Avian eggshell is made of columnar calcite crystals which protect the eggs from physical damage and microbial contamination, providing calcium sources for developing embryo [26]. Nevertheless, eggshell quality becomes to deteriorate from the middle phase of production with the characteristics of decreasing eggshell strength, increasing ratio of translucent eggs, and increasing numbers of abnormal eggs, which cause substantial economic losses and impaired animal welfare. Numerous studies have identified that Se is beneficial for health, egg and shell quality of laying hens. However, the potential molecular mechanism of selenium yeast on shell quality has not been elucidated.
Se deficiency and supplementation are closely related to Se status and antioxidants capacity. In general, plasma Se content and antioxidant enzyme were generally considered as useful biomarkers of both Se status and dietary intake [27]. Antioxidant enzyme including T-AOC and T-SOD acted as the important index for redox state in livestock and poultry. GSH-Px is a Se-dependent enzyme which has abilities for protecting tissues from oxidative damage. In our study, Se deficiency for 6 weeks leads to a significant decrease of Se status and antioxidants capacity in aged laying hens. Consistent with other studies, Se deficiency led to a significant decrease of Se content, GSH-Px [28], T-AOC [29], and SOD [30] activities in plasma in pigs and poultry. In addition, we found that 71.31% of Se content, 49.60% of T-AOC, 58.31% of GSH-Px, and 16.71% of T-SOD were decreased by Se deficient, respectively. Based on the decline degree of these indicators, Se content in plasma decreased quickly, suggesting that it was more sensitive to Se status, and could be a biomarker of Se status. Selenium yeast supplementation also enhanced the Se status and antioxidant capacity. Consistent with other studies, Se content in plasma and tissues increased with a dose-dependent trends after Se supplementation [31, 32]. After 6-week supplementation, plasma Se content in the SY0.45 group was recovered, suggesting that high dose Se supplementation may relieve the harmful effects caused by Se deficiency. Moreover, the effects of Se deposition in different tissues may be different because of complexity of Se absorption and metabolism [33]. We firstly found that the Se deposition hierarchy in tissues associated with laying are isthmus, magnum, ovary, and shell gland, successively. Meanwhile, the activities of GSH-Px, T-AOC, and T-SOD in plasma increased in supplementation period. The data are in agreement with the results previously published that antioxidant enzyme activities dose-depended increased with selenium yeast supplementation [32, 34, 35]. These findings suggested that Se content and plasma antioxidant enzyme activity can reflect the Se status and intakes, and plasma Se content can be used as an indicator of body Se status.
Studies clearly indicated that selenium yeast has crucial roles in poultry nutrition and productive. Eggshell strength is vital in ensuring the integrity and safety of the egg contents, it tends to deteriorate with the increasing of bird age [36]. In our study, Se deficiency for 6 weeks significantly decreased egg quality in aged laying hens. It has been reported that Se deficiency is detrimental to bone microarchitecture possibly through decreasing antioxidant capacity [37]. Thus, these results suggested that oxidative stress caused by Se deficiency [38] may decrease eggshell strength in conjunction with age. Moreover, 0.45 mg/kg selenium yeast supplementation for 12 weeks significantly elevated egg quality including increasing eggshell strength, and decreasing egg weight and translucent eggs. Consistent with previous studies, eggshell breaking strength was significantly increased after high does selenium yeast supplementation [39, 40]. Meanwhile, translucent eggshell was a problematic issue to affect eggshell appearance and decrease the commercial value of eggs. The reason of eggshell translucent was still unknown, it was inferred to be associated with variations of eggshell membrane [41]. The decreasing of translucent eggs suggested that selenium yeast supplementation may increase the antioxidant capacity of eggs to ameliorate the decline of egg quality. Moreover, increasing egg weight in the last phase of laying cycle is another of the problematic issues [42–44], large eggs are more difficult to handle and more prone to break during transport and collection. The results in our study showed that selenium yeast supplementation decreased the egg weight, suggested that selenium yeast supplementation in last stage of laying period played beneficial roles in egg and shell quality.
In our study, the effect of selenium yeast supplementation on shell gland of aged laying hens were obtained by whole transcriptome analysis. The results of transcriptome analysis revealed several novel genes and biological pathways regulates the ions transport, laying period, eggshell calcification and consequently the eggshell formation. Based on the enrichment pathways, molecular functions, and gene expression trends, thirteen genes were identified as potential candidate genes during eggshell formation including CEMIP, SDC3, OVAL, SPP1, SLC6A17, SLC13A5, OTOP2, CA2, POMC, PTN, PENK, WNT11 and EREG.
