Effect of Organic and Inorganic Dietary Selenium Supplementation on Gene Expression in Oviduct Tissues and, Antioxidant Capacity of Laying Hens

Some functional genes were investigated for their involvement in egg (eggshell biomineralization) formation and selenoproteins in the oviduct and liver of laying hens fed different organic and inorganic selenium source. A total of 24 hens were selected randomly from the four treatments and slaughter. Uteri and liver tissue samples were collected from hens during the active growth phase of calcication (15 - 20 h post-ovulation) for RT-PCR. The basal diets supplemented with 0.3mg/kg of different organic Se sources and sodium selenite upregulate uterine and selenoproteins mRNA levels. This research reaps the advantage of tissue sampling from specialized segments of the oviduct that consecutively form different egg components. Expression of OC-17 and OC-116, and OC-17 were signicantly higher in the uterus and magnum of laying hens, respectively. Their higher expression was observed with organic Se (bacterial selenoprotein or Se-yeast) fed-hens. The results may postulate the ecacy of organic Se in enhancing the expression of functional genes involved in the egg (eggshell biomineralization) formation and selenoproteins compared to inorganic and non-Se supplemented hens. This study proposed the ecacy of bacterial selenoprotein in the upregulation of the uterine genes and hepatic selenoproteins in laying hens. to Gallus gallus sequences Table 2 . The synthesis of rst strand cDNA was run by reverse transcription of 1 µg isolated total RNA (20 µl reaction mixture) using QuantiNova Rev Transcription Kit (cat. No. 205413, Qiagen, Hilden, Germany). The reaction was done in a Bio-Rad thermal cycler (MyCycler, Germany). Master mix was prepared as per the manufacturer’s protocols. Real-time PCR was then performed using QuantiNova SYBR Green PCR Kit (cat. No. 208054, Qiagen, Hilden, Germany) on a Bio-Rad CFX Manager™ 3.1 realtime PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Each reaction (20 µL) contained 10 µL QuantiNova SYBR Green Master Mix, 1 µL of each forward and reverse primers, 7 µL of nuclease-free water and 1 µL of cDNA. The qPCR reactions were carried out following standard cycling mode as per kit protocol. Three tissue were used from each hen to determine their stable house-keeping genes using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin (β-actin) and TATA-Box Binding Protein (TBP). The target genes were analyzed in duplicates and their expression level was determined using cycle threshold (Ct) values following standard curve method after normalization with reference genes. Genes of interest were amplied through the following thermo cycling program: reverse transcription at 95 °C for 10 minutes, rst denaturation at 95 °C for 2 minutes, then 40 cycles of denaturation at 95 0 C for 5 s, and combined primer annealing/extension at 60 0 C for 10 s. The uorescent data were acquired at the end of each annealing step during PCR cycles a construct melting assess specicity of PCR amplication. A real-time PCR was run for each pair of primer in which cDNA samples were replaced with distilled water to ascertain the absence of exogenous DNA. The eciency of amplication was determined for each primer pair using cDNA serial dilutions utilization. The fold changes for each target gene was calculated using power of 2 (-ΔΔCT) method described by 90 . The fold changes in data were shown as mean ± standard error. The values were subjected to one-way analysis of variance (ANOVA) followed by Duncan multiple range test for mean comparisons to determine signicance at P-value < 0.05 on a SAS (Statistical Analysis System, Version 9.4). The result was compared and presented as a fold change between treatments and the control group.


Introduction
Selenium (Se) is an essential trace element required for numerous physiological functions in animals, ranging from immunoregulatory function 1,2 , reproduction 3 , and protecting the tissue damage by the maintenance of the antioxidant system 4,5 . The mechanisms in which Se functions solely is applied via Se-containing proteins 6 . Selenocysteine is the major part by which Se exerts its biological role within a living system after incorporation into selenoproteins 7 . Consequently, selenoprotein levels and the yield of selenoprotein mRNA levels depend on Se availability. In chickens, more than 25 unique selenoproteins were recognized and play key roles in their catalytic activity site. Among which were identi ed in humans and animals include glutathione peroxidases, iodothyronine deiodinases, and thioredoxin reductase 8 .
