Preventive, Behavioral, Productive, and Tissue Modification using Green Synthesized Selenium Nanoparticles in the Drinking Water of Two Broiler Breeds under Microbial Stress

27/June/2019 Approved: 18/October/2019 ABSTRACT A study was conducted to investigate the influence of selenium nanoparticles (SeNPS) and inorganic selenium supplementation in the drinking water on behavior, performance, and immunity of Arbor Acres ® and Ross ® 308 broilers exposed to E. coli O157:H7 1.6 × 10 8 challenge at the 10 th day of age. 180 one-day-old female broilers were divided into six groups, each with 30 chicks. G1 and G4 were supplied with 1 mL SeNPS 100 mg.L -1 /L, G2 and G5 were supplied with 1 mL inorganic selenium 100 mg.L -1 /L, and G3 and G6 were supplied with non-supplemented water. Where, G1, G2, and G3 were Arbor Acres ® , while G4, G5, and G6 were Ross ® 308 broilers. A total of 1280 samples (160 sera, 160 intestinal swabs and 960 organ samples including liver, spleen, bursa, heart, breast muscles, and thymus) were collected in a study period of 38 days. A highly significant increase ( p< 0.01) of weight gain, feed conversion, performance index, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, immunoglobulin G and M, total antioxidant capacity, malondialdehyde, and superoxide dismutase was recorded in G4 Ross ® 308 compared to other supplemented and control groups. G1 Arbor Acres ® and G4 Ross ® 308 broilers, also revealed a highly significant decline ( p< 0.01) in total bacterial and enterobacteriaceae counts of intestine and breast muscles compared to G2 Arbor Acres ® and G5 Ross ® 308 broilers and to controls. Photomicrographs revealed a higher degree of cellular and tissue protection in G4 and G5 Ross ® 308 compared to G1 and G2 Arbor Acres ® broilers. An improvement from SeNPS supplementation was detected on behavior, performance, bacterial load, immunological, antioxidant profiles, and tissue architecture in broilers breeds with special reference to Ross ® 308 compared to Arbor


INTRODUCTION
The poultry industry worldwide has witnessed large scale intensification proportional to the growing population. Intensification in poultry industry demands proactive preventive measures and enhancing supplemental to maintain high productivity, minimize disease risk, and lessen the influence of environmental stressors. Rearing broilers under adverse conditions produce oxidative stress, including extreme freeradical formation and dispersion, cell destruction, and production losses (Schwean-Lardner et al., 2012). It was established that using complexes containing vitamins, minerals, and selenium improved productivity, increased weight gain by 8.46 %, and improved feed conversion by 3.1% (Trukhachev et al., 2016).
Selenium is a nutritionally essential for chickens, to maintain an optimum health, therefore 0.15 mg/kg diet is suggested for broilers during the growth period. Selenium is an important structure of Glutathione peroxidase (GSH-Px) which is a strong antioxidant in poultry (Oliveira et al., 2014). Selenium bioavailability depends on its form, including elemental selenium with poor absorption capabilities, inorganic selenium (selenite) which is not biologically active and rapidly excreted from the body, and organic selenium which is more active and plays an important role in biological processes (Suchý et al., 2014). However, there is a common con cern that the selenium minimum recommendation is not enough to prevent production losses due to selenium deficiency and that's why, there is contin ued research into alternative selenium supplementation levels.
Nanotechnology manipulates design, production, devices, and systems via controlling sizes and shapes. Nanomaterials have completely different physical and chemical characteristics compared to the original and raw unmodified chemical forms of selenium (Hartemann et al., 2015), these new characteristics authorize their application in medicinal and nutritional fields (Mohapatra et al., 2014). The success of nanomaterials in many fields contributes to their increased use in poultry industry to enhance productivity, immunity, and combating pathogenic micro-organisms' activity (El Sabry et al., 2018). Nano-selenium has many advantages including high absorption capability, increased surface activity, catalytic efficiency, low toxicity, and reimbursement of the body during oxidative and environmental stressors, as well as alleviate their negative impact (Nasirpour et al., 2017;Purohit et al., 2017).
The present study aimed to investigate the influence of supplementing broiler's drinking water with 1 mL of selenium nanoparticles (Green synthesized selenium nanoparticles -SeNPS -at concentration of 100 mg/ 1L) compared to 1 mL commercial inorganic selenium (Selenite -SEDICO ® at concentration of 100 mg/ 1L) on productive performance, behavior, intestinal and muscular bacterial loads (Total bacterial and Enterobacteriaceae counts), biochemical and immunological profile, and histopathological architecture of two broiler breeds (ArborAcres ® and Ross ® 308) exposed to E. coli O157:H7 challenge at the 10 th day of age.

