A completely randomized design performed to evaluate the effect of inclusion of egg powder in pre-started diet (1-7 day age) on immune system of male broiler chickens. Experimental pre-starter diets formulated to have 0, 20, 40 or 60 g/kg egg powder. The relative weight of lymphoid organs (spleen and bursa) reduced, in days 7 and 42 of age, by inclusion of egg powder in pre-starter period (P ≤0.05). The highest antibody titer against Newcastle disease vaccine (NDV) and Infectious bronchitis virus (IBV) in days 7 and 42 of age was for chickens fed diet containing 60 g/kg egg powder in pre-starter diet (P ≤0.05). At day 7 of age, birds fed diet containing egg powder had higher antibody titer against Infectious bursal disease virus (IB) compared with the control group (P ≤0.05), but at day 42 of age, there was no significant difference between treatments (P >0.05). Although parameters like titer against sheep red blood cells and immune response against dinitrochlorobenzene were not significantly affected (P >0.05), but results showed that exact mean values for these criteria increased by consuming egg powder. Generally in accordance with results obtained in this study, consuming egg powder in pre-starter diet significantly affected immune system of the male broiler chickens positively.
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Key words: egg powder; pre-starter; immune response; antibody titer; broiler.
Newly hatched chicken has an incapable digestive system to process conventional feed ingredients. This incapability comes from both morphologic and also enzymatic aspect. In this period chickens has high levels of nutritional requirements to support growth and development of gastrointestinal system and also immune system to warranty the health and ultimate production of the bird (Noy and Sklan, 2001).
An egg during the incubation period provides all nutritional requirements of embryo, and there is a very strong relation between egg weight and subsequent hatched chicken weight. The most important point after immediate access to feed is nutritional value of feed ingredient for chickens in early time after hatch. The importance of early feeding of newly hatched chicks on their performance and health was demonstrated by several studies (Jin et al., 1998; Noy and Sklan, 1999; Madsen et al., 2004). Most of feed ingredients like grains and plant origin proteins which used in diet of chickens had low utilization for chickens at early times after hatch. The main reason of this poor ability is the immature gastrointestinal tract of chickens (Madsen et al., 2004). In addition to that disability, new hatched chicken have a high level of nutrient requirement because of its speed up growth of gastrointestinal system and other organs related to nutrition and immunity. This high level of growth in gastrointestinal system needs more digestible feed ingredients to support consequent growth in all parts of chicken body like muscles, skeleton and immune system.
Also there are important interactions and antagonisms between nutrition and immunity that affect productivity of poultry. Sever deficiency of most nutrients impairs the immune response and increase susceptibility of birds to infectious diseases. Sever nutrient deficiencies are particularly deleterious to the immune system when they occur early in life during the development of the primary lymphoid organs and the maturation of immune system (Kirk, 1997).
Thus, these facts obliged nutritionists to pay special attention to nutritional requirements of chickens in early time after hatch. One of the best choices to support nutritional requirements of newly hatched chickens is application of pre-starter diets. These pre-starter diets must provide highly digestible ingredient for the young chickens. Nowadays a lot of researches have been conducted to evaluate new alternatives for feeding young chicks. Because before hatch all nutritional requirements of embryo comes from egg reserves, egg and egg by-products could be used as a good choice to feed newly hatched chickens. Egg by-products include out comes from breaking facilities and unsellable eggs. Sparks (2006) reported that these by-products are rich in fat, maternal antibodies, protein and bioactive nutrients. Other beneficial effects of consuming egg by-product showed in studies conducted by El-Deek and Al-Harthi (2009) and El-Deek et al. (2011). They concluded that dried egg powder may be used as an alternative to antibiotics due to its high content of antimicrobial proteins and eggs antibodies and could be fed to large flocks, without negative effects on performance of chickens. In the literature, there was no information regarding the antibody titers of broiler chicks fed egg powder during the pre-starter period. Therefore, the objective of this study was to evaluate egg powder as an alternative in pre-starter diet of broiler chicken and its influence on immune system of chickens.