The growing crystals interact with the shell organic matrix to form a highly ordered microstructure during shell calcification. Thus, the organic matrix interacts with minerals plays a key role in eggshell formation. We paid particular attention to genes involved in shell mineralization, including CEMIP, SDC3, OVAL, SPP1. CEMIP involved in glycosaminoglycan metabolism and calcium release from endoplasmic reticulum [45]. The up-regulated CEMIP expression (log2 fold change > 2 in both two comparison) might suggest that high dose selenium yeast supplementation can promote calcium release for eggshell formation. SDC3 protein possesses domains containing some potential glycosaminoglycan attachment sites [46]. Glycosaminoglycan is widely thought to regulate mineral deposition and determine the properties of eggshell [47]. Thus, we inferred that the up-regulation of SDC3 may play an active role in eggshell formation. OVAL is an abundant eggshell matrix protein binding calcium and plays an active role in carbonate formation [48], in coherence with our results, the up-regulation of OVAL might improve the eggshell formation. SPP1 was known as osteopontin, has mineral-binding domains [49] which involved in calcium metabolism and calcium carbonate precipitation [47] for eggshell calcification. Consistent with other studies, the up-regulation of SPP1 occurred in eggshell calcification period [50, 51]. Similarly, the pathways DEGs enriched in including calcium signaling pathway, glycosaminoglycan biosynthesis and positive regulation of bone mineralization, were also closely associated with eggshell calcification and formation. Hence, the results suggested that selenium yeast supplementation induced shell calcification by regulating the expression of CEMIP, SDC3, OVAL, SPP1 and other candidate genes beneficial for eggshell formation.
The chicken eggshell is a highly ordered structure and is mainly composed of calcium carbonate. Furthermore, the eggshell formation process requires a large amount of calcium (Ca2+) and bicarbonate (HCO3−), so the ions transport plays a crucial role. Some DEGs and pathways associated with ions transport were screened out in our study, including SLC6A17, SLC13A5, OTOP2, CA2, voltage-gated calcium channel activity and calcium signaling pathway. SLC6A17 and SLC13A5 as solute carrier family, plays an important role in transporting ions across cell membranes for synthesis of eggshell formation, it had been reported that increasing SLC6A17 expression plays an alanine transport role during eggshell formation, while SLC13A5 plays a more important role in the initiation of synthesis of eggshell formation [52]. The carbonate ion required for eggshell synthesis is catalyzed by carbonic anhydrases (CA), and uterine glandular cells possess the carbonic anhydrase activity sites. Growing evidence has demonstrated that high level of CA2 expression play a pivotal role for conversion of intracellular CO2 to HCO3− in chicken [53, 54]. As a member of the otopetrin gene family, OTOP2 gene may have similar functions to OTOP1, which regarded as modulator of cellular calcium influx [55], to transport calcium across the uterine epithelium for eggshell calcification [56]. Our findings inferred that SLC6A17, SLC13A5, OTOP2 and CA2 are the regulators of ions transport affect by selenium yeast.
Moreover, the eggshell formation also depends upon numerous physiological adaptations and processes by the uterine cells, as well as reproductive hormones. In our study, functional enrichment analysis showed that selenium yeast supplementation may affect the response to estrogen, regulation of cell proliferation, regulation of epithelial cell proliferation, and extrinsic apoptotic signaling pathway. POMC plays a role in stimulating the release of cortisol hormone and its up-regulate expression during the mineralization period [52]. Consistent with other studies, a higher expression of POMC was observed in a hard shell egg group [53]. PTN is a developmentally-regulated growth factor and its expression is induced by estrogen [57], PTN and estrogen were reported with a pivotal role in eggshell formation [52]. These findings suggested that selenium yeast may enhance the expression of reproductive hormones to affect the eggshell formation and quality. Indeed, WGCNA analysis also identified some candidate genes which affected the eggshell formation regulated by selenium yeast, including PENK, WNT11, EREG. These up-regulation DEGs had been reported in previous studies which had a vital role in eggshell formation [52].
Previous studies have found that trace elements manganese and zinc enhanced eggshell strength by improving biosynthesis of glycosaminoglycan [58] and affecting carbonic anhydrase activity [59], respectively. An increasing number of studies had explored that selenium has a good effect on eggshell quality of laying hens. However, studies on the molecular mechanism of selenium yeast affecting eggshell quality is limited. To date, it is hypothesized that selenium exhibits beneficial regulation on eggshell quality may be directly involved in the process of regulating eggshell formation or the interaction between trace elements. Overall, based on biological functions of the DEGs, we hypothesize that the molecular pathways impacted by selenium on aged laying hens are those related to eggshell mineralization, hormone regulation and ion transduction, suggesting that selenium might play a beneficial role in eggshell formation. Despite that, additional studies are needed to draw conclusive remarks about the molecular mechanisms modulated by selenium yeast in accurate phases of eggshell formation.