Ethical Approval
The procedures in the present study were approved by the Scientific Research Ethics Committee, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt with approval number 2018060.

Experimental birds' microclimate
A total of 180 one-day-old females Arbor Acres ® and Ross ® 308 broilers (ninety of each breed) were purchased from Ismailia-Egypt Poultry Company and housed in a battery system (galvanized iron cages). Birds of each breed were divided into three groups; each group consisted of 30 chicks (five replicates of six birds).
The birds were immunized be mean of mass vaccination in drinking water with Infectious Bronchitis live attenuated virus vaccine (PESTIKAL B1 SPF 1000 dose/ 100 Liter) on the 7 th day of age; Infectious Bursal disease live attenuated virus vaccine (SER-VAC D78 Strain 1000 dose/ 100 Liter) on the 14 th and 21 st days of age, and Newcastle disease live lentogenic virus vaccine (PESTIKAL 1000 dose/100 Liter) on the 16 th and 26 th days. The experiment lasted for 38 days.
At day-old the rearing temperature was 34 ºC and gradually declined by 3.5 ºC weekly until achieving 25 ºC by the end of the 3rd week. The birds were given ad libitum access to water and a standard corn-soybean basal diet (NRC 1994) based on 23% crude protein over the first fourteen days, and 21.5% crude protein for the remaining 24 days in the fattening cycle. The room was ventilated naturally using V shape windows to stimulate a stack effect. Artificial continuous lightening program for 18 h light and 6 h darkness using Blue LED lights was provided as recommended by Soliman & Hassan (2019). Mortalities, microclimatic temperature and relative humidity were monitored and recorded daily during the experiment.

Synthesis of Selenium nanoparticles
Selenium powder (Merk ® ) was used as a precursor for the SeNPS and polyvinyl alcohol (PVA) served as a capping agent for the NPS to prevent aggregation and any change in size and shape. Glucose that was commercially produced acts as a reducing agent of selenium; it is assumed that glucose makes reduction and oxidized itself into gluconic acid, this acid may also surround the NPS and act as a stabilizing agent. Firstly aqueous sodium seleno-sulphate solution was prepared by refluxing and heating at 70 ºC for an aqueous solution of Na 2 SO 3 (Merk ® ) and selenium powder (selenite) for 6h as reported in Gorer & Hodes (1994). The solution was filtered to remove any unreacted materials, then glucose powder (6%) and 1% PVA were added to the solution, refluxing persisted for another 6 h. The color of the solution changed through refluxing from colorless to pale yellow. The colored solution remained stable for months. The synthesized SeNPS were characterized by UV-visible optical absorption spectroscopy (Shimaduz RF5301PC double beam spectrophotometer), and absorption spectra of the resulting SeNPS were scanned (200-800 cm-1) using 1 cm matched quartz cells. The morphology of the NPS was observed using Transmission Electron Microscopy (HR-TEM) JOEL JEM-2010 operating at an accelerating voltage of 200 kV and equipped with a Gatan digital camera Erlangshen ES500. TEM sample was prepared by dropping a sample suspension as it is after preparation on a coppergrid coated with a carbon film. Preparing SeNPS via this method characterized by obtaining Nanocrystalline SeNPS without post annealing treatment.

Selenium and SeNPS solution supplement
One group (N=30) from each breed was assigned the following treatments: G1 and G4 were supplied with 1 mL SeNPS (green synthesized SeNPS at concentration of 100 mg/L) per each liter of drinking water, G2 and G5 were supplied with 1 mL commercial inorganic selenium (Selenite -SEDICO ® at concentration of 100 mg/L) per each liter of drinking water, finally, G3 and G6 were supplied with non-supplemented water. Where, G1, G2, and G3 were Arbor Acres ® , G4, G5, and G6 were Ross ® 308 broilers. The final concentration of both SeNPS and inorganic selenium in the supplemented water was about 0.1 mg per each liter of drinking water on a daily basis of the entire fattening cycle (38 days).
E. coli O157:H7 challenge E. coli O157: H73.6 × 10 5 CFU culture was purchased from the Animal Health Research Institute -Dokki, the culture was propagated using Mac-Conkey broth at 44 ºC / 24 h. Ten microliters from the positive tubes (yellow color development and gas accumulation in inverted Durham's tube) were dropped aseptically onto Eosine Methylene Blue (EMB) agar, and incubated at 37 ºC / 24 h (Herigstad et al., 2001). Metallic green colonies were counted and picked up. Four out of the six groups (G1, G2, G4 and G5) were challenged with E. coli O157:H7 1.6 × 10 8 on the 10 th day of age in drinking water (Kocijancic et al., 2016).

