MATERIALS AND METHODS

A total of 420 one-day-old Hubbard broiler chicks were randomly divided into four equal groups, each group contained three replicates of 35 chicks. The chicks were grown in a separate floor pens with wood shavings and exposed to 24 h constant light. Feed and water were supplied ad libitum. The ingredients and chemical composition of the basal diet (starter from 1-21 days of age, grower from 22-35 days of age and finisher from 36-42 days of age) are presented in Table 1. All experimental birds were reared as per the recommendations of the National Research Council (NRC., 1994).

Bee-pollen was obtained from the apiary of the Honeybee Research Section, Plant Protection Research Institute, Sakha, Kafrelsheikh, Egypt. Bee-pollen were homogenized to be a fine powder and packed in polyamide-polyethylene bags and stored at -16°C until use. Treatments were prepared by mixing the bee-pollen into the basal diet at the rate of 0% (control), 0.2% (T₁), 0.4% (T₂) or 0.6% (T₃).

Table 1:	Composition and calculated analysis of the experimental diets
*Each 3 kg of premix contained: vit. A 12000 IU, vit. D 2200IU, vit. E 10 mg, vit. K3 2000 mg, vit. B1 1000 mg, vit. B2 5000 mg, vit. B6 1500 mg, vit. B12 10 mg, pantothenic acid 10 mg, niacin 30 mg, folic acid 1000 mg, biotin 50 mg, choline chloride 300 mg, manganese 60 mg, zinc 50 mg, copper 10 mg, Iron 30 mg, Iodine 1000 mg, selenium 100 mg, cobalt 100 mg and CaCo3 to 3 g

Moisture, crude protein, ether extract, ash and crude fiber of bee-pollen were determined according to the method of AOAC (1994). Mineral elements were determined according to the method of AOAC (1994) by the atomic absorption spectrophotometer. The amino acids profile was determined according to the method of Duranti and Cerlletti (1979), using a Beckman Amino Acid Analyzer. The fatty acids were analysed using gas chromatography according to Bonvehi and Jorda (1997).

Individual body weight, feed consumption and feed conversion ratio were recorded weekly. Mortality of birds was recorded at the day when it occurred. At six weeks of age, six birds from each treatment were randomly chosen, weighed and then slaughtered. Blood samples were collected at the time of slaughtering from each bird. After complete bleeding and feather removal, carcass, liver, gizzard, heart, spleen, bursa and thymus were weighed and their weight was recorded as percentage of body weight.

Blood samples were divided into two equal parts. The first part of blood was collected with heparin as anticoagulant to be used for estimation of hematological parameters testes such as: Red Blood Cells (RBCs) and White Blood Cells (WBCs) according to Natt and Herrick (1952), Packed Cells Volume (PCV) and heamoglobin (Hb) according to Schalm et al. (1975) and Hesser (1960), respectively. Counts of Hetrophils (H), Lymphocytes (L) and H/L ratio were measured according to Shen and Patterson (1983). The second part of each blood sample was centrifuged at 4000 rpm for 15 min to separate blood serum. The obtained serum was kept frozen at -18°C until analysed. Serum samples were used for biochemical analysis such as: Total protein, albumin, cholesterol, triglyceride, uric acid, creatinine, Gamma Pyruvic Transferees (GPT) and Glutamic Oxalocetic Transferees (GOT) were estimated by calorimetric methods using commercial kits supplied by Biomericux (Poains, France). Serum globulin was calculated by the difference between serum total protein and albumin, since the fibrinogen usually comprises a negligible fraction (Sturkie, 1976).

Statistical analysis: Data were subjected to the one-way analysis of variance (ANOVA) using SAS^® software computer program (SAS., 2003). Treatment means were compared using Duncan's Multiple Range Test (Duncan, 1955).

RESULTS

Bee-pollen composition: Bee-pollen has 62.82% carbohydrates, 19.23% proteins, 4.09% lipids, 2.28% ash and 0.90% Fiber. The most prevalent amino acids in bee-pollen were glutamic acid, glycine, aspartic acid, alanine, leucine, valine and lysine. Palmitic (C16:0), linolenic (C18:3), oleic (C18:1) and linoleic (C18:2) were the most predominant fatty acids (Table 2).

