group (P < .01). Simsek et al. (2009) reported that a lower stocking density decreased the fat ratio of meat and increased the protein ratio, total PUFA and n-3, n-6 fatty acid ratio. In accordance with these reports, in the present study it was found that the PUFA ratios of breast muscle and kidney tissue of the control group were higher (P < .001) than those of other groups. Also, total SFA ratio of kidney (P < .05) and leg muscle (P < .01) tissues was higher in the HSD group than Control in accordance with Simsek et al. (2009)'s study.

Table 5. Effects of bee pollen on fatty acid profile of muscle and inner organ tissues under HSD conditions in Japanese quails (% fatty acids).

Characteristics (%)	Control	Stocking density (HSD)	Pollen (POL)	P (Statistical significance)
Breast muscle (M. pectoralisprofundus)
PUFA	46.56 ± 0.62^ab	42.95 ± 0.90^b	49.46 ± 0.35^a	***
MUFA	21.02 ± 1.10^b	26.07 ± 1.09^a	8.95 ± 1.28^b	**
SFA	31.61 ± 0.71	30.03 ± 0.44	30.88 ± 0.98	–
N3-PUFA	8.38 ± 0.51	8.81 ± 0.32	8.58 ± 0.49	–
N6-PUFA	38.18 ± 0.52^b	34.09 ± 0.65^c	40.87 ± 0.38^a	***
Leg muscle (M. gastrocnemius)
PUFA	46.51 ± 1.68^a	44.21 ± 1.05^b	45.24 ± 3.72^ab	*
MUFA	24.96 ± 1.85	24.12 ± 1.28	24.15 ± 4.52	–
SFA	28.14 ± 1.38^b	31.04 ± 1.06^a	30.58 ± 1.32^a	**
N3-PUFA	6.70 ± 0.68	6.51 ± 0.34	6.29 ± 0.57	–
N6-PUFA	39.80 ± 2.48	37.70 ± 0.87	38.95 ± 2.15	–
Liver
PUFA	40.00 ± 2.71^ab	35.08 ± 4.00^b	47.39 ± 1.61^a	**
MUFA	26.93 ± 2.20^a	31.97 ± 3.67^a	20.78 ± 1.41^b	*
SFA	30.66 ± 1.13	31.60 ± 1.26	31.79 ± 1.45	–
N3-PUFA	4.79 ± 0.78	5.23 ± 0.82	6.85 ± 1.74	–
N6-PUFA	35.21 ± 2.91^a	29.84 ± 3.19^b	40.54 ± 1.20^a	**
Kidney
PUFA	43.14 ± 0.82^a	34.11 ± 2.32^b	45.57 ± 1.02^a	***
MUFA	17.40 ± 1.32	19.82 ± 1.83	15.17 ± 1.27	–
SFA	36.93 ± 1.25^b	43.74 ± 3.0^a	35.27 ± 0.94^b	*
N3-PUFA	1.73 ± 0.29	1.41 ± 0.48	1.43 ± 0.56

^a,b,cMean values with different superscripts with in a row differ significantly.

*P < .05, **P < .01, ***P < .001.

As given in Table 5, PUFA ratios of breast muscle were higher in the POL than in Control and HSD groups (P < .001). The PUFA ratio of kidney tissue was significantly higher in the POL than that of HSD (P<.001). This result was found similar to the Control group. In the present study, it was determined that bee pollen had positive effects and increased n-6 PUFA levels. N-6 PUFA ratios of breast muscle, kidney (P<.001) and liver (P<.01) tissues were higher in the POL than those in HSD. Total MUFA ratios of liver tissue were higher in the HSD group than the pollen group (P < .01).

Conclusion

In summary, the present study suggested that bee pollen supplementation to 1 g/kg diet had potential protective activity on lipid peroxidation and tissue fatty acid composition of quails reared under HSD. Bee pollen supplementation increased PUFA ratios in the tissues especially n-6 PUFA. Further studies are needed to comprehensively assess biological activity, quality and effects on toxicity.

Disclosure statement

No potential conflict of interest was reported by the authors.