Identification of crucial genes and metabolites regulating the eggshell brownness in chicken.

BACKGROUND: Protoporphyrin IX (Pp IX) is the primary pigment for brown eggshells. However, the regulatory mechanisms directing Pp IX synthesis, transport, and genetic regulation during eggshell calcification in chickens remain obscure. In this study, we investigated the mechanism of brown eggshell formation at different times following oviposition, using White Leghorn hens (WS group), Rhode Island Red light brown eggshell line hens (LBS group) and Rhode Island Red dark brown eggshell line hens (DBS group). RESULTS: At 4, 16 and 22 h following oviposition, Pp IX concentrations in LBS and DBS groups were significantly higher in shell glands than in liver (P < 0.05). Pp IX concentrations in shell glands of LBS and DBS groups at 16 and 22 h following oviposition were significantly higher than WS group (P < 0.05). In comparative transcriptome analysis, δ-aminolevulinate synthase 1 (ALAS1), solute carrier family 25 member 38 (SLC25A38), ATP binding cassette subfamily G member 2 (ABCG2) and feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), which were associated with Pp IX synthesis, were identified as differentially expressed genes (DEGs). RT-qPCR results showed that the expression level of ALAS1 in shell glands was significantly higher in DBS group than in WS group at 16 and 22 h following oviposition (P < 0.05). In addition, four single nucleotide polymorphisms (SNPs) in ALAS1 gene that were significantly associated with eggshell brownness were identified. By identifying the differential metabolites in LBS and DBS groups, we found 11-hydroxy-E4-neuroprostane in shell glands and 15-dehydro-prostaglandin E1(1-) and prostaglandin G2 2-glyceryl ester in uterine fluid were related to eggshell pigment secretion. CONCLUSIONS: In this study, the regulatory mechanisms of eggshell brownness were studied comprehensively by different eggshell color and time following oviposition. Results show that Pp IX is synthesized de novo and stored in shell gland, and ALAS1 is a key gene regulating Pp IX synthesis in the shell gland. We found three transporters in Pp IX pathway and three metabolites in shell glands and uterine fluid that may influence eggshell browning.

Expression of estrogen receptor alpha in response to stress and estrogen antagonist tamoxifen in the shell gland of Gallus gallus domesticus: involvement of anti-oxidant system and estrogen.

Animals are frequently exposed to various kinds of environmental stressors and estrogen is known to play important role in stress response besides its crucial role in regulation of cellular proliferation, metabolic activity and reproduction. The study investigates the estrogen antagonist, tamoxifen (TM), mediated estrogen receptor alpha (ERα) expression, to modulate stress induced parameters in chickens. The study further explores the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) and malonaldehyde (MDA) in brain, ovary and shell gland during water deprivation (WD) and tamoxifen administration in sexually mature chicken. WD and TM administration both decrease the plasma estradiol while WD increases corticosterone. WD also elevates MDA concentration in the brain, ovary and shell gland while TM lowers it. WD and TM administration lowers the specific activity of SOD in brain and shell gland. In contrast, WD increases the specific activity of catalase, GPx and GR in the brain and shell gland, while TM decreases it. It appears that endogenous estradiol plays a crucial role in expression of antioxidant enzymes and tamoxifen acts as an antioxidant by reducing the oxidative stress in chicken. Abundant expression of ERα has been observed in the shell gland of egg laying birds while stress like water deprivation and TM down-regulates its expression. Thus, it can be concluded that expression of ERα in shell gland plays a predominant role in mediating estrogen action in response to water deprivation stress and tamoxifen.

Identification of three cell populations from the shell gland of a bivalve mollusc.

The molluscan larval shell formation is a complicated process. There is evidence that the mantle of the primary larva (trochophore) contains functionally different cell populations with distinct gene expression profiles. However, it remains unclear how these cells are specified. In the present study, we identified three cell populations from the shell gland in earlier stages (gastrula) from the bivalve mollusc Crassostrea gigas. These cell populations were determined by analyzing the co-expression relationships among six potential shell formation (pSF) genes using two-color hybridization. The three cell populations, which we designated as SGCPs (shell gland cell populations), formed a concentric-circle pattern from outside to inside of the shell gland. SGCP I was located in the outer edge of the shell gland and the cells expressed pax2/5/8, gata2/3, and bmp2/4. SGCP II was located more internally and the cells expressed two engrailed genes. The last population, SGCP III, was located in the central region of the shell gland and the cells expressed lox4. Determination of the gene expression profiles of SGCPs would help trace their origins and fates and elucidate how these cell populations are specified. Moreover, potential roles of the SGCPs, e.g., development of sensory cells and shell biogenesis, are suggested. Our results reveal the internal organization of the embryonic shell gland at the molecular level and add to the knowledge of larval shell formation.