The synthesis of selenoproteins is pretentious by nutritional form and levels of Se supplementation in the diet 9,10 . A substantial number of research reports con rm a positive connection between dietary Se supplementation and the expression of selenoproteins in animal tissues. The GPx activity signals as a biomarker of Se status 11 , and the Se-de cient diet downregulate the expression of selenoproteins in broiler muscular stomach 12 . Similarly, Zhang et al. 13 reported downregulated mRNA levels of 14 selenoproteins genes, and 9 selenoproteins genes upregulated in the low-Se diet group, but neither the DIO3 nor SEPX1 mRNA levels was affected in broiler kidney. Excess Se was reported to down-regulate the expression of GPx4 mRNA levels in chicken liver 14 . Moreover, elevation in liver mRNA of SELW1 was observed for broiler fed with Se (Na 2 SeO 3 )-supplemented diet for 90 days 15 .
The Se form and duration of its supplementation signi cantly in uence the reproductive performance in poultry 16,17 .It was observed that Se supplementation (< 8 wk) of either form or source has an insigni cant effect on hen reproductive parameters 18,19 . On the other hand, birds supplemented with organic Se for a period beyond 12 weeks enhanced egg production, fertility, hatching performance in broiler breeder hens' 20,21 , layers 22,23 , and duck breeders 24 . Nevertheless, it is obvious that Se supplementation affects reproductive performance in hens, yet very little is known on how it modulates these effects. Recently, advancements in genomic technologies, mainly using species-speci c microarrays could aid investigation and explain how gene expression pro les are affected by nutrients, thus affecting cellular function. It may serve as a tool for providing useful data on how different forms of nutrients modulate their consequences on production and reproductive performance. Laying hen oviduct is consider a conducive environment biologically for egg development and potential fertilization 25 . The biological process involved in mineralization of the hen eggshell is highly complex, and resulted in the e cient calcium mobilization and biomineralization 25 , from the bloodstream via the uterine transepithelial cells, and nally into the uterine uid, which bathes the eggshell 26 . Also, the activation of genes in the biological process of calci cation is tissue-speci c and timely 26,27 .
As reported in previous studies, sources of Se and its levels may show diverse responses in metabolic effects in animal tissues 26,28 . Besides, the Se bioavailability of either source and form is determined by the absorption pathways 29 . Different strains of microorganisms can be employed to produced organic Se via microbial reduction pathway. Among which is Stenotrophomonas maltophilia (ADS18) isolated from hot-spring water and linked with rich in organic Se-containing proteins which can be applied as Se source in poultry 11,30 . While it is evident that Se can enhance the antioxidant system, scienti c data about the effect of this new organic Se source on layers is limited, and to this edge and to our knowledge, there is no published study reported to investigate the effect of bacterial organic Se of ADS18 source on the expression of some uteri genes and selenoproteins in layers. Therefore, the present study was designed to investigate the e cacy of bacterial organic Se of ADS18 source as an alternative organic Se compared to inorganic Se; on mRNA expression of some selected selenoproteins, and uteri genes responsible for eggshell biomineralization in laying hens.

Results
Effects of dietary selenium supplementation on mRNA expression of eggshell matrix and cuticle proteins in uterus.
The relative expression of OC-17 and OC-116 showed signi cant changes (p < 0.05) in mRNA expression pro les between the experimental groups (Fig. 1a). The mRNA expression of OC-17 and OC-116 were higher (p < 0.05) in organic Se (T4 or T3) supplemented compared to inorganic Se (T2) and negative control (T1) of laying hens. Expression of OC-17 mRNA was down-regulated in hens received an inorganic Se (T2) diet compared to either hens fed with organic Se (T4 or T3), or negative control (T1). However, OC-116 mRNA expression increased (p < 0.05) in organic Se (T4 or T3) supplemented hens than sodium selenite (T2) or non-supplemented (T1) group. Furthermore, the mRNA expression of cuticle proteins (OCX-32 and OCX-36) in the uterus of the laying hens were in uenced by dietary Se treatments (Fig. 1b).
