In-Ovo injection of melittin into Alexandria chicken eggs: a way for early immune acceleration

Abstract This study intended to assess the properties of in-ovo administration of Melittin (MLT) on hatchability, chick yield, hematology, immunological indices and relative organs weight of Alexandria chickens at hatch. A total of 600 eggs with an average weight of (45.12 g), were gathered and split into five groups: a non-injected group or negative control (NC), a saline injection group or positive control (PC), and three concentrations of MLT (5, 10 and 15 µg of MLT per egg, respectively). On day 18 of incubation, eggs from the injection groups were injected into the amniotic fluid from the large end with the in-ovo injection solutions (0.2 ml per egg). Results indicated that 10 µg MLT/egg positively affected the weight and yield of chicks. In addition, our findings indicated that the in-ovo administration with 10 or 15 µg MLT/egg was superior in most of the immunological indicators (spleen and bursa relative weights, immunoglobulins IgG and IgM, T cells and B cells). In conclusion, in order to improve the immune efficiency (early immune acceleration) of Alexandria chicks, which may contribute to offering a significant boost to their future performance, this study suggests injecting eggs with 5 or 10 µg MLT/egg.


Introduction
The stages of incubation and embryonic development contribute a lot to the success of poultry farming (1).The effects of the feeding schedules, in-ovo injection of nutrients and early feeding on the performance of young chicks have been researched throughout the past few years (2-5).The in-ovo injection procedure with different nutrients help in embryonic development and results in healthier chicks and a good hatchability percentage (3), which offers to maintain improvements in commercial poultry production efficiency (6).All over the world, the using of bee products (e.g.bee venom (BV), pollen, propolis and royal jelly) dates back a very long time for their medicinal properties (7-9).Apitherapy is a subspecialty of complementary medicine that makes use of honeybee products (10).At least 18 pharmacologically active components are present in BV.In this regard, peptides such as MLT, adolapin, apamin, and mastcell-degranulating peptides are among the active ingredients in BV (11)(12)(13)(14)(15). Natural BV substance describes strongly as anti-inflammatory, antibacterial and analgesic actions and contributes to the enhancement of immune responses (15), and has anti-cancer and anti-viral potential (9).On the other hand, BV given either by injection or via drinking water increased performance and enhanced antioxidant capacity in broilers and pigs (16).The administration of whole BV and some of its ingredients, particularly MLT, which has antimicrobial properties and nonsteroidal anti-inflammatory activities in small volumes with no side effects in broiler nutrition, has been the subject of various studies (16,17).
The MLT has a number of biological properties, including those that are anti-inflammatory, anti-bacterial, anti-fungal, and anti-tumor (18) and has been found to enhance hens and rabbits' oxidative stress and immunological system (19,20).In order to safeguard neurons, it also improved the harmony of oxidation and antioxidation in mice with cognitive impairment (21).Additionally, it can enhance the barrier function, intestinal antioxidant capacity, and laying performance in quail (22).In addition, it was revealed that MLT has been demonstrated to exert anti-cancer, anti-inflammatory, antidiabetic, antiinfective, and adjuvant properties in humans (23).
There are, however, relatively few investigations on MLT's impact on hatchability and hatching traits of chicks on the day of hatch.Therefore, this study is designed to value the effects of in-ovo administration of three levels of MLT (5, 10 and 15 lg/egg) on hatchability calculation, hatchling characters, immune indices and blood biochemical factors of chicks at the hatch of Alexandria breeder hens.

Ethics statement
The

Eggs and incubation
Alexandria chickens (a local strain) were obtained in 1958 by crossing between four strains of chickens (White Leghorn, Rhode Island Red, Plymouth Rock, and Fayoumi) (24).A total of 600 eggs with an average weight of (45.12 g ± 0.14), were collected from Alexandria breeder hens 38 weeks of age and a hatching rate of 83%.Eggs were transferred into an automatic incubator (Model S380, PTO Incubation System Co., Alexandria, Egypt) with optimal conditions (37.8 � C and 60% relative humidity).Before setting, eggs were divided into 5 experimental groups (each treatment has six replicates, with 20 eggs for each).The first group was not injected and served as negative control (NC); the second group was injected with 0.2 ml normal saline (NaCl 0.9%) and served as positive control (PC); while, the third, fourth and fifth groups were injected with 5, 10 and 15 mg of MLT/egg, respectively.On day 14 of embryonic development, all eggs were candled and the unfertilized and nonviable eggs were replaced with the same average weight.