Performance indices
Average live body weight (LBW) was estimated by weighing 28 birds (the number was calculated using Solvin's formula for random sampling) from each group on a weekly basis. Weekly feed intake (FI) was calculated based on total amount consumed in relation to the bird's absolute number in each group. Body Weight Gain (WG), Feed Conversion Ratio (FCR), and Performance Index (PI) were calculated according to Soliman & Hassan (2017).

Behavior recording and observation methods
Twelve broiler chicks from each group were randomly selected for recording the behavioral parameters. Broiler's behavior was recorded for a total of 3 h a day, 1 h starting at 9:00 am, 2:00 pm, and 9:00 pm in a daily pattern for three weeks (1 st , 3 rd , and the 5 th ) using a video camera (Panasonic WV Ns202ae) connected to computer through DVR card, and they were suspended at a height of 1.5 m above the floor of each pen (Li et al., 2015).
The recorded videos were replayed in the laboratory, and the performed behaviors, duration, and frequency were recorded every five min/h (12 observations per h) using focal sampling method. They were classified into: feeding, drinking, walking, standing, resting, preening, stretching, pecking, and flapping (Villagra et al., 2014).

Sampling
Sampling was carried out via two slaughters (at the 19 th and the 38 th day of age). A total of 1280 samples were collected during the study period, including 160 sera, 160 intestinal swabs and 960 organ samples of liver, spleen, bursa, heart, breast muscles, and thymus. Blood samples were collected during slaughters, centrifuged at 4000 rpm for 15 minutes. Nonhemolyzed clear sera were obtained, stored at -20 °C for the biochemical and antioxidant assay . Liver, spleen, bursa, heart, and thymus were kept on formalin 10% for histopathological examination. Intestinal swabs, and 3 g of breast muscles were collected in 9 mL buffered peptone water for bacteriological assessment.
Serum immunoglobulin IgG and IgM concentrations were measured by using immunoturbidimetric assay using Sysmex CS-5100 System.

Bacteriological examination
Intestinal swabs, as well as breast muscles samples, were prepared according to APHA (2012). Tenfold serial dilution sup to 10 -6 were prepared. Total Bacterial Count (TBC) and Total Enterobacteriaceae Count (TEC) were applied on standard plate count (SPC) and Eosine Methylene Blue Agar (EMB), respectively at 37 ºC for 24 -48 h by using the drop plate technique as recommended by Soliman et al. (2016) and Kim & Lee (2016). Plates were counted using Dark-field colony counter (Murray et al., 2015).

Histopathological examination
Representative tissue samples from liver, heart, lymphoid organs (spleen, bursa of Fabricious, and thymus) were fixed in 10% buffered formalin saline solution until further processing. Specimens were cut into 5-mm thick sections, put into tissue cassettes, dehydrated by transferring through a series of alcohols increasing concentrations, cleared in two changes of xylene, infiltrated with different grades of melted paraffin in the oven, and finally the sections were cut at 5 μm thickness using rotator microtome. After cutting, sections were floated on warm water bath at 38°C for stretching, mounted on clean slides using egg albumin, and dried on a slide warmer at 38°C (Luna, 1968 andDarboux, 1994). The sections were stained using Hematoxylin and Eosin (H & E). The histological structures of the examined organs were observed using light microscope under (x10) and (x20) magnification.

Statistical analysis
Statistical analysis was carried out using the statistical package for social sciences (SPSS version 20.0) software package (Argyrous, 2005). E. coli O157:H7 and total bacterial counts were expressed as logarithms using Microsoft Excel. The obtained data were analyzed statistically using two-tailed Analysis of Variance (ANOVA). In the two-tailed ANOVA, factors including breeds, groups, age of broiler, and slaughter times were used as between-subjects' effects along with their interactions. Mixed model ANOVA was used as the principal statistical mean for the current data. The principle and interaction effects were tested for their statistical significance at 0.05 and 0.01 significance levels. The statistical model was emphasized as following: Y ijk = µ+α 1 +ß j +(αß) ij +e ijk Where, Y ijk was the measurement of any dependent variable; µ was the overall mean; α 1 was the fixed effect of slaughter time; ß j was the fixed effect of the age of the birds; (αß) ij was the interaction effect of slaughter time by age of the birds; e ijk was the random error. The error term, e ijk was approximately NID (0, σ 2 e ), normally independently distributed with mean of 0 and variance of, σ 2 e .