The expression of OCX-32 mRNA was up-regulated in only the organic Se treated group (T4 or T3), signi cantly different in inorganic (T2) and non-Se supplemented (T1) group. The mRNA expression of OCX-36 was highest in Se-yeast (T3) group followed by bacterial selenoprotein (T4), and sodium selenite (T2) hens, while least in non-Se supplemented (T1) laying hens, respectively.
Effects of dietary selenium supplementation on mRNA expression of eggshell matrix and cuticle proteins in magnum.
The effect of dietary Se supplementation of eggshell proteins gene expression in the magnum of laying hens measured by real-time PCR is shown in Fig. 1c, d. The expression of OC-17 mRNA was up-regulated (p < 0.05) only in hens supplemented with bacterial organic Se (T4) (Fig. 1c). However, there was a downregulation of the same gene in hens fed Se-yeast (T3) and sodium selenite (T2) with no signi cant (p > 0.05) between them, and inferior to the non-supplemented (T1) group. The OC-116 mRNA was downregulated with all the dietary Se treated groups compared with the non-Se supplemented group. Despite the down-regulation of OC-116 mRNA with bacterial selenoprotein group (T4), yet not statistically different (p > 0.05) from negative control (T1), however, both (T1 and T4) are superior (p < 0.05) than (T3) and (T2), respectively. Moreover, the results of the quantitative expression of OCX-32 and OCX-36 in magnum showed that the mRNA levels were in uenced by dietary Se treatments (Fig. 1d). The expression of OCX-32 mRNA was up-regulated in all dietary Se treatments with the highest expression in bacterial organic Se (T4). However, there was no signi cant (p > 0.05) increase in OCX-32 mRNA expression between Se-yeast (T3) and sodium selenite (T2) fed hens, but superior (p < 0.05) than negative control hens (T1). No signi cant (p > 0.05) difference was observed among all the dietary Se treated groups (T2 -T4), but signi cantly higher (p < 0.05) from non-Se supplemented (T1) hens of OCX-36 mRNA expression.
Effects of dietary selenium supplementation on mRNA expression of hepatic selenoproteins in the liver of laying hens.
The relative expression of some selenoproteins of hens supplemented with organic and inorganic dietary Se sources is shown in Fig. 2a, b. The hepatic expressions of GSH-Px1, GSH-Px4, DIO1, DIO2, TXNRD1, and SELW1 genes were studied. The expression of GSH-Px1 and GSH-Px4 (Fig. 2a) was in uenced by dietary Se supplementation. Organic Se (T4 or T3) supplemented hens showed higher expression of both GSH-Px1 and GSH-Px4, with a signi cant difference (p < 0.05) than the inorganic and non-supplemented groups, respectively. A signi cant difference (p < 0.05) was higher in T4 than T3, and other treatment groups for GSH-Px1. Similarly, organic (ADS18 or Se-Yeast) Se showed higher (p < 0.05) fold changes in mRNA expression of GSH-Px4 than inorganic (sodium selenite) and control group, respectively. However, hens who received a diet supplemented with inorganic Se (T2) also showed an increase in GSH-Px1 and GSH-Px4 mRNA expression compared to the negative control, although not statistically different (p > 0.05).
The fold changes in the relative expression of DIO1 and DIO2 mRNA levels in the liver tissue are shown in Fig. 2b. Higher fold change in relative gene expression of both genes was observed in T4 (ADS18) with signi cant differences among other treatment groups. Only bacterial selenoprotein (T4) was shown to be signi cantly (p < 0.05) different compared to other treatment groups, however, T3 and T2 were slightly above the negative control but not signi cantly different (p > 0.05) in mRNA expression of DIO1. Similarly, a signi cant increase (p < 0.05) in the DIO2 mRNA expression was observed in the liver of hens supplemented with bacterial organic Se compared to hens fed Se-yeast (T3), sodium selenite (T2), and negative control (T1), respectively. However, the Se supplemented groups hens recorded higher fold changes in the liver mRNA expression of DIO1 and DIO2 regardless of Se forms.