Body weight or body weight gain: The live body weight at one day of age for all treatments was nearly similar. It was found that, there were significant (p<0.01) differences in average values of either live body weight or body weight gain between broiler chicks fed basal diet and those having bee-pollen in their diets at all experimental periods. At the end of the experimental period, the highest values (g) of live body weight (2312.33) or body weight gain (2270.91) were obtained from chicks fed diet contained 0.6% bee-pollen (Table 3).

Table 2:	Chemical composition of bee-pollen

Table 3:	Effect of different levels of bee-pollen on average weekly live body weight (g) and body weight gain (g) of broilers chicks
Means of each column followed by the same letter are not significantly different at the 5% level according to Duncan's Multiple Range Test, ** and NS indicate p<0.01 and not significant, respectively

Table 4:	Effect of different levels of bee-pollen on feed consumption (g) and Feed conversion ratio of broiler chicks
Means of each column followed by the same letter are not significantly different at the 5% level according to Duncan's Multiple Range Test, **’*and NS indicate p<0.01, 0.05 and not significant, respectively

Feed consumption and conversion: The feed consumption was decreased significantly (p<0.01) in birds received bee-pollen diets compared with the control group during the all experimental periods. Birds in control group consumed feed more than birds fed on bee-pollen (0.2, 0.4 and 0.6%) in their diets (3893.49 g bird^–1 vs. 3769.42, 3789.87 and 3812.91 g bird^–1), respectively (Table 4). Feed conversion was improved in parallel with the increasing of the bee-pollen supplementation level.

Carcass: The relative weight of carcass increased significantly (p<0.01) in groups fed bee-pollen diets compared to the control group. The highest values (%) of relative weight of gizzard (2.21) and liver (2.07) were obtained from chicks fed on the basal diet while the highest values (%) of relative weight of heart (0.47), thymus (0.40), bursa (0.11) and spleen (0.15) were obtained from chicks fed on bee-pollen at 0.6% level (Table 5).

Hematological parameters and blood constituents: The hematological parameters and blood constituents of broilers chicks were affected significantly by dietary bee-pollen supplementation (Table 6 and 7). The values of PCV, Hb, RBCs, WBCs, H and L in chicks fed on the basal diet were lower than that of chicks fed bee-pollen at any level in their diets while the highest value of H/L was obtained in birds fed control diet. The highest values (g/100 mL) of total protein (5.30), albumin (2.90), globulin (2.40) and the lowest serum uric acid (2.18), creatinine (0.70), triglycerides (176.90), cholesterol (196.00), SGOT (63.0 UL^–1) and SGPT (67.30 UL^–1) levels were obtained from chicks fed diet supplemented with 0.6% bee-pollen.

Mortality: The mortality percentage (2.16%) of broiler chicks fed different levels of bee-pollen during the whole experimental period was almost similar the control group, therefore results were not tabulated.

DISCUSSION

Broiler receiving 0.6% bee-pollen (T₃) in their diet had significantly (p<0.01) highest body weight and body weight gains during the experimental periods. The birds received 0.2, 0.4 or 0.6% bee-pollen showed an increasing in body weight gain by 8.14, 8.86 and 11.65% compared to the control birds, respectively. These positive improvements could be due to the nutritive value of the bee-pollen as a rich source of protein (19.32%), essential amino acids (e.g., Leucine and lysine), fat (4.09%), unsaturated fatty acids (e.g., oleic, linoleic and linolenic), carbohydrates (62.82%) and minerals (e.g., Na, K, Ca, Mg, P, Zn, Mn, Fe and Cu) (Table 2).

Table 5:	Effect of different levels of bee-pollen on relative weight of carcass and organs
Means of each column followed by the same letter are not significantly different at the 5% level according to Duncan's Multiple Range Test. **, * and NS indicate p<0.01, 0.05 and not significant, respectively

Table 6:	Effect of different levels of bee-pollen on hematological parameters of broilers chicks at 6 weeks of age
PVC: Packed cell volume, Hb: Heamoglobin, RBC: Red blood cells, WBC: White blood cells, H: Hetrophils, L: Lymphocytes and H/L: Hetrophils ratio lymphocytes. Means of each column followed by the same letter are not significantly different at the 5% level according to Duncan's Multiple Range Test. ** and NS indicate p<0.01 and not significant, respectively

Also bee-pollen increased the intestinal absorptive capacity through the longer and thicker villi and it stimulates the digestive and absorptive functions of broilers (Wang et al., 2007) in association with a significant improvement in body weight gain due to higher protein anabolism (Attia et al., 2011a) and bee-pollen has many of enzyme which support the digestive system (Hascik et al., 2012). These results are in agreement with the findings of Wang et al. (2007), Han et al. (2010), Hascik et al. (2012), Attia et al. (2014a) and Coskun et al. (2014) for broiler chicks and El-Hanoun et al. (2007), Shewika (2009), Attia et al. (2011b), El-Neney and El-Kholy (2014) for rabbit, Wang and Cheng (2005) for pigs, Zhang et al. (2010) for calves and Abbass et al. (2012) for Nile tilapia.

