Evaluation of Nano Selenium on Rabbit growth and Immunity

Since rabbit production makes up only a very small percentage
of the total animal production in the world...

wide range of applications. Many of these benefits result from the ultrafine dimensions of Nano-particles, which enable them to reach new locations in the body, and thus can perform their biological functions more efficiently [4]. According to many literatures, consumers are more concerned about food produced by the application of Nanotechnology [5]. Nano-Se has attracted more attention because of its high bioavailability, high catalytic efficiency, strong adsorbing ability and low toxicity compared with selenite in rats (Jia, et al.), mice [6] and goats [7]. The immune system provides body defense against invading microorganisms as well as being importantly involved in tissue repair after injury. Immune responses can be divided into nonspecific (or innate) and specific (or acquired) immunity [8]. Non-specific immunity exists from birth and operates in a generalized fashion without regard to the exact identity of the pathogen. It serves as the body's first line of defense against invasion.
There are a wide variety of innate immune defenses, ranging from anatomic (e.g., the integrity of the skin; tears that wash out bacteria), to physiological (e.g., lysozymes in mucus cleave bacterial cell walls; acid in the stomach kills pathogens), to phagocytic (e.g., macrophages engulf and destro bacteria) [9]. Both non-specific and specific immune responses are regulated by mediators such as cytokines. Cytokines are a diverse group of non-antibody proteins that act as mediators between cells. They were initially identified as products of immune cells that act as mediators and regulators of immune processes, but many cytokines are now known to be produced by cells other than immune cells and they can have effects on non-immune cells as well. Cytokines are currently being used clinically as biological response modifiers for the treatment of various disorders. Molecular understanding of signaling mechanisms orchestrating the immune responses is required to define new targets for future treatments of bacterial infections [10]. Staphylococcus aureus (S. aureus) is an adaptive opportunistic pathogen, capable to persist and replicate under various conditions. This microbial species causes a wide range of diseases in both men and animals [11].
In rabbits, problems of staphylococcosis arise when S. aureus bacteria infect small dermal lesions and invade subcutaneous tissue [12], provoking a number of lesions including pododermatitis, visceral abscesses and mastitis [13]. Occasionally, abscesses in internal organs, most commonly lungs, liver and uterus, are observed [14]. This research work studies the evaluation of Nano selenium as a feed additive in enhancing the processes of growth and immunity in rabbit (Oryctolagus cuniculus). Growth will be evaluated through growth parameters and measurements. Evaluation of immunity will be carried out based on molecular studies by examining some immune genes expression (interleukin 6 (IL6), interleukin 1beta (IL-1β) and tumor necrosis factor (TNF-Α α)) as a proinflammatory cytokines after Staphylococcus aureus experimental infection (Challenge) using the high technology of real-time PCR (qPCR).
Immunity will be assessed also on a biochemical bases exemplified by nitric oxide and lysozymes activity measurements . Finally, comet assay will be carried out to assess if there is a hazardous effects of using nano selenium in comparison to bulk selenium on rabbit tissues DNA?

Ethical Approval and Place of Experimental Work
Rabbits were handled according to the EU Directive 86/609/ EEC on the protection of animals used for experimental and other scientific purposes. The experimental work of the present study was carried out at the Animal Reproduction Research Institute, Giza, Egypt.

Experimental Animals, Batteries , Feed and Management
Sixty female White New Zealand rabbits were purchased from El-fayoum farm, 8 weeks old, with average weight of 1 to 1.5 kgs and housed in batteries in a special lab. The rabbit room was naturally ventilated by wired windows and provided with sided fans. Each cage had its feeder and automatic nipple drinker, feed and water were offered. All animals were kept under the same managerial, hygienic and environmental conditions and were maintained and treated according to the accepted standards of animals treatment

Feed and Feed Supplements
The experiment was conducted, and ration was purchased with factorial arrangement of treatments, being selenium additive-free rabbit ration. Ingredients and chemical analyses of the basal diet are presented in Table 1 following NRC (1977) recommendations.