The mRNA expression of TXNDR1 and SELW1 genes trialed livers of hens fed different forms of dietary Se was shown in Fig. 2c. The signi cant difference in TXNDR1 mRNA fold changes was discerned in the liver of the T3 (Se-yeast) treatment group and signi cantly (p < 0.05) differed with negative control only. However, there was no signi cant (p > 0.05) difference noted in the liver mRNA levels among all the dietary Se supplemented groups. Furthermore, a signi cant fold change of hepatic SELW1 mRNA level was found in all the Se supplemented groups, with no in uence compared to the basal diet with no Se supplementation. However, hens received bacterial organic Se (T4) proved a signi cant difference (p < 0.05) compared to other treatment groups, nonetheless superior fold change in Se supplemented group (T2, T3, and T4) than the negative control group (T1).

Discussion
One of the most critical roles of the eggshell is to enfold and shelter the egg contents through its mechanical properties for optimum economic success in layer production 31 . The eggshell ultrastructure of hen is a highly ordered structure with unique mechanical properties crystal morphology and organic matrix 32,33 . Calcium carbonate contains 95% in its calcitic polymorph and 3.5% macromolecules of the organic matrix. Among many factors that determine its formation are physiological changes and the complex stages of egg calci cation involved uterine cells and uid constituents 32,26 . The formation of complex bio-ceramic eggshell arises from direct acellular uterine uid interaction of ions such as Ca 2+ and HCO 3 − and precursors of organic constituents 32,34 , with an uninterrupted action of cells 35 . Soluble precursors like proteins and minerals were released by uterus cells into the acellular uterine uid 33 . A solid layer is formed as a result of interaction between the developing crystal and organic shell matrix with greatly systematic microstructure and texture as eggshell by extraordinary mechanical properties 32,26 . The current study compared gene expression in the oviduct of laying hens supplemented with dissimilar Se sources. Egg formation and yolk ovulation are stimulated by reproductive hormones either during the active calci cation stage, thus, regulating the calcium metabolism 33 . Furthermore, genes either connected to the biomineralization process and, or supply the shell precursors could be upregulated. The chicken uterus plays a key role in the daily calci cation of the shell during 19 hours process though the egg remains in there. Moreover, a compact layer is formed due to the interaction between the developing crystal and organic shell matrix with largely systematic microstructure and texture as eggshell by great mechanical properties 32,34 . The proteins potentially involved in the biomineralization process were mainly focused on in this study. Numerous research has demonstrated their roles by interactions between these proteins and crystal formation 26 . However, studies on different Se sources on the expression of reproductive genes were not wholly researched, and to the best of our knowledge, there was no published data reported on the e cacy of bacterial organic Se on laying hens. In the current study, dietary Se supplementation affects the mRNA expression of all the examined genes by either up or down-regulation depending on the type of tissue. Physical egg quality factors such as shell thickness, egg shape, and elasticity are determined by the mRNA expression of OC-116 jointly with OCX-32 genes 32,36 . Devoid of these matrix proteins could result in the cessation of the mineralization process completely 37 . Fragile, shape stiffness, and thickness of eggshells are connected to irregular OC-116 gene expression or OC-116SNP variants 38 . Yin et al. 39  Similarly, 52 observed a higher expression of the OCX-32 gene in the distal oviduct (isthmus and uterus) and proximal oviduct (magnum and uterus), thus, con rm its secretion from the glandular epithelium of the shell gland 53 . Similarly, Jonchère et al. 53 and Brionne et al. 33 established that OCX-36 is shell gland speci c, and increase through the calci cation of eggshell. On the other hand, OCX-32 was highly expressed in isthmus than ovary and magnum, although lower than the uterus 31 . Similarly, there is a discovery of OCX-36 expression in isthmus and uterus and expected to participate in natural defense mechanisms because of its similarity to lipopolysaccharide-binding proteins and bactericidal permeability-increasing protein 31 . The ndings of Hrabia et al. 54 suggest that growth hormones may participate in the development and activity, and expression of some oviduct speci c proteins (OCX-32 and OCX-36) in the chicken. Further studies, however, are required to elucidate the fundamental mechanisms behind these responses.