Nesfatin-1: Localization and expression in avian gonads and its modulation by temporal phase relation of neural oscillations in female Japanese quail, Coturnix coturnix japonica.

In a search for new appetite-controlling signals, the peptide nesfatin-1, expressed in the brain and peripheral tissues of rodents and humans has been reported to regulate feeding by reducing food intake. Recently it has also been reported that nesfatin-1 might be involved in regulating the reproductive axis in fishes and mammals, but its expression and physiological role if any, is not yet known in birds. In the present study, localization and expression of nesfatin-1 was observed in the testis, ovary and shell gland of poultry species Japanese quail, Coturnix coturnix japonica. Our earlier studies have reported that serotonin precursor 5-HTP and dopamine precursor l-DOPA given 8h apart induces gonadal suppression, when given 12h apart leads to gonadal stimulation while other relationships were found ineffective. In the present study intense ir-nesfatin-1 was observed in the regressed ovary (stromal cells) and shell gland (endometrium) of 8-h Japanese quail while in 12-h quail, weak and scarce immunostaining for nesfatin-1 was detected in the hyperactive ovary and shell gland compared to control. These findings led us to conclude that, an inverse relationship exists between ovarian activity (both in the control and simulated conditions) and nesfatin-1 expression. Present avian study, first of its kind, also suggests the role of nesfatin-1 in reproductive regulation possibly via appetite control and energy balance in female Japanese quail and needs to be investigated further in relation to food intake.

Circadian regulation of calcium and phosphorus homeostasis during the oviposition cycle in laying hens.

Maintenance of calcium and phosphorus homeostasis in laying hens is crucial for preservation of skeletal integrity and eggshell quality, though physiological regulation of these systems is incompletely defined. To investigate changes in mineral and vitamin D3 homeostasis during the 24-h egg formation cycle, 32-wk-old commercial laying hens were sampled at 1, 3, 4, 6, 7, 8, 12, 15, 18, 21, 23, and 24 h post-oviposition (HPOP; n ≥ 4). Ovum location and egg calcification stage were recorded, and blood chemistry, plasma vitamin D3 metabolites, circulating parathyroid hormone (PTH), and expression of genes mediating uptake and utilization of calcium and phosphorus were evaluated. Elevated levels of renal 25-hydroxylase from 12 to 23 HPOP suggest this tissue might play a role in vitamin D3 25-hydroxylation during eggshell calcification. In shell gland, retinoid-x-receptor gamma upregulation between 6 and 8 HPOP followed by subsequently increased vitamin D receptor indicate that vitamin D3 signaling is important for eggshell calcification. Increased expression of PTH, calcitonin, and fibroblast growth factor 23 (FGF23) receptors in the shell gland between 18 and 24 HPOP suggest elevated sensitivity to these hormones toward the end of eggshell calcification. Shell gland sodium-calcium exchanger 1 was upregulated between 4 and 7 HPOP and plasma membrane calcium ATPase 1 increased throughout eggshell calcification, suggesting the primary calcium transporter may differ according to eggshell calcification stage. Expression in shell gland further indicated that bicarbonate synthesis precedes transport, where genes peaked at 6 to 7 and 12 to 18 HPOP, respectively. Inorganic phosphorus transporter 1 (PiT-1) expression peaked in kidney between 12 and 15 HPOP, likely to excrete excess circulating phosphorus, and in shell gland between 18 and 21 HPOP. Upregulation of FGF23 receptors and PiT-1 during late eggshell calcification suggest shell gland phosphorus uptake is important at this time. Together, these findings identified potentially novel hormonal pathways involved in calcium and phosphorus homeostasis along with associated circadian patterns in gene expression that can be used to devise strategies aimed at improving eggshell and skeletal strength in laying hens.

Physiological changes in the regulation of calcium and phosphorus utilization that occur after the onset of egg production in commercial laying hens.