Selenium Additive
In the present study, selenium bulk (Sodium Selenite ) was supplied by the Egyptian National Research Center. The use of Green DOI: 10.26717/BJSTR.2020.32.005244 synthesis to generate selenium Nanoparticles was demonstrated so, the procedure is free from the drawbacks of using any chemicals.
Green synthesis of selenium Nanoparticles (Se NPs) was achieved according to Ramamurthy,et al. [15], by a simple biological procedure using the reducing power of iron present in the fenugreek (Trigonella foenum-graecum L, Fabaceae family ) seed extract. The plant is cultivated worldwide as a semi-arid crop (Table 1). Fenugreek seeds were purchased from a super market then washed well and left overnight to dry, dried seeds were crushed well and turned into power, five grams of the powder were added to 500 ml distilled water (DW) and left for 24 hrs to obtain extract.
Filtration of the solution was carried out by using filter paper to obtain a pure extract. A mixture of 0.02 gm of selenium + 2ml ethanol + 1ml ascorbic acid was prepared and two ml of the fenugreek extract were added to the mixture prepared. The solution was shacked well then placed in a dark bottle at room temp for 24hrs. After 24hrs, the extract was turned from white into red color ( Figure 1) and selenium was expected to be converted into Nano selenium. Chetosane added to Nanoselenium as stabilizer ( Figure 1).

Scanning Electron Microscope Analysis
Scanning analysis using scanning electron microscope (SEM); JEM-2100) was carried out in the National Research Centre, images were taken for the analysis of size and shape of SeNPs. The size of selenium Nanoparticles was ˂50 nm.

Experimental Design
Selenium was added to the ration in the designed rates, Rabbits (no. 60) were housed in grow-out batteries with access to water and commercial feed, they were randomly divided into 5 groups (Control and 4 treatments). Se was added to the ration in two concentrations ( 0.1 and.3 mg/kg). Feeding was continued for two months.

Growth Parameters Estimation
Body weight (BW) was measured at the beginning of the experiment (8th week of age).Growth performance was calculated as weight gain through an equations: Weight gain (WG %) = (final weight -initial weight) × 100/ initial weight

Challenge Test
To the evaluate the immunity function in rabbits after feeding for 2 months on bulk and Nano-forms of selenium supplemented feed, a challenge test was carried out through Staphylococcus aureus experimental bacterial infection in rabbits and examination of some immune genes expression.

Experimental Infection
Rabbit groups were immunized by experimental infection with sub lethal pathogenic bacteria Staphylococcus aureus (8×10 8 CFU).

Clinical Investigations
Rabbits were clinically investigated for disease symptoms and investigated for the internal organs and tissue lesions.

Samples Collection
Blood Sampling and Analysis of Some Immune Response:

Estimation of Serum Nitric Oxide and Lysozymes Levels:
Measurement of nitric oxide was assessed according to the assay described by Ramadan, et al. [16].

Molecular studies (IL6, IL1-β and TNF-Α Genes expression):
Gene expression was assessed using Real-Time PCR, RNA Extraction was carried out using Gene-Jet RNA Purification Kit following the manufacture's recommended protocol, cDNA Synthesis: using

Cell Gel Electrophoresis (SCGE)/ Comet Assay:
The comet assay was carried out according to the technique described by Singh, et al. [17], a protocol that can be used to detect DNA damage.

Statistical Analysis
The obtained data in the present study were statistically analyzed using the computer program (SPSS version 15 for Windows), and comparison were made using one-way ANOVA.

Results
The scanning electron microscope (SEM) image of Nano-Se is shown in Figure 2. It can be seen that the particle sizes is ˃ 50 nm in diameter, they were spherical in shape and there were some agglomerations among the NPs.

Rabbit Mortality Rates
No mortalities were recorded along the course of the experiment in all the control and the challenged groups.