The present study investigated the expression of selenoproteins in the liver of laying hens fed with two forms of organic Se from bacteria and yeast compared with an inorganic source (sodium selenite). The main organ and site primarily for nutrients (carbohydrate, protein, and fat metabolism) homeostasis is the liver 55 . The up-regulation and down-regulation of selenoproteins mRNA expression is dietary Se ingestion dependent 56 . Selenium has been reported to exert its physiological and biological roles primarily mediated via the activity of selenoproteins 15,57 , and lead to chemical and biological dysfunction with its de ciency 57 . In the current study, the mRNA levels of the hepatic selenoproteins in hen's liver signi cantly upregulated in organic Se compared to inorganic or non-supplemented group, which implied that organic Se may exhibit antioxidant properties, and ultimately reduced oxidative stress 58 . Contrary to inorganic Se, it is passively absorbed into the system with typical lower absorption rates 59  The iodothyronine deiodinase (DIO) family plays an essential role in thyroid metabolism 72 , and thioredoxin reductase (TrxR) genes which constitute a major cellular redox system in all living organism 73 . Furthermore, the qPCR analysis revealed that relative higher mRNA levels of DIO1, DIO2, TXNDR1, and SELW1 genes were expressed in the liver as well, an organ more responsive to changes in the levels and form of dietary Se 74 . As proved by previous studies, 75,74 , the ndings con rmed that Se sources and intake alter the mRNA levels of laying hens selenoproteins, and the effects vary greatly between different selenoproteins and tissues, although only liver tissue was investigated in this study. Lin et al. 75  SelW in layers liver only 15 . Conversely, supplementation of the inorganic form of Se (sodium selenite) leads to higher mRNA expression of GSH-Px1, SELW1, SEP15, and TXNRD1 levels in lamb liver, where GSH-Px 4 was unaffected by the treatment 74 . The results showed that DIO1, DIO2, TXNRD1, and SELW1 transcripts were upregulated in all the liver of Se supplemented groups of hens. In agreement with these ndings, TXNRD and GPX were observed to function in reducing free radical-mediated peroxidation and redevelopment post-Se supplementation to male Wistar rats 77 . Accordingly, higher Se supplementation may be responsible for preserving optimal activities of GPX and TXNRD, and partial detoxi cation against the negative effects of Cd in male rats 78,79 , and broilers 80 . A recent trial on the toxicity of Pb revealed that Se might alleviate the downregulation of GPX4, 2 and 1, DIO1, DIO2, TXNRD2-3, selenoprotein U, I, O, M, K, W, T, S 15 Sepx1, and Sepn1 expression in chicken cartilage tissue 81,82 . Similar results with Se-yeast and SeMet as organic Se sources upregulate GSH-Px1 and TXNDR1 mRNA expression in broiler breeders compared with sodium selenite 28,83 . Furthermore, SELW1 may participate in the protective role against H 2 O 2 , oxidative stress, and metabolic pathways 84,74,82 . Comparable data were published in rat testes 85 , and pig liver 57 . It is noteworthy, that the ndings showed a de nite trend of up-regulating selenoproteins (GSH-Px1, GSH-Px4, DIO1, DIO2, and SELW1) mRNA expression signi cantly (p < 0.05) with bacterial organic Se supplementation, except for TXNDR1 with Se-yeast hens compared to the negative control. Furthermore, the ndings suggested that DIO2 mRNA may be more sensitive to regulation by bacterial organic Se status than others, and perhaps the different response of mRNA expression to dietary Se source might occur between selenoproteins (GSH-Px1, GSH-Px4, DIO1, DIO2, TXNDR1, and SELW1). The noted variance between organic and inorganic Se could be attributed to the higher bioavailability of organic forms, thus, stimulate more selenoproteins gene expression 86 .