The in-ovo injection solution
According to Elmalky et al. (17), the lyophilized MLT we employed in this investigation contains 70.76, 7.48, and 3.82% MLT, apamine, and phospholipase A2, respectively.It was separated from bee venom that was obtained from honeybees and collected by the electric shock method according to Mohanny (25).The MLT separation and purification process was performed according to the method of Schmidt et al. (26).Then, to obtain a solution of MLT, an appropriate weight was dissolved in the saline solution immediately before injection.The injection solution of MLT was prepared immediately before injection and gently warmed to reach the incubation temperature.

The in-ovo injection procedure
According to Hassan et al., (3), a mini grinder (Model CT13428, CROWN, China) was used to create a suitable hole on the broadside of the eggshell at day 18 of incubation.Eggs from all injection sets were injected (0.2 ml/each egg) into the amniotic fluid using a 21gauge needle, in accordance with Uni & Ferket's instructions (27).The injection site was sanitized with ethanol 70%.After injection, the area was sealed with a wax gun.Parallel to that, eggs of the NC group were taken out of the incubator for nearly 20 min to equalize the conditions for the injection process.Then, all eggs were moved to the hatcher (37.5 � C and 70 ± 3% relative humidity), in covered trays after the injection process.Throughout the incubation period, all eggs were exposed to common routine procedures.

Hatchability traits
At hatching, for each replicate/treatment, scientific hatchability percentages were calculated (number of hatched chicks/total fertile eggs � 100).Also, the chick yield was calculated (chick weight mean/egg weight mean � 100).Estimates of relative organ weights were expressed as a percentage of live body weight according to Attia et al..

Blood parameters
Six chicks were taken randomly from each treatment and slaughtered for blood sample collection via the jugular vein to record hematology and biochemical traits.Blood was taken in anticoagulant-free, dry, clean centrifuge tubes to separate the serum.Before conducting the chemical analyses, the clear samples of serum were meticulously collected, put in Eppendorf tubes, and stored at À 20 � C in a deep freezer.The hematological factors were evaluated in line with Schalm's recommendations (28).The assessments of white blood cell count (WBCs, 10 3 /mm 3 ) lymphocytes (LYM), monocytes (MON), heterophils (H), eosinophils (ESIN), red blood cells (RBCs), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in the complete blood samples, were analyzed using a Hema Screen18 automated hematology analyzer (Hospitex Diagnostics, Sesto Fiorentino, Italy).The pH value for chicken blood was estimated using a pH meter (model Cyber Scan 500) standardized with buffer solutions of 4.0 and 7.0 according to the method of AOAC (29).
Each animal received a separate, sterile tube for the analysis of serum metabolites.The serum was then separated by centrifuging the tubes for 15 minutes at 3500 rpm after they had coagulated at room temperature for 30 minutes.The serum samples were kept safe until exploration at À 20 � C. Spectrophotometric analysis of the serum's total protein, albumin, and glucose content was performed using commercial kits delivered from Biodiagnostic Co. (Giza, Egypt).Serum globulin level (g/dl) was calculated by the difference between total protein and albumin since the fibrinogen usually comprises a negligible fraction (30).The IgG, IgM, and IgA immunoglobulins were measured using the method of Akiba et al., (31).The percentage for differential lymphocytes (T cells and B cells) was calculated according to Schalm (32).According to Lucky's instructions, blood film was made (33).A dry, unfixed blood smear was stained for one minute after having ten drops of the May-Grunwald staining stock solution added to an equal quantity of distilled water.Without rinsing, the dye was decanted.The film was treated with diluted Giemsa's mixture (10 drops of the dye were then added to 10 ml of distilled water) as a counterstain.After 20 minutes, the film was washed in a water current and inspected using an oil immersion lens.Triiodothyronine (T 3 ) and thyroxine (T 4 ) levels in the blood were tested using a special kit for poultry according to (34).