Clinical Investigation after the Experimental Infection
Experimentally infected rabbits showed some illness exhaustion, and lack of appetite for food, examination of the internal organs revealed cases of lung hematoma and some hemorrhagic patches in 40% of the cases. Macroscopic examination revealed deeply red consolidated area in the lungs of the infected rabbits ( Figure 3).

Growth Performance in Rabbits
The effects of feeding diets supplemented with selenium and Nano-Selenium for two months on weight gain in rabbits are presented in Figure 4. The effects of dietary Se supplementation on the growth performance of NZW rabbits showed that, among the tested groups of conventional and Nano-Se supplementation, it was noticed that; the weight gain of rabbits increased than the control group which was fed on ration without any supplementation.
There was a positive relationship between the increase in the concentration of Se and the weight gain in groups fed on Se supplemented feed (Either bulk selenium or Nano selenium) with observation that Nano-Se supplementation enhanced the growth rates more than bulk selenium in respect to the two concentrations  Table 3). Values expressed as means ± SE (n = 3). Different superscript letters indicate significant differences for each pair comparison.

Figure 4:
The weight gain in New Zealand (NWZ) rabbits after two months feeding on diets supplemented with selenium (0.1 mg/kg. and 0.3mg/kg.) and Nano-Selenium (0.1 mg/kg. and 0.3mg/kg.). Values are expressed as mean ± SE.

Lysozymes Analysis and Nitric oxide Production Analysis
Data of Lysozymes and nitric oxide production in the present study is illustrated in Figure 5. It was noticed that no specific or

Expression of IL6, IL1-β and TNF-Α Genes Response to the Bacterial Infection
Real time PCR results were analyzed, for all samples, dissociation curves were acquired for quality control purposes.
In addition, amplification products were visualized by agarose gel electrophoresis. For gene expression quantification, we used the comparative Ct method. First, gene expression levels for each sample were normalized to the expression level of the housekeeping gene encoding GAPDH within a given sample (∆Ct); the difference between the treated groups (Fed on selenium bulk and Nano) compared to the control (feeding without any selenium supplementation) was used to determine the ∆∆CT. The log 2 ∆∆CT) gave the relative fold change in the gene expression of the test versus the control condition. The relative changes in mRNA transcript levels for IL6, IL 1β and TNF-α were presented in Figure   6. The results clearly demonstrated that the mRNA expression of the three genes under investigation was increased in tissues of rabbits fed on ration supplemented with Nano Se in comparison to rabbits fed on feed supplemented with bulk selenium within the same concentration of selenium used either 0.1 or 0.3 mg/kg, but the values were comparable between the 0.1 mg/kg Nano Se group and the 0.3 mg/kg bulk se group. All Se treated groups revealed better expression of studied genes when compared to the control group with observation that groups fed on Nano Se gave higher expressions over the other groups ( Figure 6).