2. Material and Methods
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2.1. Birds, diets and experimental design
Sample of egg powder was obtained from Golpoodr Company (Gorgan, Iran). Egg powder had been prepared from both egg white and egg yolk which spray-dried at 55 °C. Chemical composition (crude protein, ether extract, crude fiber, Phosphorous and Calcium) of egg powder and other feed ingredients which used in this experiment were analyzed using AOAC (1990). Amino acid content of egg powder was analyzed using near infrared reflectance spectroscopy method (FieldSpec® Pro, Analytical Spectral Devices, Boulder, CO, USA). Nutrient content of egg powder are shown in Table 1. Experimental period was divided in four phases: pre-starter (from hatch to day 7), starter (from day 8 to day 10), grower (from day 11 to day 24) and finisher period (from day 25 to day 42). Experimental treatments were four levels of egg powder inclusion in pre-starter diet of male broiler chicken. Egg powder levels in pre-starter diet of birds were 0, 20, 40 or 60 g/kg of diet. In the starter period (8-10 day) all birds consumed the diet which control group consumed in the pre-starter period. Experimental diets were formulated according to ROSS 308 management manual and all of them had equal amino acids and metabolisable energy (Table 2). Each treatment had four replicates with 20 male broiler chickens. Feed and water was ad libitum all over the experiment. Birds housed over wired floor pens under ambient temperature and were provided with continuous light.
2.2. Internal organ size
At 7 and 42 d of age, four birds in each pen were individually weighed, slaughtered, and eviscerated. Subsequently, internal organs include spleen and bursa were manually removed and individually weighed. Internal organ weights presented as percent of body weight.
2.3. Immune System Parameters
2.3.1. Humoral immune responses
Haemagglutination inhibition test against Newcastle disease virus: Newcastle disease virus (NDV) vaccine (killed vaccine, Formosa Biomedical Inc., Taipei, Taiwan) administered at days 1 and 35 after hatch and peripheral blood was collected from the wing vein in the following week post-challenge each time.
Sheep Red Blood Cell: at 36 day of age sheep red blood cell (SRBC) suspension (5% v/v in sterile PBS) was injected in breast muscle of 4 birds per treatment. Total antibody titers to SRBC were determined by agglutination according to Van der Zijpp and Leenstra (1980) in serum from the birds. Therefore, 7 days after immunization (day 42) antibody titers against SRBC were measured and expressed as the log2 of the reciprocal of the highest serum dilution giving complete agglutination.
Infectious bronchitis virus titer: Chickens vaccinated against infectious bronchitis virus (IBV-Mass type, H120 strain, Merial Company)at first day after hatch. Blood samples were collected by wing venipuncture at days 7 and 42 after hatch. The harvested sera were heat inactivated at 56 °C for 30 min. A commercial ELISA kit (Flockchek) from IDEXX Laboratories, Inc. USA, was used to determine IBV antibodies in sera of chickens, according to the manufacturer's instruction.
Infectious bursal diseases virus titer: Infectious bursal diseases virus (IBD, Polymed, Tabic) vaccinations in drinking water were administered to 16-day-old chickens. Blood samples were collected at 7 and 42 days of age to evaluate the IBD-ELISA antibody titers according the commercial kit manual (Biocheck® Inc., CA, USA).
2.3.2. Cell mediated immune responses
Contact sensitivity to 2,4 dinitro-1-chlorobenzene: Cell mediated immunity test was eriformed using2,4 dinitro-1-chlorobenzene (DNCB) (Ajax Chemicals, Sydney). It was dissolved in a mixture of acetone and olive oil (4:1) (v/v) for each time of use. The test was performed as described by Chauhan and Verma (1983). Briefly, at day 35 birds (4 birds per replicate) were sensitized by painting DNCB, in 10 μl of solvent, on to a marked ring, 1 cm diameter, on the skin of the breast. To facilitate drying of the solvent, warm air was blown on to sensitizing sites.
The response to DNCB was elicited a week after sensitization by applying 50 μg of DNCB, in 10 μl of solvent, on to the skin of the pelvic regions at day 42; 10 μl of solvent were applied on the opposite side of the chickens to act as a negative control. Skin thickness at these two sites was measured with a micrometer after 24 h and differences stated as response to DNCB.