Statistical analysis
Statistical Package for Social Sciences (SPSSV R ) software's GLM technique was used to examine data from all response variables (35).One-way ANOVA was used in CRD to evaluate all the data.According to Duncan, the substantial variations between treatment means were assessed (36).The statistical model that was utilized was as follows: where Y ij represents the measured number of the dependent variable, represents the overall mean, T i represents the effects of the treatments, and e ij represents experimental random error.
Relative change percentages were used to measure the proportional difference between MLT in-ovo injection treatments and NC values.

Hatchability traits and relative organs weight
The effects of in-ovo injection of MLT on hatchability, chick weight and yield traits of chicks at hatching are present in Table 1.The egg weights used in different studied groups (44.65 À 45.45 g) have insignificant differences.The injected median level (10 mg MLT/egg) has a significant positive effect on chick weight (36.75 g) among the MLT injected groups, however, it has an insignificant higher value compared to NC and PC groups in that respect (35.77 and 35.33 g, respectively).The corresponding values of chick yield percentages showed similar trends (80.85, 78.78 and 78.76%, respectively).On the other hand, the 5 and 10 mg MLT/egg groups showed highly significantly higher values of hatchability percentages compared to the result of the 15 mg MLT/egg group (92.50, 92.50 and 84.74%, respectively), however, they have an insignificant higher value compared to NC and PC groups in that respect (91.25 and 91.50%, respectively).
The effects of in-ovo injection of MLT on the relative organs weight of chicks at hatching are present in Table 2.The outcomes exhibited insignificant differences among all studied treatments in the relative heart (0.58 À 0.73%), liver (1.82 À 2.80%) and gizzard (4.25 À 5.46%) weights.However, the injected median level has significantly higher relative intestine weight (4.59%) among the NC and other MLT injected groups, however, it has an insignificant higher value compared to the PC group in that respect (3.96%).The chicks of 5 and 10 mg MLT/egg groups have highly significantly lower relative yolk sac weight (9.20 and 9.01%, respectively) among all studied treatments, while the chicks of and PC groups have highly significantly higher relative yolk sac weight (19.69 and 17.73% respectively).The results of Table 4 showed that the 10 mg MLT/egg group has highly significant higher values of T 3 (2.59nmol/L) and T 4 (14.80 ug/dL) compared to all other studied groups.However, the T 3 /T 4 ratio value of the PC group has a significantly higher value (22.67%) compared to all other studied groups.In addition, the blood glucose levels of the PC, 10 and 15 mg MLT/egg groups have highly significant higher values (133.67,133.33 and 129.00 mg/dl, respectively), compared to NC and 5 mg MLT/egg groups (101.67 and 108.00 mg/dl, respectively).