UV-Vis spectrophotometry of SeNPS
The absorption spectrum of yellow colored SeNPS ( Figure 1) showed appearance of the transition point at around 367 nm, without a clear maximum reflecting the formation of SeNPS. The stabilization of the yellow color during the entire reaction time supported the stabilizing influence of PVA. Morphology and structure of the synthesized SeNPS was observed using TEM imaging, and the image ( Figure 2) revealed a spherical shape for individual nanoparticles, with size at 25 nm.

Behavioral observations
Broiler's behavior (Table 1) exhibited a highly significant increase (p<0.01) in the frequencies of feeding, drinking, walking, resting, and in the duration of drinking and walking in G1 and G4 supplemented with SeNPS compared to G2 and G5 supplemented with commercial inorganic selenium and to the controls. A highly significant decrease (p<0.01) was recorded in the duration of feeding and standing among all treated groups in both broilers' breeds compared to the control. A highly significant increase (p<0.01) was declared in walking and drinking duration of G1 and G4 compared to all other supplemented and control groups.
SeNPS (Table 1) were able to produce a highly significant decrease (p<0.01) in standing duration of G1 and G4 when compared to each other, a nonsignificant decrease in standing duration of G2 and G5, and a non-significant decrease in standing frequency among all groups of both breeds.
Resting frequency showed (Table 1) a highly significant increase (p<0.01) in G1 broilers compared to G2, G5, and G4 with no significant difference between theses latter three groups. The selenium effect was inconsistent having no effect on resting frequency in either breed.
SeNPS was able to significantly decrease (p<0.01) the frequencies of preening, stretching, and pecking in G1, G4, G2, and G5 compared to their controls (Table   1). On the other hand, it was able to increase the frequency of flapping in the same groups compared to their controls.

Biochemical profile
The biochemical profile (Table 2) revealed a highly significant decrease (p<0.01) in TP, ALB, ALT, and AST among treated groups of both experimented broilers' breeds compared to their control. TP revealed (Table 2) a highly significant elevation (p<0.01) in G4 Ross ® 308 compared to G1Arbor Acres ® and to the other treated groups. ALB showed a highly significant increase (p<0.01) in G5 Ross ® 308 broilers treated with inorganic selenium compared to G2 Arbor Acres ® broilers treated with the same dose and concentration of inorganic selenium, and to G1 and G4 treated with SeNPS.
On a time scale, TP showed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the19 day old broilers in G1, G2, G3, and G4, and a highly significant increase on the 38 day old broilers compared to the 19 day old broilers in G5 and G6 (Table  2). ALB revealed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, G2, G4, and G5, no significant differences between the two slaughter times in G3 Arbor Acres ® control, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G6 Ross ® 308 control (Table 2).

Preventive, Behavioral, Productive, and Tissue Modification using Green Synthesized Selenium Nanoparticles in the Drinking Water of Two Broiler Breeds under Microbial Stress
eRBCA-2019-1129 A highly significant decline (p<0.01) was detected ( Table 2) in ALT of G1 and G2 Arbor Acres ® broilers compared to their control with no significant differences between these two groups, and a highly significant decline (p<0.01) of ALT in G4 and G5 Ross ® 308 broilers compared to their control, with a highly significant increase (p<0.01) in G4 than in G5 broilers. A highly significant decrease (p<0.01) was revealed in AST of G1 treated with SeNPS compared to G2 treated with inorganic selenium and G3 control Arbor Acres ® broilers, and in AST of G4 treated with SeNPS compared to G5 treated with inorganic selenium and G6 control Ross ® 308 broilers.
ALT showed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, G3, and G4, no significant differences between the two slaughter times in G5, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G2 and G6 (Table 2). AST showed a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in all treated and control groups (Table 2).
Urea (Table 2) showed a highly significant increase (p<0.01) in G1 Arbor Acres ® and G5 Ross ® 308, nonsignificant increase in G2 Arbor Acres ® , and a highly significant decrease (p<0.01) in G4 Ross ® 308 compared to the controls. A highly significant increase (p<0.01) of CREAT levels was noticed in G1, G2, G4, and G5 compared to their controls.
Urea revealed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G3, G5, and G6, no significant differences between the two slaughter times in G1, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G2 and G4 (Table 2). CREAT showed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, no significant differences between the two slaughter times in G6, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G2, G3, G4 and G5 (Table 2). G1=Arbor Acres ® with 1 mL nano-selenium/1L drinking water, G2=Arbor Acres ® with 1 mL selenium/1L drinking water), G3=Arbor Acres ® control, G4= Ross ® 308 with 1 mL nano--selenium/1 L drinking water, G5= Ross ® 308 with 1 mL selenium/1L drinking water, and G6= Ross ® 308 control.

