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 calcification involved uterine cells and fluid constituents 32,26. The formation of complex bio-ceramic eggshell arises from direct acellular uterine fluid interaction of ions such as Ca2+ and HCO3− 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 fluid 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 calcification 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 calcification 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 efficacy 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 observed the gene expression in the oviduct (magnum and uterus) of pre-laying and laying hens were primarily involved in the growth and development, and the progress of egg formation, respectively.
The chicken ovocleidin gene (OC-17 and OC-116) is expressed in the uterus as one of the potential eggshell matrix proteins 25,37. They were both characterized as soluble and insoluble eggshell matrix proteins. Both were described during the mineralization process as framework proteins to align calcite crystals 32. The primary function of OC-17 protein is an antimicrobial and regulates the biomineralization process 40,41. Ovocleidin-116 is a major component of the chicken eggshell matrix extracellular phosphoglycoprotein, plentiful in uterine fluid during the active phase of calcification stage, and consequently is suspected to function in the process of eggshell mineralization 42,43,44. Hincke et al. 42,46established OC-16 function in eggshell and bone strength mineralization, and Sah et al. 25 reported that it regulates the arrangement of calcite crystals in eggshell. It is noteworthy, that the findings showed higher mRNA expression of OC-17 and OC-116 in the uterus tissue than in magnum, regardless of the treatment groups. This was confirmed by published data from 31,47,37,33 who reported higher mRNA expression of OC-116 genes in the uterus tissue. On the contrary, Yin et al. 31 observed OC-17 is differentially upregulated in the isthmus than ovary and magnum, however lower than the uterus. Furthermore, in chicken uterine fluid, there has been an interaction between organic matrix and inorganic minerals resulting in tough and calcification of eggshell 37.
The shell of an egg is considered a physically protective wall for the contents against external (microbial) inversion rich in proteins with antimicrobial properties 33. These antimicrobial properties are contained in the liquid egg-white, and potentially present chemical defensive mechanisms to the egg 48. The lumen is sheltered from bacteria-free, thus protect the forming egg or embryo by antimicrobial proteins released into the uterine fluid. Among the examined antimicrobial proteins expressed in the tissues during the shell, calcification includes ovocalyxin-32 and 36. Ovocalyxin family (OCX-32 and OCX-36) constitutes mostly organic matrix proteins 26,33, highly expressed by uterine glandular cells, eggshell membrane and, egg vitelline membrane especially during the active calcification phase 49. Ovocalyxin-32 is the main determinant of eggshell quality in avian species whereas ovoaclyxin-36 presents an antimicrobial property integrated into the eggshell 50. Also, antimicrobial properties were discovered in the recombinant of OCX-32 51. Besides, OCX-36 belongs to the lipopolysaccharide-binding proteins and Bactericidal Permeability Increasing (BPI) family and recognized in mammals for its participation in anti-bacterial defense 33. Members of these family might be lethal to Gram-negative bacteria via binding to the lipid A portion of the lipopolysaccharide cell wall. The present study note that mRNA expression of OCX-32 and OCX-36 were up-regulated in the uterus than magnum tissue irrespective of the dietary Se treatment. In conformity with our results, 47,31 reported highly expressed OCX-32 precursors in the uterus with egg. 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, confirm 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 specific, and increase through the calcification 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 findings of Hrabia et al. 54 suggest that growth hormones may participate in the development and activity, and expression of some oviduct specific 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 deficiency 57. In the current study, the mRNA levels of the hepatic selenoproteins in hen’s liver significantly 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. Those results are consistent with previous studies. The foremost selenoproteins discovered and abundant in the