Immune indices
The protein profile results (Table 5) showed that the total protein (2.59 À 3.20 g/dL) and globulin (0.97 À 1.24 g/dL) values of all studied groups have insignificant differences.However, the 10 mg MLT/egg group has a highly significant higher value of albumin (2.07 g/dL) compared to all other studied groups.On the other hand, the relative spleen weight of the PC, 10 and 15 mg MLT/egg groups have highly significantly higher values (0.05, 0.06 and 0.04%, respectively), compared to NC and 5 mg MLT/egg groups (0.02 and 0.03%, respectively).However, the relative Bursa weight of the 10 mg MLT/egg group has a significantly higher value (0.31%) compared to all other studied groups.The immune indices showed highly   significant (P � 0.001) differences among all studied groups for IgG, IgM, IgA and T cells values, while it was significant (P � 0.05) for B cells value.The 10 mg MLT/egg group enhanced significantly IgG and IgM parameters since this group has the highest value of both (44.00 and 19.23 g/dL, respectively) compared to all other studied groups.However, the 5 mg MLT/egg group has a significantly higher IgA value (7.47 g/dL) compared to NC, PC and 10 mg MLT/egg groups (5.97, 4.14 and 4.87 g/dL, respectively), with insignificant differences with the value of 15 mg MLT/egg group (6.66 g/dL).In addition, the 15 mg MLT/egg group has a highly significant higher value of T cells (16.67%) compared to all other studied groups.However, the 15 mg MLT/egg group has a significantly higher value of B cells (15.50%), compared to all other studied groups, except for the insignificant differences with 10 mg MLT/egg group value (14.00%).Table 6 illustrates the significant changes or improvements (%) of MLT treatments compared to the NC group.It illustrated that the 10 mg MLT/egg group improved spleen and bursa relative weights, IgG and IgM (0.04%, 0.18%, 133.7% and 108.3%, respectively).On the other hand, the 15 mg MLT/egg group enhanced T cells and B cells (177.8 and 36.80,respectively).
In general, the comparison between MLT and NC indicated that the chicks of in-ovo injection with 10 mg MLT/egg were superior in most immunological indicators (spleen and bursa relative weights, IgG, IgM, T cells and B cells).However, the chicks of inovo injection with 15 mg MLT/egg group shared the superiority with them (spleen relative weights, IgG, T cells and B cells).In addition, the chicks of in-ovo injection with 5 mg MLT/egg were superior in most immunological indicators (IgG, IgA and T cells).While the negative effect on the percentage of hatchability was observed only for those injected with 15 mg MLT/egg.