Immunoglobulin concentration
IgG and IgM revealed (Table 3) a synchronized highly significant increase (p<0.01) in G1, G2, G4, and G5 compared to the controls with more significant increase (p<0.01) in G4 and G1 treated with SeNPS, respectively compared to other groups with no significant differences between G1 and G2. The overall average concentrations of IgG and IgM were significantly higher (p<0.01) in Ross ® 308 (Mean=1625.8 mg/dL) compared to Arbor Acres ® broilers (Mean=1607.1 mg/ dL).
IgG showed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G1 and G2, no significant differences between the two slaughter times in G6, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G3, G4 and G5 (Table 3). Meanwhile, IgM revealed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G1 and G2, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G3, G4, G5, and G6 (Table 3).
TAC showed a highly significant decrease (p<0.01) on the 38 day old broilers compared to Means carrying different superscripts in the same column are significantly different at (p≤0.05) or highly significantly different at (p<0.01). Means carrying the same superscripts in the same column are non-significantly different at (p<0.05).
IgG=Immunoglobulin G, IgM=Immunoglobulin M, TAC=Total Antioxidant capacity, MDA= Malondialdehyde, and SOD=Superoxide Dismutase, SE=Standard error. the 19 day old in G2, no significant differences between the two slaughter times in G3 and G6, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, G4 and G5 (Table 3). MDA revealed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G2, no significant differences between the two slaughter times in G6, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, G3, G4 and G5 (Table 3). SOD showed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G3 and G6, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, G2, G4 and G5 (Table 3).

Bacterial Counts
TBC revealed a highly significant decrease (p<0.01) in intestinal swabs of G5 compared to G4 Ross ® 308 broilers, and in G1 compared to G2 Arbor Acres ® broilers, with no significant differences between G3 and G6 control of the two breeds, and a highly significant decrease (p<0.01) in TBC of breast muscles of G1 compared to G2 Arbor Acres ® broilers, and a significant decrease (p≤0.05) in G4 compared to G5 Ross ® 308 broilers (Table 4).
TBC of intestinal swabs showed no significant differences in G4, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, G2, G3, G5 and G6 (Table 4). TBC of breast muscles showed a highly significant decrease (p<0.01) on the 38 day old broilers compared to the 19 day old in G3, G4, and G6, and a highly significant increase (p<0.01) on the 38 day old broilers compared to the 19 day old in G1, G2, and G5 (Table 4).TEC of intestinal swabs and breast muscles showed a highly significant increase on the 38 day old broilers compared to the 19 day old in all treated and control groups (Table 4).

