Effect of IN-OVO injection with Nano Iron -Particles on Physiological Responses and Performance of Broiler Chickens under Saini Conditions

A total of 600 fertile eggs, in a completely randomized design were used to investigate the effects of Iron nano-particles IN-OVO injection on productive performance, immune status and physiological responses in broiler chickens. The eggs were divided into 6 groups that assigned as: T1 (control; without injection), T2 (injected with 0.1 ml saline 9.0%; sham control), T3; (injected with 0.1 ml of 20 ppm Fe-NPs organic, T4 (injected with 0.1 ml of 20 ppm Fe-Nano inorganic), T5 (injected with 0.1 ml of 20 ppm Fe organic) and T6 (injected with 0.1 ml of 20 ppm Fe-inorganic). At 7th day of incubation, the corresponding doses were in- ovo injected in 0.1 ml solution into the air sac. The results showed that: Hatchability was highly significant (P< 0.01) in T1, 0.1 ml of 20 ppm Fe-NPs, 0.1 ml of 20-ppm Fe-NPs-Alimet chelate, 0.1 ml of 20 ppm Fe-Aliment chelate and 0.1 ml of 20-ppm Fe-Aliment chelate. The egg weight was higher (P< 0.01) in T2. There was an increase (P< 0.01) in chick weight in controls, other Fe-NPs organic or Fe-NPs- inorganic and Fe organic in comparison with other treatments. In addition, chick body weight to egg weight ratio in controls, Fe-Nano organic and FeNPs- inorganic was higher (P < 0.01) than in the other groups. T3 has shown the highest (P< 0.01) relative weight compared to the other treatments. Serum Fe content and liver function were (P< 0.01) higher in by using Fe-NPs, Fe-NPs alimet inorganic and Fe-organic than other treatments. The treatments of Fe-NPs- organic and Fe-Aliment chelate, chickens' blood hemoglobin increased significantly compared with the other treatments. These results suggest that Fe-NPs, Fe-NPs-Alimet chelate and Fe-Alimet chelate improved embryonic growth and development.


INTRODUCTION
Minerals play a vital role for maintaining homeostatic conditions in living organisms. Nanotechnology (the use of nano-particles of diameters between 1 and 100 nm) is nowadays applied in science, engineering, and agriculture (Scott andChen, 2002 andOberdorster andDonaldson, 2007). Nano-particles activities depend on their physical and chemical characteristics. Nanoparticles can show unique biological behavior, yet, the main mechanism of their action is still unknown (Shimizu, et al., 2009). These particles have features, such as large surface area (increasing physical, chemical, and biological activities) and higher solubility and mobility (Dimanet al., 2018 andToyooka, et al., 2009). High surface to volume ratio allows the functionalizing of nanoparticles with different ligands, coatings and other useful tools for lots of biomedical applications. High surface to volume ratio allows the functionalizing of nanoparticles with different ligands, coatings and other useful tools for lots of biomedical applications. Thus, it allows the functionalizing of nanoparticles with different ligands, coatings and other useful tools for lots of biomedical applications. However, the new physical and chemical properties of novel engineered nanoparticles make them extremely attractive for use in applications like medical sciences (Park, et al., 2010). Nano-particles have many novel properties compared with the bulk materials. Thus, inorganic nano-particle elements are widely used to enhance the productive performance of livestock, Ma et al., (2006). Embryonic development relays upon the availability of the required nutrients within the egg. Nutrient management inovo may provide an alternative method for poultry industry to increase hatchling weight. Chicks are affected by the nutrients in yolk remaining in the peritoneal cavity post hatching (Romanoff, 1960). Thus, a continually and precisely regulated supply of trace elements derived from stores within the egg is essential to ensure avian embryonic survival.
However, the high-metabolic rate, fast-growing rate of chicken embryos could be liable tomineral deficiency that lead to metabolic disorders (Tonaet al., 2004).
On the other hand, embryonic development relays upon the availability of the required nutrients within the egg. Nutrient management in-ovo may provide an alternative method for poultry industry to increase hatchling weight. Chicks are affected by the nutrients in yolk remaining in the peritoneal cavity post hatching (Romanoff, 1960). Thus, a continually and precisely regulated supply of trace elements derived from stores within the egg is essential to ensure avian embryonic survival. The high-metabolic rate, fast-growing rate of chicken embryos could be liable tomineral deficiency that lead to metabolic disorders (Tonaet al., 2004).
Iron (Fe) is essential for a variety of physiological processes in livestock (e.g. DNA synthesis, oxygen transport, etc.) as illustrated by Lozoffet al., (2006); Whitnall and Richardson, (2006) and Li and Zhao, (2009). NRC (1994) recommended 50-120 ppm daily intake of iron for poultry. Iron in the form of nano-particles has been reported to be less toxic than inorganic iron salts (Nikonovet al., 2012). Additionally, they have prolonged effects on biological activities (Kovalenko and Folmanis, 2006). Iron nanoparticles are more stable in air and have the ability to be degraded or metabolized in vivo, making them excellent candidates for a large number of applications (Bronstein et al., 2007).
Iron oxide nanoparticles (IONPs) are frequently used in biomedical applications, yet their toxic potential is still a major concern. While most studies of biosafety focus on cellular responses after exposure to nanomaterials, little is reported to analyze reactions on the surface of nanoparticles as a source of cytotoxicity. Results showed that IONPs had a concentration-dependent cytotoxicity on human glioma U251 cells, and they could enhance H2O2-induced cell damage dramatically. However, many studies have been conducted to evaluate the potential toxicity of iron oxide nanoparticles, Das, et al., (2007).
The goal of present study was to investigate the effects of in-ovo injection of iron, iron nanoparticle and iron chelates nanoparticles methionine during broiler embryonic devolvement on productive performance, physiological and immunological responses and the absorption of iron.

