Effect of Bee Pollen on The Immunity and Tibia Characteristics in Broilers

This study was carried out to evaluate the effect of bee pollen (BP) levels on the IgG and IgM titers, weight of lymphoid organs, and on the tibia morphometric measures and mineralization in broilers at 21 and 42 days of age. Four hundred birds were used in an entirely randomized design with four treatments (0, 0.5, 1 and 1.5% of BP feed inclusion) and five replicates. At 21 and 42 days of rearing, blood samples were collected for IgG and IgM analysis, as well as lymphoid organs (bursa, thymus and spleen) and the tibiae. There was no effect (p>0.05) of the BP inclusion on IgG titers, bursa and spleen weights, tibia morphometric measures and mineralization at 21 and 42 days, IgM titer at 42 days or thymus weight at 21 days. However, IgM titers at 21 days and the thymus weight at 42 days linearly increased with BP dietary inclusion. It was concluded that up to 1.5% BP can be included in broiler feeds until 21 days of age to enhance bird immunity.


INTRODUCTION
Bee pollen is an agglomerate of flower pollen collected by bees mixed with nectar and secretions from the hypofaryngean glands (Carpes et al., 2008).Bee pollen sold in Brazil must present the following physicalchemical characteristics: maximum moisture 30%, maximum ashes 4%, minimum ether extract 1.8%, minimum crude protein 8%, total sugar from 14.5 to 55%, minimum crude fiber 2%, and pH from 4 to 6 (Brasil, 2001).
Bee pollen is rich in oligoelements, including Fe, Cu, Zn, Cr and Mn; it contains active substances such as catalase, peroxidase, superoxide dismutase and antioxidants.Its use has been associated with increased humoral immune cells and phagocytes number and activity, increased red blood cells number, accelerated antibody production and delayed disappearing antibody (Zuo & Xu, 2003).
In experiments with broilers, Wang et al. (2005a) included 1.5% bee pollen in diets for broilers from one to 42 days of age and reported that there was an increase of the absolute and relative weights of the spleen, bursa and thymus.However, Zhang et al. (2005) studied the oral administration for 30 days of bee pollen to rats, at doses of 165, 330 and 660 mg/kg body weight, and did not notice any differences in spleen weight, but the authors reported higher antibody titers, higher macrophage and natural-killer cells activity.
There is evidence that the bee pollen improves bone mineralization due its high vitamin D content, which increases calcium absorption.According to Hamamoto et al. (2006) and Yamaguchi et al. (2007b), bee pollen has stimulatory effects on bone formation and inhibitory effects on bone resorption.When evaluating the effect of the dietary inclusion of bee pollen extracts on femoral components, Yamaguchi et al. (2006) verified that there was an increase in calcium and DNA content, due the cellular proliferation, and of alkaline phosphatase in rats' femur with the administration of 5 and 10 mg/100 g body weight.
This study was carried out to evaluate the effects of different bee pollen dietary inclusion levels on IgG and IgM titers, lymphoid organs weight, and tibial morphometric measures and mineralization in broilers at 21 and 42 days of age.

MATERIAL AND METHODS
Four hundred one-day-old male (M) and female (F) chicks were used, with mean initial weight 52.44 ± 1.76 g.Birds were housed in a masonry shed divided into 21.85 m² experimental pens at a density of 10.8 birds/m 2 .
A completely randomized experimental design was applied, with four treatments of five replicates of 12 birds (M and F) per experimental unit.Treatments consisted of the inclusion of increasing ground bee pollen (BP) levels in the diet.The inclusion levels assessed were 0, 5, 10 and 15 g/kg.
Birds were fed a starter diet from one to 21 days of age and a grower diet from 22 to 42 days of age (Table 1).The experimental feeds were isonutritive and were formulated to meet broilers' requirements as recommended by Rostagno et al. (2005), except for crude protein (CP), apparent metabolizable energy (AME), calcium (Ca) and phosphorus (P) levels, which corresponded to 97% of the recommendations.Water and feed were supplied ad libitum throughout the experimental period.
On days 21 and 42 of the rearing period, two birds per pen were feed fasted for 12 hours and then weighed.Blood samples were collected by jugular vein puncture for IgG and IgM analysis using the turbidimetry and nephelometry methods, respectively.Birds were then sacrificed by cervical dislocation and their tibiae, bursa, thymus and spleen were collected.Lymphoid organs were weighed and their relative weights were determined as a function of live weight.

