Extraction of bacterial selenium content
In the current study, Se enriched bacterial strains identified as Enterobacter cloacae (ADS1), Klebsiella pneumoniae (ADS2), and Stenotrophomonas maltophilia (ADS18) were used as a source of bacterial organic Se. The stock culture of ADS1, ADS2, and ADS18 strains prepared at the Laboratory of Microbiology, Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia (UPM) and the sonicated Se-enriched bacterial cells were produced according to procedure described by Dalia et al. . The extraction of seleno protein from Se-enriched bacterial cells was carried out using dialysis technique The dialysis process was performed using dialysis sacks of flat width 25 mm, 12,000 Da, (Sigma-Aldrich) against deionised water, which was changed every 12h for a total of 96 hours to separate inorganic Se from organic form . The content in the dialysis tube was lyophilised and then used as source of bacterial Se.
Birds and experimental procedure
The birds handling and use in this study was carried out in compliance with the research policy guidelines of UPM on Animal Welfare and Ethics. A total of 180 one-day-old (Cobb 500, Arkansas, USA) female broiler chicks averaging 40 ± 0.13 g were obtained from a commercial hatchery. The chicks were individually wing-banded on arrival at the farm. The birds were randomly divided into five treatments fed the basal diet (Table 1), each with six replicates of 6 birds per replicate. The dietary treatments consisted of the basal diet supplemented with (0.3 mg/Kg feed sodium selenite), (0.3 mg /kg feed ADS1 Se), (0.3 mg /kg feed ADS2 Se), and (0.3 mg /kg feed ADS18 Se) in addition to the basal diet treatment served as control group. Starter diet was offered from 0 to 3 weeks old and finisher from 4 to 6 weeks old. Water and feed were given ad libitum to all the chickens. Experimental birds were housed in UPM- farm (Ladang-2) using semi-closed system. Lightening was 12h per day. All the birds subjected to vaccination against bronchitis (IB) and Newcastle disease (ND) on day 7, and against infectious bursal disease on day 14 through the intraocular route.
On completion of the experiment, 12 birds from each dietary treatment (2 birds from each replicate) were selected at random, then weighted and sacrificed. The slaughter procedure was conducted at the Department of Animal Science slaughter house, Faculty of Agriculture, Universiti Putra Malaysia. The animals were humanely slaughtered by a licensed slaughter man. The procedure involved severing the carotid artery, jugular vein, trachea and esophagus. Blood samples were taken directly from the neck vein into a vacuum tube (BD Vacutainer®, NJ. USA) containing anticoagulant (EDTA) and directed to hematological analysis using hematology analyzer (CELL DYN 3700, Abbott USA).
Plasma immunoglobulin concentration
At days 21 and 42, 6 birds per treatment were randomly selected and blood samples were collected into vacutainer tubes containing ethylene diamine tetra acetic acid (EDTA). Blood samples were mixed gently before storage in ice, followed by centrifuging at 3000 rpm for 15 min at 4 °C, and the plasma was stored at −80 °C until antibody analysis.
Plasma IgA and IgM were determined using (Chicken IgA ELISA, Immunology Consultants Laboratory, Inc. USA) and (Chicken IgM ELISA, Immunology Consultants Laboratory, Inc., USA), while Chicken IgG determined using (CEA544Ga, Enzyme-linked Immunosorbent Assay Kit, Cloud-Clone Corp., USA). All the analysis performed according to the procedure recommended by the manufacturer. The absorbance was measured at 450 nm wavelength using micro-plate reader (Infinite® 200 PRO, TECAN).
Twelve representative samples from each batch of feed (starter and finisher diets) were collected randomly and kept at −20°C until further analysis. Total excreta collection was performed on days 17, 18, 19 and 20 for starter diet and on days 38, 39, 40, 41 for finisher diet. Feed and fecal samples were analyzed for Se concentration using ICP.MS. Determination of Se retention was calculated using mass balance method  as follows:
Se retention (%) = (Ingested Se − Excreted Se) × 100
Ingested Se = daily feed intake × analytical feed Se concentration
Excreted Se = daily feces weight × feces Se concentration.
Histomorphology of small intestine
Intestinal morphology was done employing the method stated by Choe et al. . The intestinal samples of 6 birds/ treatment were collected at days 21 and day 42. Approximately 5 cm segments of the ileum (midway between the Meckel‘s diverticulum and ileo-ceacal junction), middle portion of the duodenum (apex section), and jejunum (midway between the end point of duodenal loop and Meckel‘s diverticulum) were cut gently and washed with phosphate buffer saline (PBS) and fixed in 10% neutral buffered formalin. Then, the intestinal samples were dried for 16 h in an automatic tissue processor (Leica ASP 3000, Tokyo, Japan) and embedded in paraffin wax following a paraffin embedding system (Leica EG 1160, Japan). Each sample was cut at 4 μm with a rotary microtome machine (Leica RM 2155, Japan). Sections of size 4 mm were fixed on glass slides, heated at 57oC until dried, and stained with hematoxylin and eosin (Appendix F). The distance from the tip of the villi to the villus crypt junction represented the villus height, while, crypt depth was described as the depth of the invagination between 2 villi and was determined employing Image-Pro Plus software as described by Touchette et al. . A total of 5 villi sections per slide were evaluated in each of 6 replicate slides per intestinal sample (30 measurements for each sample) and studied with light microscope (Dialux, LeitzWetzlar, Germany) fitted with a digital camera (Laice, Germany).
An ANOVA was carried using six replicates per means. Differences between treatments were studied using one-way ANOVA . Duncan test was used to establish the significant differences among the treatment groups at a significant level (P < 0.05). The data of Se retention was analysed using GLM procedure suitable for Completely Randomized Design . Treatment differences were established by orthogonal contrasts
(1) Basal diet vs. Se supplemented diets,
(2) Sodium selenite vs. bacterial organic Se,
Values of P < 0.05 were accepted as significant.