In comparison to the control group, feed consumption was decreased significantly (p<0.01) in chicks supplemented with bee-pollen diets during the all experimental periods. At all investigated periods, birds received bee-pollen (0.2, 0.4 or 0.6%) showed an improvement in feed conversion ratio compared to the control group. The best feed conversion ratio was obtained in the 0.6% bee-pollen supplementation (T₃) for the entire experimental period. The current results are in contrast with those observed by Attia et al. (2014a) and Abou El-Naga (2014) for broiler chicks, Hedia et al. (2007), Attia et al. (2011a) and Attia et al. (2014b) for rabbit, Wang and Cheng (2005) for pigs, Zhang et al. (2010) for calves and Abbass et al. (2012) for fish. The improvement of feed conversion and lower feed intake in the present study might be due to the vitamins, amino acids, hormones and minerals contained in bee-pollen, as well as enzymes and coenzymes added by bees during formation of pellets, which are necessary for good digestion and cell growth (Wang et al., 2007). On contrary, Han et al. (2010) and Hascik et al. (2012) reported that bee-pollen increased feed intake of broiler chickens.

In comparison to the control group, feeding broiler chicks on bee-pollen diets (0.2, 0.4 and 0.6%) showed an increase in the relative weight of carcass. The increase in the weight of carcass might be due to the increasing in the growth performance and digestibility. The relative weight of gizzard and liver were insignificantly decreased in broiler received bee-pollen compared with the control. There were no significant differences in the relative weight of heart between the broilers that fed 0.4 or 0.6% bee-pollen while it was significantly (p<0.05) decreased in the birds received 0.2% bee-pollen compared to the control group. These results are agreed with Hascik et al. (2012) who found that the weights of carcass and giblets were insignificantly increased in the male chickens fed bee-pollen (400 mg kg^–1) compared with the control group. In addition, Attia et al. (2014a) reported that the supplementation of bee-pollen in diets of broiler increased the dressing percentage while the percentage of the gizzard, liver and heart were un-affected compared with the control treatment. El-Neney and El-Kholy (2014) found that the addition of bee-pollen to diets of rabbits increased the carcass traits. On the other hand, Song et al. (2005) and Wang et al. (2007) noticed that the liver and pancreas weights of broilers fed with bee-pollen were higher than those weights of the control group. However, addition of bee-pollen did not influence the carcass yield of the birds (Ke et al., 2009) and the rabbits (Dias et al., 2013; Attia et al., 2014b).

The relative weights of lymphoid organs were significantly increased in broiler received diet contained bee-pollen, except of spleen weight that was insignificantly increased. In general the highest relative weights of thymus, bursa and spleen were found in the birds gave 0.6% bee-pollen.

Table 7:	Effect of different levels of bee-pollen on blood constituents of broilers chicks at 6 weeks of age
Means of each column followed by the same letter are not significantly different at the 5% level according to Duncan's Multiple Range Test. **, * and indicate p<0.01 and 0.05, respectively

These results indicated that supplement bee-pollen in chicks diet promoted the growth of thymus, bursa and spleen that resulted in positively influence the immunity of broiler. Bee-pollen can also improve the cell immune response, the antibody production speed and reinforce the immunological system (Song et al., 2005). Bee-pollen enhanced lymphocytes percentage which leads to produce more T-cells in rabbit (El-Neney and El-Kholy, 2014). T-cells undergo maturation in the thymus gland and play a major role in cell-mediated immunity (Stephen, 2007). These positive effects may be due to the bee-pollen which has nucleic acids that stimulate the natural killer cells and T-lymphocyte activities such as: Vitamin A fand E and microelements such as Fe, Se, Zn and Mn, which promote the proliferation and differentiation of immune system cells (Wang et al., 2005). These results are in agreement with Wang et al. (2005), who reported that the absolute weight of spleen increased in broiler fed 1.5% bee-pollen. Also bee-pollen could boost the early development of thymus and bursa, retard the bursa degeneration and promote the immune response of spleen in chickens. The spleen weight of broiler feed diets contained bee-pollen (0.5, 1 and 1.5%) was higher from the 7th up to 42nd day of age (Cheng, 2009). In another experiment, Dias et al. (2013), Attia et al. (2014b) and El-Neney and El-Kholy (2014) noted that spleen percentage was higher in rabbits treated with bee-pollen.