Effect of Bee Pollen on The Immunity and Tibia Characteristics in Broilers
Oliveira MC de, Silva DM da, Loch FC, Martins PC, Dias DMB, Simon GA Tibiae were cleaned of adherent soft tissues and the right tibiae were weighed and their length and diameter were weighed using a manual caliper.The weight/length index was also calculated by dividing bone weight, in mg, by its length, in mm.The left tibiae were evaluated for ashes, calcium and phosphorus content.
In order to determine ash content, tibiae were weighed before and after the drying in a forcedventilation oven at 55 o C for 48 hours.Subsequently, tibiae were defatted, ground, and analyzed using the methodology of Silva & Queiroz (2002).
The results were submitted to analyses of variance using the system of statistical and genetic analyses (SAEG, 2007) at 5% probability level and the significant results were submitted to the polynomial regression model to estimate the best BP level.

RESULTS AND DISCUSSION
There was no effect (p>0.05) of BP inclusion in the broilers diets on IgG titers at 21 and 42 days of age and on IgM titers at 42 days of age (Table 2); however, IgM titers at 21 days of age linearly increased (p<0.05) with the BP inclusion levels (Figure 1). 1 coefficient of variation, obtained with log-transformed means (log X).
2 Linear effect (Ŷ = 1.93 + 11.52x, r2 = 0.30) Chicks were vaccinated against infectious bursal disease at 7 and 21 days of age and against Newcastle disease at 14 days of age, via drinking water, which may explain the results above.IgM is the first antibody to appear in response to an infection or vaccination, reaching its highest concentration in 4 to 5 days, approximately, and then it diminishes after 10 or 12 days.As the immune response progresses, the cells that produce IgM, start producing IgG or IgA.On day 21, IgM titers were high in response to vaccination at 7 and 14 days of age (Di Fábio & Rossini, 2008).
When evaluating the inclusion of 1, 0.5 and 0.1% BP in the diets of broilers vaccinated against Newcastle disease (ND), Wang et al. (2005a) observed that the lowest antibody titers against ND were obtained in the control group, with no BP, indicating that BP improves the immune response to vaccines or challenges up to 28 days of age, but thereafter, antibody titers were similar in all treatments.Evaluating the same BP feed inclusion levels (1, 0.5, and 0.1%) in broilers vaccinated against ND, Wang et al. (2005b) also reported that there was no influence of dietary BP inclusion on the humoral response of birds at 42 days of age.
BP inclusion did not influence (p>0.05)absolute or relative weights of the spleen and bursa on days 21 and 42, and of thymus on day 21; however, there was a linear increase (p<0.01) in thymus weight determined when birds were 42 days old (Table 3). 2 Linear effect ( Ŷ = 11.96+ 2.12x, r 2 = 0.43).
BP is rich in many nutrients that stimulate immune cell proliferation and differentiation.According to Wang et al. (2005b), selenium (Se) increases thymus cortical thickness, vitamin C is important to maintain thymus reticular cells, polysaccharides in BP promote T lymphocytes production, cell proliferation and activity in the spleen, as well as in the thymus and bursa, which produce T and B lymphocytes, respectively.In addition, BP contains superoxide dismutase , an antioxidant enzyme that prevents cell senescence in these organs.All of these effects result in a better immune status in animals.

Table 2 -
IgG and IgM titers of broilers fed diets containing different bee pollen levels

Table 3 -
Weights of lymphoid organs of broilers fed diets containing different bee pollen levels 1 Coefficient of variation.