II. MATERIALS AND METHODS Experimental Design and Management
A total of 600 fertile broiler eggs obtained from cobb500™ parent stock were randomly divided into six equal groups. Eggs were individually weighed with an average of 60.83± 0.80g. Eggs were set in the hatchery and injection site was disinfected with ethyl alcohol, sealed with wax after injection then transferred to hatching baskets. The eggs were divided into 6 groups that assigned as: T1 (control; without injection), T2 (injected with 0.1 ml saline 9.0%; sham control), T3; (injected with 0.1 ml of 20 ppm Fe-NPs organic, T4 (injected with 0.1 ml of 20 ppm Fe-Nano inorganic), T5 (injected with 0.1 ml of 20 ppm Fe organic) and T6 (injected with 0.1 ml of 20 ppm Fe-inorganic). At 7 th day of incubation, the corresponding doses were in-ovo injected in 0.1 ml solution into the air sac. Iron oxide nanoparticles were prepared according to Reimers and Khalafalla (2011), suspended in Kno DMEM cell culture medium and dispersed by an ultrasonic bath. The injection was performed at day 7 of incubation into the air sac. Eggs were candled on 7 th day of hatchery and 17 th day to remove infertile eggs. Post-hatch, a total number of 360 one-day-old chicks were randomly distributed into six equal| (n = 60 / treatment) groups with three replicates (20 chicks/ each) according the corresponding treatments.
Experimental chicks were kept under similar managerial, hygienic and environmental conditions. The chicks were housed in cages from hatch up to 5 weeks of age. Average of indoor ambient temperature (AT, ᵒC) and Relative Humidity (RH, %) were recorded using electronic digital thermo-hygrometer. Average of AT and RH was 35.7 ±0.98ᵒ C and 24.2 ±1.32 %, respectively. Feed was offered ad libitum according to NRC (1994) recommendations. Fresh water was made available all the daytime. Live body weight and feed intake were recorded weekly before offering feed. At the end of the trial, five broiler chicks from each group were picked randomly for blood sampling.
Blood samples (n= 30) were randomly withdrawn from 5 chicks immediately before slaughtering of chicks (at day 35) from the (brachial) wing vein into tubes containing EDTA as anticoagulant and centrifuged at 3000 rpm for 20 minutes for the separation of plasma and kept at (−20°C) until further analysis.