liver are GPx (GPx1-4), which reserves enzymatic properties with the majority being involved in peroxides catabolism 60. Hou et al. 61 reported Se-enriched Saccharomyces cerevisiae (SSC) supplementation significantly increased GPx-1 and GPx-4 expression levels in broiler chicken muscle compared with control, Saccharomyces cerevisiae, and sodium selenite group. Recently, Chen et al. 62 reported selenide chitosan sulfate (Se-CTS-S) up-regulate GPx-1 and GPx-4 mRNA levels in hepatocytes and liver of chickens compared with chitosan (CTS), chitosan sulfate (CTS-S), selenide chitosan (CTS-Se), and sodium selenite (Na2SeO3). Meng et al. 4 observed higher GPx1 and GPx4 mRNA levels in the liver of laying hens supplemented with nano-Se and Se-yeast, respectively. Wang et al. 63 reported dietary Se-yeast supplementation caused an up-regulation of selenoproteins gene expressions in the liver (10) and muscles (11) of rainbow trout (Oncorhynchus mykiss). Similarly, organic bacterial Se showed a significant increase in liver mRNA expression of GSH-Px1, GSH-Px4, DIO1, and TXNDR1 compared to sodium selenite supplemented broilers (Dalia et al., 2017). Chen et al. 65 reported higher expression of GPx-1 and GPx-4 mRNA levels with organic Se supplementation of Se-enriched Saccharomyces cerevisiae compared to other groups in Arbor Acres broilers. Khan et al. 66 observed the upregulation of mRNA expression of GPx1 and GPx4 and downregulation of heat shock proteins genes (HSP60, HSP70, and HSP90) in the chicken heart with Se-enriched probiotics. Luan et al. (2016) observed lower selenoproteins transcript levels in chicken erythrocytes fed a Se-deficient diet, though, with high expression of GSH-Px, TXNDR1, selenoprotein P1 (SELP), and selenoprotein synthetase (SPS2) compared to other selenoproteins. GSH-Px family (GSH-Px1, GSH-Px2, GSH-Px3, and GSH-Px4) are plentiful in the liver catabolizing peroxides as their functions. For instance, GPx1 is a potential antioxidant enzyme with a significant role in the detoxification of lipid hydroperoxides and H2O2, whereas GPx4 inhibits atherosclerosis by reducing oxidative stress 66. It has been reported by previous data that GSH-Px and SELW1 mRNA levels increased with dietary Se intake in poultry 57, and sheep 68. However, studies on dissimilar Se sources on the expression of these genes was not completely explored, as well as no findings on the efficacy of bacterial organic Se on laying hens. The bioavailability of Se sources or forms and levels differs with the type of tissue and animal species with regards to absorption, deposition, and metabolism could directly or indirectly alter the antioxidant enzyme activities 69. Therefore, it might be attributed that ADS18 or Se-yeast supplementation is connected to the regulation of GSH-Px’s, thus reduced body oxidative stress through the transcription level of GPx1 and GPx4 mRNA in laying hens’ liver. It could perhaps by the results obtained shows superior sensitivity of GSH-Px1 than GSH-Px4 to regulation by Se status, and different responses to the mRNA expression to dietary Se between both selenoproteins might differ. Moreover, it can protect from Se-deficiency disorder 70. Furthermore, mRNA expression of GPx1 and GPx4 can be employed as molecular biomarkers for evaluating Se status as well as the requirements 71.
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 findings confirmed 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 reported downregulation of DIO1, DIO2, DIO3, TXNRD2 selenoproteins induced by Se deficiency in chicken’s thyroid gland. Liu et al. 76 observed downregulation of the SEPW1 mRNA level in pig’s liver fed a high-Se diet of 3.0 mg Se/kg against the 0.3 mg Se/kg diet. Similarly, hepatic expression of GPX1, SEPW1, and SEPW15 mRNA levels were decreased by dietary Se deficiency in chicks liver and muscle (Huang et al., 2011), 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 findings, 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 detoxification 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 H2O2, 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 findings showed a definite trend of up-regulating selenoproteins (GSH-Px1, GSH-Px4, DIO1, DIO2, and SELW1) mRNA expression significantly (p < 0.05) with bacterial organic Se supplementation, except for TXNDR1 with Se-yeast hens compared to the negative control. Furthermore, the findings 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, H2Se, 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 significant 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.