The Packed Cells Volume (PCV), Hemoglobin Concentration (Hb) and Red Blood Cells (RBCs) in birds fed bee-pollen diet (0.2, 0.4 or 0.6%) were significantly higher than these values of the control group. Bee-pollen was found to cause increases in hemoglobin and serum iron (Xie et al., 1994), the RBC count, Hb and PCV values (Attia et al., 2014a) in broiler chickens, hemoglobin, red blood cells count and hematocrite in rabbits (El-Neney and El-Kholy, 2014) and the number of red blood cells in human (Iversen et al., 1997). The highest value of them were detected in chicks fed 0.6% bee-pollen. These results indicated that feeding on bee-pollen increased Hb and RBCs lead to help increase energy levels. Since the hemoglobin in red blood cells that carry oxygen for energy metabolism, this may explain the relationship between bee-pollen and energy. The positive impact on PCV and Hb and RBCs values could be due to minerals such as Fe and Cu contained in bee-pollen (Table 2) and vitamins such as folic acid and vitamin C. These minerals and vitamins have a role in red blood cell formation and maturation (El-Wafa et al., 2002) which in turn caused an increase in hemoglobin and hematocrite, so there are positive relationships between RBC’s and hemoglobin and hematocrite (Sturkie, 1986).

White Blood Cells (WBCs) were found to be significantly increased in birds fed bee-pollen diet contained 0.2, 0.4 or 0.6% when compared with the control group. The increasing of white blood cells in a certain level is a good indicator of increasing the immunity efficiency. This result confirmed by the findings of Attia et al. (2014a) for broiler chicks and Hedia et al. (2007), Attia et al. (2014b) and El-Neney and El-Kholy (2014) for rabbits.

Hetrophils (H) and Lymphocytes (L) were increased significantly (p<0.01) in broiler fed 0.4 and 0.6% bee-pollen compared to the control group. The lymphocyte is considered the main type of white blood corpuscles and a good indicator of the increase in immune efficiency (Swiderek et al., 2006). Bee-pollen can enhance immunity (Song et al., 2005; Liu et al., 2010; Attia et al., 2011a, b; Popiela-Pleban et al., 2012). The improvements in the immune response could be due to the presence of minerals, antioxidant which present in flavonoids and vitamins in bee-pollen which have a role in enhancing immune system. The relationship between antioxidant and immune cells may be explained with the fact that the immune cell function is linked to the release of Reactive Oxygen Species (ROS) even though this ROS is involved in the microbicidal and cytotxic activity of immune cells, excessive amount of it is harmful for immune cells as it attack cellular components and lead to cell damage or death. This ROS can be scavenged by antioxidants leading to improve immune cell functions (De la Fuente and Victor, 2000; Victor et al., 2003; Hughes, 1999; Barillari et al., 2005). Antioxidants are very important to immune defense and animal health, consequently, to their production capacity (Chew, 1995). Natural antioxidant was found to stimulate cell-mediated immunity of rabbits (El-Kholy et al., 2008). Similar results were obtained by El-Neney and El-Kholy (2014) who noticed significant increase in lymphocytes type in rabbit received 200, 300 and 400 mg bee-pollen/kg body weight daily compared to the control.

Broiler fed diets containing bee-pollen (0.2, 0.4 or 0.6%) had insignificantly lower H/L ratio than those fed the control diet. The H/L ratio was found to be associated with increased levels of stress in the bird (Vijayan and Rema, 1997). The H/L ratio is a good measure of the chicken's perception in its environment and increasing H/L ratio indicated that the birds were under acute stress (Al-Daraji et al., 2002). The H/L ratio has been found as general biomarker relevant to immune function in poultry (Shini, 2003).