Effect of ovo injection by Fe-Nano, Fe-Nano-Alimet chelate, Fe-Aliment chelate and Fe-Aliment chelate on hatchability traits
Table (1) shows the egg performance when injected with different forms of supplementary Iron. . There was a significant difference (P<0.01) between control and sham control with respect to hatchability percent. There seem to be a need for NaCl solution because of a deficiency of this mineral in the egg, which might explain the positive effect of saline injection. Sodium chloride is a mineral salt and it seemed to close a gap in the requirements of egg growth to this mineral. It might also have a positive effect with respect to buffering the medium inside the egg, which led to facilitating the growth performance, livelihood of the embryo and therefore the hatchability percent improved as a result. It seems that these explanations are logic since there was no significant difference between sham control (saline solution injection) and injection of different forms of Iron either as in nano particle form or not and the form of being organic or inorganic. The different forms of Iron in nano particle or in the organic or inorganic forms showed the same significant difference as the saline solution injection did. The same explanations might, therefore, apply. The check weight/egg weight ratio of control and sham control were not significantly different (74.5 and 74.8 for control and sham control, respectively). The injection of different forms of Iron positively enhanced this ratio. The ratios were 85.2, 4.6 and 84.4 for T3, T4 and T5, respectively).The inorganic form of Iron (T6) was similar to both controls. Saki et al. (2014) found no significant effects on hatchability percent among the groups fed 50 and150 ppm Fe-Aliment chelate relative to control one. This may be explained by the deficiencies or excesses of individual trace elements that can cause impaired growth, abnormal development, thus, affecting all of the major organ systems and in extreme cases, death of the embryo (Richards and Steele, 1987). Appropriate amounts of each trace element are required to support embryonic growth and development, Richards, (1989). In mammals, Fe link to amino acids increased the transfer of Fe across the placenta and into the embryo, Ashamead and Graff, (1982).
The form of nano Fe in any form depends on the presence of protein and it would be interesting to investigate the relationship between protein and Fe atoms. Foye, et al., (2006) found that Fe atoms adhered easily to protein and that the co-existing system of protein and iron could directly scavenge ROS (OH•, O•− 2 and H2O2). Nano-particles can evade conventional physiological ways of nutrient distribution and transport across tissue and cell membranes, as well as protect compounds against destruction prior to reaching their targets. In-ovo administration of nanoparticles, may be seen as a new method of nano-nutrition, providing embryos with an additional quantity of nutrients. Growth performance at 7 day of age: Effects of in-ovo injection of nano forms of Fe-Nano particles (either organic or inorganic) on average weight gain and feed efficiency ratio of broiler during the first week of age are shown in table (2). Body weight (gm) values during the first week gradually increased significantly (P<0.01). The control group showed the lowest body weight over the period of first seven day period (90.55 gm). Sham control showed higher significant body weight (120.5 gm) over this period compared to regular control. It was lower than the treatment of the injection of nano-Iron in either form (132.4 and 123.9 gm for T3 and T4, respectively). The injection of regular Fe salt in both forms (organic and inorganic) showed lower (105.99 and 118.9 gm for organic and inorganic forms of regular Fe injection, respectively) body weight than both controls. Therefore, the percent increments of T2, T3 and T4 were 33 45 and 3.38%, respectively, than that of the T1 treatment. Results of feed conversion ratio (FCR) (gm feed/gm gain) revealed a significant difference (P<0.01) among the experimental treatments. It was monitored in this study, that T2, T3, T4, T5 and T6 recorded the best FCR; these results match up the increase in feed intake and reduction of daily weight gain.

Blood analysis.
The effects of in-ovo injection of broiler eggs on plasma iron definitions in chicks on 35day of age are shown in Table (4). The results indicate that the effect of in-ovo injection of broiler eggs with nano forms of Fe-Nano, Fe-NPs-Alimet chelate, Fe-Aliment chelate and Fe-Aliment chelate recorded significant increased (P<0.01) the values of WBC's, HGB, MCHC and HCT, while it was insignificant in RBC's and MCV , MCH, RDWCV and RDWSD compared to control treatment (Table 3).
On the other view, it was found through the results in  (Bertechini, et al., 2012) and chickens for fattening (Shinde, et al., 2011).The greatest mean increase was +22% and +31.9% for broiler muscle and liver, respectively. In addition, hemoglobin in two treatments of 100-ppm Fe-NPs-Alimet chelate and150-ppmFe-Alimet chelate significantly increased compared with other treatments.
The results of Warner et.al. (2006) indicated that the absolute amount of iron per liver increased steadily up to hatching time. Their results showed that the highest liver weight was observed in treatment having 25 ppm of Fe-NPs. The treatments 25 ppm Fe-Nano, 100 ppm Fe-NPs-Alimet chelate and 150 ppm Fe-Alimet chelate have shown higher Fe content in serum and liver compared with those in other treatments.
Effects of in-ovo injection on broiler eggs on plasma iron definitions in chicks on 35 day of age are shown in Table (5) .The data showed major variations in TP for T1, T2, T3 and T5 compared to T4 and T6, where, they were increased by (11.8, 12.57 and 2.96 %) related to T1, while the lowest value was for T6 (by 1.97 %) and no significant difference. The same trend was observed in A, G and A/G ratio. Since the albumen is synthesized mainly in liver, that liver function was enhanced by the injection of Iron in its different forms, and that the albumin is a main source for amino acid formation, the protein synthesis increased leading to more formation of muscles, which in turn leads to increased final body weight. This is clearly manifested in the results obtained in this study (Table 3).

IV.
CONCLUSION These results suggest that under semi-arid conditions, the in-ovo injection of 20-ppm iron nanoparticles (Fe-NPs), 20-ppm iron nanoparticles Alimet chelate (Fe-NPs-Alimet chelate) and 20-ppm Fe-Alimet chelate as injection contributed to embryonic growth development. Iron nanoparticles and Alimet chelate form, as the active in gradient of feed additives, premixes, and compound feed, due to the high surface activity and penetration into cell can actively influence the intracellular metabolism by stimulating various processes.
The nano form of Fe are not harmful to the embryo (injected with 20 ppm) and can be used to improve the posthatch performance of broiler.  n. s * * * a, b: Means within a column with different superscripts are significantly different (P< 0.01). Sig. = Significance, * (P< 0.01), n. s = not significant. Sig.= Significance,** (P< 0.01), n.s= not significant  40.20 b ± 01401 a, b, c Means within the same row with no common superscript differ significantly. ** P≤ 0.01, NS= non-significant