Comet Assay Data Analysis
Comet assay or single cell gel electrophoresis has been shown to be a simple and sensitive genotoxic approach for evaluating DNA damage (Frenzilli, et al.). In this study, comet assay was carried out to assess the hazard effects on DNA after using Nano selenium in comparison to bulk selenium. Results indicated that there are a rate of comet cells among different rabbit groups (Figure 7) ranged between 13.15% and 17.08% with a comparable values (Figure 8).
There is an observation that the percent of comet cells was higher in rabbits group which were fed on ration supplemented with Nano selenium (Concentration = 0.1 mg/kg feed) but still near the rate in other groups even the control ( Figure 8 & Table 4). Concerning the tail length and DNA percent in comet tails, Table 4 illustrates that the tail length was lower in comet cells of rabbits fed on feed supplemented with se than the control which had a feed without se.
Moreover, the shortest tail length appeared in cells of animals fed on nano se concentration (0.3mg/kg). The DNA percent in the tail showed irregular image and had no specific trend.   Ebeid, et al. [21] reported that feeding male California rabbits on diets supplemented with organic Se (0.15 or 0.30 ppm) resulted in an increase in live body weight and daily weight gain compared with their control counterparts and these results are consistent with the results of this work. The different physiological effects of Nano-Se and sodium selenite may be attributed to the different absorption process and metabolic pathways. It has been reported that the superior performance of nanoparticles may be attributed to their smaller particle size and larger surface area, increased mucosal permeability, improved intestinal absorption and tissue depositions, while nanoparticles show new transport and uptake characteristics and exhibit higher absorption efficiencies [22].
Mohapatra, et al. [23] found that feeding diets supplemented with nano Se up to 0.30 mg/k g from 9 to 20 weeks of age positively affected feed conversion ratio of pullets but when the level of added nano Se reached 0.60 mg/kg FCR was negatively affected, this in part is matching our results where, nano Se raised the growth rates but was positively consistent with the increase of concentration.
The improvement in the performance of rabbits due to Se supplementation may be due to that decreases the oxidative stress and helps in the scavenging of ROS from the tissues, because Se is a cofactor for many antioxidant enzymes such as glutathione peroxidase and thioredoxin reductase [24]. However, data of this work in investigating growth rates resulted from feeding on nano Se feed additive was inconsistent with the study of Dokoupilova, et al. [25] who noted that added Se (Se-enriched yeast) did not affect FCR of growing rabbits. Also, Dorra, et al. [26] found that addition of nano-Se had no significant effect on body weight gain (WG) of rabbits at all age intervals and the whole experimental period. In general, these differences in results and opinions could be due to the species-specific differences or the differences in diet contents and the nano material types and levels [27]. Infected rabbits had clinical symptoms of weight loss, coughing and purulent nasal discharge, haemorrhage in trachea and purulent pneumonia was found in dead rabbits [30], These results are consistent with our findings in the concern. Blood hematological parameters was found to be affected by Se addition where nano se increased the WBC counts, hemoglobin levels, RBCs and neutrophils values if compared to bulk se. When comparing the effect of SeNPs with the key feature of pathogen restriction by targeting and disrupting the glycoside bonding of the peptidoglycan substrate [35].
Lysozyme activity is known to be part of a non-specific immune re-sponse and is found in leukocytes. It is a natural enzyme that acts as an antimicrobial and has had an immune modulating effect. It serves as a non-specific defensive mechanism, representing the behaviour of macrophages [36]. The activation of phagocytic activity would therefore be reflected in the activity of the serum lysozyme.
The present study has revealed that, dietary administration of Se additive for 60 successive days, either nano or bulk showed revealed no specific trend of elevating of the values of lysozymes, moreover the control had higher values in all groups. Carroll, et al. [37] in their study reported that, Although normal rabbit serum and plasma serum also contain identical (though reduced) levels of lysozyme, they differ significantly in their bactericidal ability. These results clearly indicate that the predominant antibacterial element in rabbit serum is not lysozymes and that the exact source of serum bactericide in these animals is not the same. They continued to indicate this explanation as Further evidence that lysozyme is not the primary bactericidal agent in rabbit serum comes from an examination of the purified enzyme in the standard killing assay.
When tested at concentrations equivalent to those in 2 to 5% serum or plasma, rabbit lysozyme exhibits little bactericidal activity.
In another study of Weksler, et al. [38] they said that, differences observed for rabbit serum and plasma serum indicate that activity is "released" from cellular elements during coagulation or immune injury, and several investigators have presented evidence suggesting that platelets are the source of rabbit serum bactericides.