Productive performance
WG/g revealed a significant difference (p≤0.05) between G1 and G2 and between G4 and G5 with no significant differences between G3 and G6 controls (Table 5).WG also revealed a highly significant increase (p<0.01) on the 5 th , 3 rd , 4 th , 2 nd , and 1 st week of age respectively in G1, G2, and G3 Arbor Acres ® broilers, at on the 4 th , 3 rd , 5 th , 2 nd , and 1 st week of age respectively in G4 Ross ® 308 broilers, and at on the 5 th , 4 th , 3 rd , 2 nd , and 1 st week of age respectively in G5 and G6 Ross ® 308 broilers (Table 5).
FI/g (Table 5) showed a highly significant increase in G4 compared to G5 and G6 Ross ® 308 control, and no significant differences between G1 and G2 Arbor Acres ® broilers with a highly significant difference between the last two groups and G3 Arbor Acres ® control. FI revealed (Table 5) a highly significant increase (p<0.01) on the 5 th , 4 th , 3 rd , 2 nd , and 1 st week of age respectively in G1, G2, G5, and G6 broilers, and on the 4 th , 5 th , 3 rd , 2 nd , and 1 st week of age respectively in G3 Arbor Acres ® control and G4 Ross ® 308 broilers.
FCR% showed a significant difference (p<0.01) between G1 and G2 with no significant differences between G2 and G3 Arbor Acres ® control, and a highly significant difference (p<0.01) between G4 and G5 with no significant differences between G4 and G6 Ross ® 308 control (Table 5). Meanwhile, on a weekly basis, FCR revealed a highly significant increase (p<0.01) on the 4 th , 3 rd , 2 nd , 1 st , and 5 th week of age respectively in G1, a highly significant increase (p<0.01) on the 4 th , 3 rd , 2 nd , 5 th , and 1 st week of age respectively with no significant differences between 2 nd and 3 rd weeks in G2, a highly significant increase (p<0.01) on the 4 th , 5 th , 2 nd , 3 rd , and 1 st week of age respectively with no significant differences between the1 st and 3 rd weeks in G3, a highly significant increase (p<0.01) on the 5 th , 2 nd , 3 rd , 4 th , and 1 st week of age respectively in G4, a highly significant increase (p<0.01) on the 4 th , 5 th , 3 rd , 2 nd , and 1 st week of age respectively with no significant differences between the 1 st and 2 nd weeks in G5, and a highly significant increase (p<0.01) on the 4 th , 3 rd , 5 th , 1 st , and 2 nd week of age respectively with no significant difference between the 3 rd and 5 th weeks in G6.
PI showed a significant difference (p<0.01) between G1, G2 and G3 Arbor Acres ® broilers, and a highly significant differences (p<0.01) between G4 and G5 with significant differences (p≤0.05) between G4 and G6 Ross ® 308 control. PI (Table 5) showed also a highly significant increase (p<0.01) on the 5 th , 4 th , 3 rd , 2 nd , and 1 st week of age respectively with no significant difference between the 3 rd and 4 th weeks in G1, a highly significant increase (p<0.01) on the 5 th , 3 rd , 4 th , 2 nd , and 1 st week of age respectively in G2, a highly significant increase (p<0.01) on the 5 th , 4 th , 3 rd , 2 nd , and 1 st week of age respectively with no significant difference between the 4 th and 5 th weeks and between the 2 nd and 3 rd in G3, a highly significant increase (p<0.01) on the 4 th , 5 th , 3 rd , 2 nd , and 1 st week of age respectively in G4, and a highly significant increase (p<0.01) on the 5 th , 4 th , 3 rd , 2 nd , and 1 st week of age respectively in G5 and G6.

Histopathological architecture examination
Liver photomicrographs revealed in G1 ( Figure  3a) and G2 (Figure 3b) thickening of liver capsule due to fibrinous exudation and mononuclear cell infiltration, hepatic cells showed degeneration with mild vacuolation of cytoplasm and mononuclear cell infiltration, as well as, mild hemorrhage in G2 broilers ( Figure 3b). Meanwhile, the liver of G4 (Figure 3d) showed congestion of central vein, the hepatocytes showed mild degeneration, cytoplasmic vacuolation and mononuclear cell infiltration. The liver of G5 (Figure 3e) showed mild perihepatitis due to fibrinous exudation and mononuclear cell infiltration, hepatic cells showed mild degeneration, mild hemorrhage and mononuclear cell infiltration compared to Arbor Acres ® control ( Figure 3c) and Ross ® 308 control (Figure 3f).
Heart examination showed moderate fibrinous pericarditis which extend to myocardium resulting in myocardial degeneration with cytoplasmic vacuolation and mononuclear cell infiltration in G1 as revealed (Figure 4a) and G2 side by side to the presence of congestion and mild hemorrhage of cardiac muscle in G2 only (Figure 4b) compared to G3 control (Figure 4c). G4 Ross ® 308 broilers revealed (Figure 4d) mild pericarditis, mild myocardial degeneration with mild congestion, cytoplasmic vacuolation and mononuclear cell infiltration. Heart of G5 (Figure 4e) showed mild fibrinous pericarditis, myocardial degeneration with mild hemorrhage cytoplasmic vacuolation and mononuclear cell infiltration compared to G6 (Figure 4f).
The spleen of G1 and G2 (Figures 5a and 5b) revealed congestion of splenic sinus, mild hemorrhage with hemosedrosis, and moderate lymphoid depletion compared to G3 (Figure 5c). Meanwhile, G4 in Figure 5d revealed no pathological changes, and G5 showed moderate lymphoid depletion of spleen and hemorrhage (Figure 5e) compared to the normal picture in Ross ® 308 broilers of G6 (Figure 5f). Histopathological examination of the bursa of fabricious revealed severe thickening and hyperplasia of follicular epithelium with severe depletion of lymphoid follicles which are replaced by edematous fluid and fibrous tissue in G1 (Figure 6a). Severe hyperplasia of follicular epithelium with severe lymphoid depletion of lymphoid follicles were revealed in G2 (Figure 6b) in comparison to G3 control (Figure 6c). On the other hand, G4 in Figure 6d showed normal histologic structure of lymphoid follicles with increased interfollicular fibrosis, G5 (Figure 6e) showed moderate lymphoid depletion with mild necrosis of lymphoid follicles with increased interfollicular fibrosis compared to G6 control ( Figure  6f).
Photomicrographs of thymus showed moderate depletion of lymphocytes in the cortex and medulla in G1 (Figure 7a