Serum biochemical parameters have been used as indicators of the nutritional and physiological status of birds. Serum total protein, albumen and globulin increased significantly (p<0.01) in birds received 0.2, 0.4 or 0.6% bee pollen compared with the control group. The highest values of them were recorded in chicks fed diets containing 0.6% bee-pollen. The increase in the plasma protein and albumin may be due to the high concentrations of protein and amino acids contained bee-pollen (Bell et al., 1983). Also bee-pollen improved liver function. It is well known that the changing in the plasma albumin level reflects the change in liver function, where the liver is the site of albumin synthesis while the globulin formed by lymphatic tissues (Jones and Bark, 1979). These results are in agreement with the findings of Attia et al. (2014a), who noted that bee pollen has a positive effect on serum total protein, albumen and globulin in broiler chicks. Also, Hedia et al. (2007), Attia et al. (2011a, b) and El-Neney and El-Kholy (2014) observed the same trend in rabbit.

Serum uric acid level showed significant (p<0.001) decrease in broiler received 0.2, 0.4 or 0.6% bee-pollen compared with the control. The levels of creatinine were similar in broiler received 0.2, 0.4% bee-pollen and control diet while birds fed diet containing 0.6% bee-pollen recorded significantly decreased in the creatinine level. These results showed that the bee-pollen dietary improved kidney health in terms of filtration rate. Significant increases in serum uric acid and creatinine levels are indicative of nephrotoxicity in broiler chickens (Huff et al., 1988). These results are agreement with the findings of Nagyova et al. (1994), Hu et al. (2003) and Attia et al. (2014a), who reported that bee-pollen reduced urea-N and creatinine concentrations in broiler chickens fed diet supplemented with bee-pollen compared with the control treatment. Also, Hedia et al. (2007), Attia et al. (2011a, b, 2014b) and El-Neney and El-Kholy (2014) found the same results in rabbit.

The results showed that there was a significantly (p<0.001) decrease in average values for serum triglycerides and cholesterol levels with increasing the duration of adding bee-pollen to the broiler diet. The decreasing in serum triglycerides and cholesterol values may be due to unsaturated acids; oleic, linoleic and linolenic (14.20,10.14 and 16.64% of fatty acids, respectively ) in bee-pollen (Table 2) that inhibits accumulation of lipid peroxidation product. The decrease in plasma lipids and cholesterol could be due to phospholipids and PUFA particularly linolenic fatty acid which represented by 1.19% in bee-pollen (Xu et al., 2009). The dietary monounsaturated fatty acids (e.g., oleic acid) were very effective in lowering blood cholesterol concentration and may be important in preventing coronary heart disease (Hornstra, 1980). Data obtained herein are in agreement with those obtained by Attia et al. (2014a), who noticed that bee-pollen can decrease the level of triglycerides and cholesterol in broiler chicks. Also, Hu et al. (2003) found the same results in rats.

Concerning liver function, the Serum Glutamic Oxaloacetic Transaminase (SGOT) and the Serum Glutamic Pyruvic Transaminase (SGPT) decreased significantly (p<0.001) in birds fed bee-pollen diets (0.2, 0.4 or 0.6 %) compared to the control group. These findings suggested that bee-pollen improved liver function and reduced liver damage. The protective effect of bee-pollen upon liver could be due to antioxidant contents of some flavonoids such as quercerin and rutin which play a role as antioxidant against oxidative material which caused damage to liver (El-Neney and El-Kholy, 2014). According to Attia et al. (2014a) the level of SGOT was reduced because of the administration of bee-pollen in broiler^’s diet. On other animals, Wojcicki et al. (1985) found that the protective effect of pollen extracts against allyl alcohol damage of the liver in mice reduced SGOT, SGPT and alkaline phosphatase activities. Supplementation of fish diet with honey bee-pollen (2.5%) decreased the serum GPT level, contrary to the serum GOT level when compared to the control group (Abbass et al., 2012). Also, Hedia et al. (2007), Attia et al. (2011a, b, 2014b) and El-Neney and El-Kholy (2014) found that supplementation of bee-pollen to the diet of the rabbit caused significant decrease in serum GOT and GPT compared to the control group.

CONCLUSION

It was concluded that bee-pollen is confirmed as an interesting resource, able to improve performance, carcass traits, immunity and blood parameters of broiler chicks. Standing on the present results, the best concentration of bee-pollen in broiler diets is 0.6% during starter, grower and finisher periods.