The role of lysozyme in host defense against infection remains relatively elusive although elevated circulating lysozyme concentrations were evidenced after antigen stimulation [39]. In the same way, Pawlikowska, et al. [40] reported that lysozymes concentrations increased before the efficient production of specific antibodies. Georgieva, et al. [41] in their study to evaluate the changes in the concentrations of major blood proteins associated with experimental E. coli infection in New Zealand rabbits revealed that E. coli infected rabbits exhibited considerable increases of the serum lysozymes concentrations on days 11 and 18, confirming the role of this protein in the innate immunity and that lysozyme can be considered as a late major positive acute phase proteins (APPs) with its low values, data from our work is consistent with those studies and it could be proved that the low values of lysozymes in this study may be due to that lysozymes is a late major positive acute phase proteins (APPs) and there are other immune elements that had the main role as a bactericidal agent.
The relative changes in mRNA transcript levels for some innate immune genes exemplified by IL6, IL1β and TNF-α were analyzed.
Results clearly demonstrated that the mRNA expression of these genes was unregulated or increased in spleen and liver of the rabbits fed on nano Se more than bulk organic Se when compared with the control group. These results revealed feeding on nano Se supplemented diet, at 0.3mg/kg showed the highest expression status compared with the other protocols. Such amelioration of proinflamatory cytokines expression as an immune response to bacterial infection can be ascribed to the activation effect of Se on signaling pathways that react with the signal transduction of Tolllike receptors in monocytes and thereby stimulate the secretion of pro-inflammatory cytokines [42]. In parallel with our result, Schnupf, et al. [43] reported that, during the natural infection, Shigella infection of rabbit ileal loops led to a large increase in mRNA abundance for the pro-inflammatory cytokines IL-1b, TNFa, IL-6, IL-4, IFN-c and IL-8.
Using nano ZnO as a feed additive in rabbits, they said that the administration of zinc nanoparticles to growing rabbits as an alternative to conventional zinc sources could elicit favorable influences on growth performance, serum parameters and splenic expression of IL6. Also, a study conducted by Sundaresan, et al. [44] reported that the expression of IL6 in the ovary and oviduct In this work, assessment of genotoxicity by the single cell gel electrophoresis (SCGE)/ comet assay was carried out to assess if there is any hazard effect in using nano se supplement. Dietary supplementation of nano se versus bulk organic se for rabbits produced comparable rates of comet cells like the control after 60 days post immunization. With regard to tail length and DNA percent in comet tails, Table 4 shows that the tail length in comet cells of rabbits fed on feed complemented with se was lower than the control which had se-free feed. In addition, the shortest tail length was observed in cells of animal that fed on nano-se (concentration 0.3 mg / kg). The DNA percentage in the tail displayed an unusual picture and had no clear pattern. As SeNPs have been shown to be a promising agent to regulate chronic toxicity caused by exposure to heavy metals. Prasad, et l. [49] studied the effects of T-synthesizing SeNPs. Arjuna leaf extract on human lymphocytes treated with arsenite (As+III) [50][51][52][53][54][55].
Studies of cell viability using MTT assay and DNA damage using comet assay showed the protective effect of SeNPs against As+IIIinduced cell death and DNA damage. This approach may be used in the future to mitigate ROS-induced arsenic-mediated toxic hazards, especially in areas with arsenic-contaminated groundwater and the prevalence of arsenicosis. However, if nano-selenium has been used as a protective agent against induced cell death and DNA damaging materials, it is a fact that it does not have the character of causing cell death or DNA damage. Finally, We may infer that Nano-se supplementation is capable of causing positive changes in the blood composition, making it possible to boost the growth efficiency of growing NZW rabbits and improve its immunity more than supplementation with bulk Se [56][57][58][59][60].

Conclusion
From this study it could be mentioned that administration of Se nanoparticles to growing rabbits as an alternative to conventional Se sources could improve growth performance, immune system function utilized by some hematological parameters, serum No and expression of some proinflamatory cytokines like IL 1-β, IL6 and TNF-α. From the economic point of view 0.3 mg/kg of Se nanoparticles is the optimal concentration for use. Comet assay revealed that using nano selenium showed no hazardous effect on liver and spleen cells, but still a great efforts in research is needed to improve our knowledge of SeNPs involvement in the functionality of organs and the potential role of SeNPs in unwanted disorders.

Conflict of Interest
Authors hereby that there is no conflict of interest concerning this work. also to prof. Iman Abumourad for her support in the molecular work.