Discussion
The present study indicated that the higher MLT dose (15 mg MLT/egg) has a reduction effect on the hatchability percentages; however, the 10 mg MLT/egg dose has a positive effect on the weight and yield of chicks.The result was in line with the previous findings of Khalil et al., (37) showed that in-ovo administration with 10 lg BV/egg (lower dose) of Alexandria strain significantly increased hatchability percentage compared to the control and 20 lg of BV/egg groups.Also, they found that the highest chick yield was found in the control group then comes the lower and higher doses of BV (20 lg BV/egg).In general, the inovo injection studies of bee products have various effects on broiler chicken weight (3, 38).In general, numerous factors, including genetics, egg weight, incubator environment, storage period, and weight loss in the incubator, influence the weight of the chick at hatch (39).
The present results indicated that yolk consumption was higher in MLT treatments, which is in line with the findings of Khalil et al., (37), observed lower yolk sac weight percentages with chicks of in-ovo 10 and 20 lg BV/egg groups in comparison to the control group (17.71, 14.70 and 19.70%, respectively).The present outcomes displayed that the in-ovo injection of 10 lg MLT/egg has a more significant enhancement for relative intestine weight compared to other MLT treatments, which may encourage great efficiency to take advantage of the nutrients and reflect this in boosting the growth of Alexandria chicks.
Generally, it should be pointed out that the obtained hematological and blood biochemical values in this study within the wide range of physiological  standards specified for chickens (40), however, several factors affect the blood profiles of healthy birds (e.g.physiological, environmental conditions, age, diet contents and fasting).However, no significant effect of strain on blood profiles (41).Finally, the chicken's blood traits had daily rhythms (42).In explanation, the reduction in RBCs suggests a decrease in both the amount of carbon dioxide that would be returned to the lungs and the amount of oxygen that would be transported to the tissues (40).Whereas, low WBC numbers put people at greater risk of disease infection, while high counts allowed for the production of antibodies and increased disease resistance (43), and improved capacity for adjusting to regional environmental and disease-prone situations (40).As a broad biomarker important to immunological function in poultry, the H/L ratio is regarded as a sensitive hematological indication of stress response among chicken populations (44).According to Chineke et al., (45) indicated high PCV and Hb readings are significant in the diagnosis of anemia and also act as helpful measures of the bone marrow's capacity to make RBCs.They showed either an increase in RBC number or a decrease in circulating plasma volume.Furthermore, Isaac et al., (40) reported that PCV is involved in the transport of oxygen and absorbed nutrients since the increase of PCV shows better transportation.Accordingly, the chicks injected with 10 mg MLT/egg, regardless of the insignificant hematological values are in good health condition compared to other studied groups.
The present results showed that T 3 and T 4 values of 10 mg MLT/egg group have significantly highest value compared to all other studied groups.However, Khalil et al. (37) exhibited that BV in-ovo injection at levels of 10 and 20 lg/egg decreased significantly both T 3 and T 4 concentrations in Alexandria chicken serum at hatching day.The thyroid gland secretes T 3 and T 4 hormones, and normal levels are necessary for a wide range of metabolic activities influencing the growth, development of organisms, upkeep and role of both the cell-mediated immune responses and antibody (46), and also the basic metabolic level of birds and mammals (47).T 3 has been shown to be the primary physiological thyroid hormone controlling oxygen uptake and daily activity, especially in young chickens (48), and compared to T 4 , it is metabolically more active (49).The present glucose results support that glucose is one of the energy sources in the chicken body, which is in line with the findings of Klasing et al. (50), explicated that glucose with triglycerides are directly linked to the body's ability to sustain its energy supply in order to carry out its physiological and biochemical processes.It seems that Alexandria chicks of the 10 MLT/egg group, in relation to significantly higher glucose levels (Table 4) and lower relative yolk sac weight (Table 2), indicated that they had more metabolic activity than those in other treatments.Hematology traits were used for diagnosis and monitoring of disease and good indicators of the physiological status of animals and poultry (51,52).Animals with healthy blood composition are more likely to perform well (40).
The present results of relative spleen and bursa weights are in line with the findings of Khalil et al. (37), which showed that the spleen and bursa relative weights of Alexandria chicks increased significantly by the in-ovo injection of BV in comparison to the control group.Also, in a combination of MLT via drink water and Thepax via formulated diets, Elmalky et al., (17) reported that there was no significant difference among experimental groups regarding liver, gizzard, heart, spleen, or intestine relative weights of Ross 308 broiler chicks at the end of the experimental period (35 days of age).Also, it is clearly shown that all groups treated with the high dosage of MLT had highly significantly the highest relative weights of bursa in comparison to those of the NC group.Chickens' developing adaptive immunity depends on the development of their spleen and bursa glands (53), and both among other lymphatic systems (54).To provide protection from infections (body defense), the lymphoid system plays a great role with different etiological agents (55), which reflect in healthy chickens.On the other hand, B lymphocytes maturate and differentiate in avian bursa (56).The B lymphocytes (85-95%), T cells (4%), and other nonlymphoid cells make up the bursal follicles (57).The T cells were discovered to be responsible for killing infection cells and rejecting foreign tissue grafts exiting the thymus (55).
The natural antibodies were identified in poultry (58,59), which can take the IgM, IgG, or IgA types (60).It has been suggested that they can operate as a first line of defense against infection by directly neutralizing bacteria or viruses or by enhancing certain immune responses (61).Moreover, Bao et al., (62) indicated that plasma natural antibodies levels were heritable and might be used as a measure to assess the health of chickens.The 10 mg of MLT/egg group in the present study enhanced significantly IgG and IgM parameters while reducing IgA value.Mariyum Gondal (63) concluded that honey bee venom is very effective in producing immunity in a chick's body against different viral diseases.In a mouse model, Leiva et al. (64), demonstrated the efficiency of IgY to neutralize the deadly action of bee venom.

Conclusion
This study recommends the injection of 5 or 10 mg of MLT per egg to enhance the immune efficiency (early immune acceleration) of Alexandria chicks, which may be contributing to giving a great boost to their future performance.
experiment was carried out at the Poultry Research Center, Faculty of Agriculture, Alexandria University and Livestock Research Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications, New Borg El-Arab City, Alexandria, Egypt.The directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010, on the protection of animals and birds employed for research purposes, was complied with, the authors certify.

Table 2 .
Effect of Melittin in-ovo injection on relative organs weight (%) of Alexandria chick's strain at hatch.

Table 3 .
Effect of Melittin in-ovo injection on hematological traits and pH of Alexandria chick's strain at hatch.

Table 5 .
Effect of Melittin in-ovo injection on relative weight of lymphoid organs (spleen and bursa), protein profile, and immune indices of Alexandria chick's strain at hatch.

Table 6 .
The changing values (%) in Melittin in-ovo injection treatments compared to control (NC) treatment.