DISCUSSION
Selenium has been considered an essential element required for multiple functions in broiler as productivity, growth, fertility, and prophylaxis via increasing immunoglobulin concentrations as reported by Del Puerto et al. (2017). Based on the European Union recommendation (2004) selenium has to be supplemented at 0.5 mg/kg dry matter to contribute a notable enhancement of performance, immunity, and antioxidant activity.
Broiler's behavior attested to an improvement in Ross ® 308 and Arbor Acres ® broilers supplemented with 1 mL SeNPS (100 mg/1 L) as represented by higher feeding frequency, drinking duration and frequency, walking duration and frequency, and resting frequency. These finding were in agreement with Omidi et al. (2018) who reported that reduced selenium levels may create oxidative stress, contributing to an increase in the risk of behavioral disturbances. In  addition, selenium deficiency is linked to temperament deviations of animals (Ferencik & Ebringer, 2003).
The biochemical profile in Ross ® 308 and Arbor Acres ® broilers supplemented with 1 mL SeNPS (100 mg/1 L) revealed significant increase in TP, ALB, ALT, and AST serum concentration with a special reference to Ross ® 308 broilers groups despite the variation noticed inside each group between the two slaughter times on the 19 th and 38 th days. The intergroup variation might be attributed to broiler's resistance and breed. The results were in agreement with those of Liu et al. (2017) who supplemented broilers with 0.0, 0.2, 0.4, 0.8, 2.0, 4.0, and 8.0 mg SeNPS in two mL 0.9% saline for 14 days, and found a significant increase in ALP, AST, and ALT serum concentrations. Aparna et al. (2017) also agreed with our findings, they used 0.075, 0.1125, 0.1875, and 0.225 mg SeNPS/kg feed to 150 broiler chicks and found an improvement in glutathione expression, enhancement in liver enzymes, and serum proteins.
Selenium is known to have immune regulatory functions that reduce the negative impact of stressors. The current study revealed a notable increase in IgG and IgM concentrations in Ross ® 308 and Arbor Acres ® broilers supplemented with 1 mL SeNPS (100 mg/1 L), with more significant increase in Ross ® 308 broilers. The increase was significantly notable on the 19 th day in treated Ross ® 308 broilers and on the 38 th day in treated Arbor Acres ® broilers. The results were in agreement to those of Ahmadi et al. (2018) who reported a significant increase in immunoglobulin of one-day old male Ross 308 chicks supplemented with 0.1, 0.2, 0.3, 0.4, and 0.5 mg SeNPS/kg feed. Bakhshalinejad et al. (2018) were also in agreement with our results, they used 0.4 mg SeNPS/kg for 1200 one-day-old male Ross broilers and found an enhancement in IgG concentrations. Zamani Moghaddam et al. (2017) proved a significant increase of immunoglobulin in broilers supplemented with 0.3 mg organic selenium /kg feed.
Glutathione (GSH) and SOD are considered major antioxidant defenses against oxygen reducing metabolites. Meanwhile, TAC and MDA usually increased relevant to oxidative and environmental stressors of broilers (Gangadoo et al., 2018). Nanoselenium at rate of 1 mL (100 mg/1 L) was able to a meliorate TAC, SOD, and MDA activity in the recent study to alleviate the impact of microbial stress induced  by E. coli O157:H7 1.6 × 10 8 challenge on the 10 th day of age. The results were in agreement with Jiang et al. (2017) who reported that 0.3 ppm selenium-Yeast with 1.5% soybean or linseed oil was able to enhance carcass quality and oxidative stability even in the presence of stressors. Bakhshalinejad et al. (2018) reported outcomes supporting the current results, they used 0.4 mg SeNPS/kg in male Ross broilers and revealed a significant increase in GSH-Px, TAC, and MDA activity and formation.
TBC and TEC were significantly reduced in the intestine and breast muscles in the current study after supplementing Ross ® 308 and Arbor Acres ® broilers with 1 mL SeNPS (100 mg/1 L). An intergroup variation in TBC and TEC counts with significant increases on the 39 th day were attributed to overwhelming microclimatic conditions. The reduction of overall pathogenic bacterial counts might be attributed to the ameliorated immunity conditions. The results agreed with those of Barko et al. (2017) who reported that SeNPS was able to modify intestinal microbiota to compete against pathogenic micro-organisms and restrict their survival. Underwood (2014) recommended a dose of 0.9 mg SeNPS/kg to encourage the beneficial growth and proliferation of opportunistic microorganism in broilers' gut to overcome the growth of the pathogenic ones. The recorded antibacterial action of 1 mL SeNPS (100 mg/1 L) was supported by the in-vitro antibacterial action recorded by Gangadoo et al. (2016) using SeNPS against poultry pathogens including Escherichia coli. Trang et al. (2017) conducted an in-vitro study to evaluate the antibacterial action of SeNPS, in agreement with the current results, they found dose-dependent antibacterial action by using 10 µgSeNPS/mL against Staphelococcus aureus, Listeria monocytogens, E. coli O157:H7, and Salmonella.