Injection of phytohaemagglutinin-P: The in vivo cell mediated immune (CMI) response to phytohaemagglutinin-P (PHA-P) was studied at 42 d of age. The thickness of comb measured using micrometer (4 birds per replicate). The comb was injected with 100 μg of PHA-P (Sigma, St. Louis, MO) suspended in 0.1 ml of phosphate buffer saline (PBS). The comb swelling was measured after 24 h of injection. The cell mediated response was determined by subtracting the comb thickness of first measurement from the second (Corrier and De Loach, 1990).
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2.4. Statistical analysis
Collected data were subjected to variance analysis using a completely randomized design. Basic statistics and variance analysis were performed to test the significance between the treatments. To evaluate the differences between the control and samples, significant means were further analyzed using Duncan's multiple range tests. Data were analyzed by ANOVA using PROC GLM (SAS Institute, Cary, NC). In all cases, P-values ≤ 0.05 were considered significant.
3.1. Internal organs
As presented in Table 3, application of egg powder in pre-starter diet of male broilers affected relative weight of immune related organs. According to these results increasing levels of egg powder in pre-starter diet of birds reduced relative weight of bursa at 7 and 42 d of age. Chickens which consumed the control diet had highest bursal weight and the group of chickens consumed 40 and 60 g/kg egg powder in their pre-starter diet had lowest bursal weight at both ages (P ≤0.05). Spleen showed the same trend as bursa and increase in level of egg powder inclusion in pre-starter diet of broilers reduced the relative weight of spleen in both 7 and 42 d of age (P ≤0.05). There was not any difference between different levels of egg powder inclusion and only control group differed from all experimental groups.
3.2. Immune System Parameters
According to results presented in Table 4, most of parameters related to immune system significantly affected by consuming egg powder in their pre-starter diet. Mean values presented for DNCB and SRBC test showed that by increasing the level of egg powder in pre-starter diet, immunity response against DNCB and SRBC were increased but this increase was not significant (P >0.05). Change in skin thickness response to DNCB in control group was 1.32 mm and for birds consumed 60 g/kg egg powders in their pre-starter diet it was 2.24 mm. The same trend has seen for SRBC test.
According to mean values of haemagglutination inhibition (HI) titers of NDV presented in Table 4, in both 7 and 42 day HI test was affected from consuming egg powder in pre-starter diet. Increase in inclusion level of egg powder in pre-starter diet of male broiler chickens significantly (P ≤0.05) enhanced immune response to NDV. Although anti-NDV titer reduced a little in 42 day age in comparison to 7 day of age, differences between experimental treatments were clearer in day 42 of age.
Immune system response to PHA injection to comb of chickens measured as increase in thickness of comb. Although there was a little difference between control and experimental treatments in case of response to PHA injection, the differences were not significant (P > 0.05) and there was almost no difference between chickens consumed diets with different levels of egg powder.
Antibody titer against IBDV measured in days 7 and 42. Level of antibody titer in serum of male broiler chicken was affected by level of egg powder in their pre-starter diet. Although IBD vaccine administered in day 16 after hatch, antibody titer at day 7 of age measured to determine the level of antibody in maternal flock. In day 7, antibody titer against IBD significantly affected by consuming egg powder in pre-starter diet and chickens consumed egg powder in their pre-starter diet had higher level of antibody titer against IBD in comparison with control group. Antibody titer against IBD in day 42 was higher than day 7, because of vaccination against IBD administered at day 16 after hatch. Although the level of antibody was higher in day 42 but there was not any difference between antibody titer of chickens consumed control or experimental diets (P >0.05).
Antibody titer against infectious bronchitis virus (IBV) was measured in days 7 and 42 of experiment. Infectious bronchitis vaccine administered at first day after hatch. Results presented in Table 4 showed that in both days antibody titer affected by consuming egg powder in pre-starter diet (P ≤0.05). At day 7 of experiment all chickens consumed egg powder in their pre-starter diet had higher levels of anti-IBV in their blood in comparison with control group. At day 42 of experiment only birds consumed 60 g/kg egg powder in pre-starter diet significantly (P ≤0.05) had higher level of antibody titer and other experimental groups had no difference with control group.