Similarly, Surai et al. 87 and Meng et al. 4 suggested the mechanisms of action behind nano-Se are by the mediation of the gut microbiota in converting nano-Se into selenite, H 2 Se, or Se-phosphate with the synthesis of selenoproteins. It has been established that organic Se compounds such as; SeMet, SeCys, and Se-methyl-Se cysteine among others differ in terms of their bioavailability to the body 88 . Moreover, the current study notes a signi cant change in mRNA expression of liver selenoproteins in the hens regardless of Se source or form. Though, the mechanisms behind how dissimilar Se sources can regulate the expression of selenoproteins are yet uncertain and required further exploration.
In conclusion, the current study showed that the expression of uterine genes and selenoproteins was upregulated by basal diets supplemented with 0.3 mg/kg of different organic sources of Se and sodium selenite. Compared to inorganic and non-Se supplemented hens, the bacterial selenoprotein proves stronger at increasing the expression of functional genes involved in the formation of eggs (eggshell biomineralization) and selenoproteins.

Materials And Methods
Animal Ethics and husbandry management.
All procedures concerning animals' care, handling, and sampling were carried out in compliance with the ARRIVE guidelines.

Samples and data collection
Collection of tissues.
A total of 24 hens were selected randomly from the four treatments (a bird from each replicate), slaughter, and dissected. Before slaughter, hen's abdominal palpation was used to assume the egg presence in the uterus. The carcass was skinned ventrally, and uteri samples were collected from hens at the active growth phase of calci cation (15 -20 h post-ovulation) for RT-PCR. It is aimed at targeting higher expression of genes responsible for eggshell biomineralization an egg. Sections of the uterine tissues were scrapped for total RNA isolation, transferred into 5 ml capped tubes, and immediately snapped frozen in liquid nitrogen and stored at -80 0 C before extraction of RNA. Furthermore, a portion of liver was cut, wrapped in aluminum foil paper for antioxidant enzymes genes and activity assay snapped freeze in liquid nitrogen before storage in -80 0 for RNA extraction.
Total RNA isolation and puri cation.
Total RNA was isolated from frozen tissues (uterus, magnum and liver) ( QuantiNova SYBR Green Master Mix, 1 µL of each forward and reverse primers, 7 µL of nuclease-free water and 1 µL of cDNA. The qPCR reactions were carried out following standard cycling mode as per kit protocol. Three tissue were used from each hen to determine their stable house-keeping genes using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin (β-actin) and TATA-Box Binding Protein (TBP). The target genes were analyzed in duplicates and their expression level was determined using cycle threshold (Ct) values following standard curve method after normalization with reference genes. Genes of interest were ampli ed through the following thermo cycling program: reverse transcription at 95 °C for 10 minutes, rst denaturation at 95 °C for 2 minutes, then 40 cycles of denaturation at 95 0 C for 5 s, and combined primer annealing/extension at 60 0 C for 10 s. The uorescent data were acquired at the end of each annealing step during PCR cycles with a construct of melting curve to assess the speci city of PCR ampli cation. A real-time PCR was run for each pair of primer in which cDNA samples were replaced with distilled water to ascertain the absence of exogenous DNA. The e ciency of ampli cation was determined for each primer pair using cDNA serial dilutions utilization. The fold changes for each target gene was calculated using power of 2 (-ΔΔCT) method described by 90 . The fold changes in data were shown as mean ± standard error. The values were subjected to one-way analysis of variance (ANOVA) followed by Duncan multiple range test for mean comparisons to determine signi cance at P-value < 0.05 on a SAS (Statistical Analysis System, Version 9.4). The result was compared and presented as a fold change between treatments and the control group.
Statistical analysis.
For reference gene validation, relative expression levels of all the target genes were calculated by the comparative 2 −ΔΔCq approach 91,90 , in Microsoft Excel (2016), using the two most stable reference genes (GAPDH and β-actin). From the Excel, normalized relative quantities (NRQ) values were further analysed with One-way analysis of variance (ANOVA) using the Proc GLM procedure of SAS software (SAS Institute Inc., Cary, NC), and Duncan Multiple Range Test was used to separate level of signi cance (P < 0.05) between the treatment means. The results were presented as mean ± SEM.