The influence of selenium on productive performance in different broiler breeds are somewhat changeable. In our study, supplementing 1 mL SeNPS (100 mg/1 L) had an enhancing effect on the productive performance, despite of the individual variation among groups in relation to age. Similar results had been established to other studies as Senthil et al. (2015) and Selim et al. (2015), suggesting that addition of SeNPS both in diets or in drinking water was more effective compared to inorganic selenium to get better performance for broilers. However, our results were in contrary with the results recorded by Rao et al. (2013) and Chen et al. (2013), who recorded in their studies a non-significant difference in growth performance of broilers supplemented with SeNPS.
Our results revealed that 1 mL (100 mg/1 L) of inorganic selenium and selenium nanoparticles (SeNPs) were not able to enforce enough protection against E. coli pathognomonic lesions and in Arbor Acres ® broilers represented as severe perihepatitis and pericarditis that extended to the myocardium resulting in myocarditis. Changes recorded in the liver and heart in the present study are consistent with those of Manimaran et al. (2003). Also, the recorded histopathological lymphoid depletion in bursa, thymus, and spleen was in agreement with the results recorded by Nakamura et al. (1990) who reported that E. coli infection induce damage in the immune systems of the chickens including lymphocyte depletion in both the bursa and the thymus.
Meanwhile, our results revealed a higher degree of protection against E. coli lesions in Ross ® 308 broilers supplemented with 1 mL (100 mg/1 L) SeNPs, as we recorded mild hepatic lesion, mild pericarditis, and no pathological changes were recorded in the spleen, bursa, and thymus. On the other hand,1 mL (100 mg/1 L) of inorganic selenium in Ross ® 308 broilers gave moderate protection against E. coli lesions as mild perihepatitis, mild pericarditis, and mild to moderate lymphoid depletion of the spleen, bursa, and thymus. These results came inconsistent with Jia et al. (2005) who reported that selenium is an essential trace element that is commonly used as an antioxidant for animals, but there is a very narrow margin between the safe and toxic nutrient levels. Also, Qu et al. (2017) reported that, a new source of selenium is selenium nanoparticles (SeNPs) which exhibit the following properties: antioxidant, antibacterial activities, lower toxicity, better absorption and bioactivity. In addition, SeNPs decreased the oxidative stress in the hens, and improved the avian immune system status by alleviating the immunocompetent cells ability for an antigen challenge.
In the same line, selenium is a necessary trace element, which has antioxidant activity, this has been sustained by both clinical and laboratory experiments (Boostani et al. 2015).

CONCLUSION
The addition of 1 mL SeNPS (100 mg/L) rather than inorganic selenium (100 mg/L) per liter of drinking water was able to enhance some aspects of behavioral patterns, biochemical profile, immune and antioxidant functions, and decreased TBC and TEC inline with microbial stress from challenge and surrounding microclimate in Arbor Acres ® and Ross ® 308 broilers with enhanced influence in Ross ® 308 rather than Arbor Acres ® broilers.
By the end of this study, results provided too many new question marks to be answered as: can lower doses and concentrations of SeNPS in drinking water produce the same stimulatory influence on the broiler breeds used and other breeds? Can lower doses and concentrations of SeNPS in drinking water minimize the influence of stressors other than biological?

AUTHORS' CONTRIBUTIONS
ESS designed the experiment, participated in the preparation, executing the experiment, and in writing the manuscript. AAA participated in executing the experiment, and in writing the manuscript. RTH conducted the histopathological examination and participated in writing the manuscript, RAH participated in executing the experiment and in writing the manuscript, OMAB prepared SeNPS, ran the characterization, and participated in writing the manuscript. MSH participated in writing the manuscript.