As bursa and spleen have direct relations with immune system, with regard to results presented in Table 3 application of egg powder in pre-starter diet of male broiler chickens significantly reduced the relative weight of these organs in both 7 and 42 d of age. According to different studies carried out on nutritional value and positive effects of egg on immune system, reduction in relative weight of organs related to immune system could be explained. Egg is rich in fat, high quality protein, maternal antibody and bioactive nutrients (Schaafsma et al., 2000; Anton et al., 2006; Sparks, 2006). Also in a study carried out by Burley and Vadehra (1989) positive effects of egg component on immune system reported by inhibition of haemagglutination by viruses. Because egg powder was highly digestible, birds could consume egg powder efficiently. Growth and development of gastrointestinal tract and immune system of these birds occurred in a good manner and so health of birds warranted and challenges like antigens were not affected immune system of chickens.
Results presented in Table 4 showed that many of immune system responses enhanced due to feeding egg powder in pre-starter diet. Effect of consuming egg powder in pre-starter diet on immune system responses was very sharp and clear. Looking for a good reason to explain these effects leads us to nutritional value of egg for chicken prior to hatch period. An egg during the incubation period provides all nutritional requirements of chicken embryo, and there is a very strong relation between egg weight and subsequent hatched chicken weight. According to studies (Sulistiyanto et al., 1999; Sklan et al., 2000) regardless of type of feed, presence of feed in early times after hatch had a great effect on health and production of chickens. By presence of feed at first birds could consume the antibodies absorbed in yolk sac to improve their immune system and protect the bird against undesirable antigens. And the second, presence of feed help the chickens to provide essential nutrients to speed up growth of internal organs, especially digestive tract, to support the final growth and production of the bird (Noy and Sklan, 1999).
The most important point after immediate access to feed is nutritional value of feed ingredient for chickens in early time after hatch. The importance of early feeding of newly hatched chicks on their performance and health was adequately demonstrated by several studies (Jin et al., 1998; Noy and Sklan, 1999; Madsen et al., 2004). Most of feed ingredients like grains and plant origin proteins that used in diet of chickens had low utilization for chickens at early times after hatch. The main reason of this poor ability is the immature gastrointestinal tract of chickens (Madsen et al., 2004). In addition to that disability, new hatched chicken have a high level of nutrient requirement because of its speed up growth of gastrointestinal system and other organs related to nutrition and immunity.
Many studies documented the nutritional value of egg and egg products. Eggs are known to be rich in fat, maternal antibodies, protein, bioactive nutrients and lysozyme (Schaafsma et al., 2000; Anton et al., 2006; Sparks, 2006). Also positive effects of egg component on immune system and inhibition of haemagglutination by viruses showed in a study by Burley and Vadehra (1989).
Eggs contain antibodies against all challenges to which hens has been exposed and this is attractive since they do not cause inflammatory responses they can provide protection against enteric infections (Burley and Vadehra, 1989; Davis and Reeves, 2002). Also egg yolk is a reservoir of antibodies with many proven uses and applications (Li-Chan, 1998; Anton et al., 2006; Sparks, 2006).
With regard to facts and discussions presented above, all positive effects of consuming egg powder in pre-starter diet of chickens and all results obtained in this study could be explained. Egg powder was an ingredient with high levels of utilization for chickens early times after hatch. High levels of digestibility and utilization of egg powder, relieved incapability of gastrointestinal system of newly hatched chickens and by supplying required nutrients growth and development of gastrointestinal system and other organs of young chicken could be warranted. In addition to high utilization, egg powder prepared in low temperature (55° C) and because of that most of bioactive components exist in egg saved their functionality and impressed health of birds and enhanced chicken immune system responses. By supplying all requirements of young chickens and elevating immune system responses, egg powder consumption in pre-starter diet supported and warranted the ultimate health and production of birds.
Results presented in this study indicated that egg powder is an ingredient of choice for inclusion in pre-starter diet of fast growing broiler chickens. Immune modulating effects of egg powder in the pre-starter diets were observed in this trial and it seems consuming egg powder in pre-starter diet of broiler chickens could enhance immune response of birds.