At the onset of egg production, physiological changes governing calcium and phosphorus utilization must occur to meet demands for medullary bone formation and eggshell mineralization. The objective of this study was to identify these changes and determine if they are influenced by dietary supplementation with 1α-hydroxycholecalciferol (AlphaD3™, Iluma Alliance). Commercial laying hens fed either a control or AlphaD3-supplemented diet beginning at 18 weeks of age were sampled at 18 (n = 8) and 31 weeks (n = 8/diet) to evaluate mRNA expression associated with calcium and phosphorus utilization in kidney, shell gland, ileum, and liver, circulating vitamin D3 metabolites, and bone quality parameters in humerus, tibia, and keel bone. Though diet did not heavily influence gene expression at 31 weeks, several significant differences were observed between 18- and 31-week-old hens. Heightened sensitivity to hormones regulating calcium and phosphorus homeostasis was observed at 31 weeks, indicated by increased parathyroid hormone receptor 1, calcium-sensing receptor, calcitonin receptor, and fibroblast growth factor 23 receptors in several tissues. Increased renal expression of 25-hydroxylase and vitamin D binding protein ( DBP ) at 31 weeks suggests kidney participates in local vitamin D3 25-hydroxylation and DBP synthesis after egg production begins. Biologically active 1,25(OH)2D3 was higher at 31 weeks, with correspondingly lower inactive 24,25(OH)2D3. Increased expression of plasma membrane calcium ATPase 1 and calbindin in kidney, shell gland, and ileum suggests these are key facilitators of calcium uptake. Elevated renal inorganic phosphorus transporter 1 and 2 and sodium-dependent phosphate transporter IIa at 31 weeks suggests increased phosphorus excretion following hyperphosphatemia due to bone breakdown for eggshell formation. Diet did influence bone quality parameters. Bone mineral density in both humerus and tibia was higher in AlphaD3-supplemented hens at 31 weeks. Tibial bone mineral content increased between 18 and 31 weeks, with AlphaD3-supplemented hens increasing more than control hens. Moreover, control hens exhibited diminished tibial breaking strength at 31 weeks compared to hens at 18 weeks, while AlphaD3-supplemented hens did not. Together, these results indicate supplementation with AlphaD3 enhanced bone mineralization during the medullary bone formation period and elucidate the adaptive pathways regulating calcium and phosphorus utilization after the onset of lay.

Dietary mulberry-leaf flavonoids improve the eggshell quality of aged breeder hens.

Eggshell quality is subject to a significant decline in the late laying period, which results in huge economic losses. The purpose of this study was to investigate the effects of dietary mulberry-leaf flavonoids (MF) on the eggshell quality of aged breeder hens. A total of 270 (60-week-old) Qiling breeder hens were randomly assigned to 3 treatments with supplemental dietary MF doses (0, 30, and 60 mg/kg). The results showed that dietary MF improved the eggshell thickness and strength, following the reduced broken egg ratio (P < 0.05). Histological analysis showed that dietary MF increased glandular density and luminal epithelium height in the shell gland (P < 0.05). MF treatment reduced the apoptotic index of the shell gland, following by improved antioxidant capacity (P < 0.05). The protein expression of Caspase 3 was down-regulated, and Nrf2 was up-regulated by dietary MF (P < 0.05). Furthermore, calcium (Ca) content in the serum and shell gland, as well as the activity of Ca2+-ATPase in the shell gland were increased by dietary MF (P < 0.05). Ca transport-related genes (ESRα, ESRß, KCNA1, OPN, CABP-28K and CDH6) in the shell gland were upregulated by dietary MF treatment (P < 0.05). In conclusion, dietary MF could ameliorate the eggshell quality of aged hens by improving antioxidative capability and Ca deposition in the shell gland of uterus.

The effect of Artichoke (Cynara scolymus L.) on the expression of calcium-binding proteins in the eggshell gland of laying hens.

The purpose of the present work is to investigate the effect of dietary-supplemented artichoke (Cynara scolymus L.) on the mRNA expression of calbindin 1 (Calb1), osteopontin (Spp1), albumin (Alb) and CALB1 protein in the eggshell gland (ESG) of laying hens. A total of 80 ISA Brown hens (each at 40 weeks of age) were randomly divided into two groups: a control and a treated group. All poultry received 130 g/day of compound feed for laying hens but the treated hens' diet was also supplemented with 3g/kg of dried and milled artichoke (Cynara scolymus L.). The increase of the Ca content in blood of the treated hens was established. Significantly decrease of Spp1 mRNA transcripts was found in the eggshell gland of the treated hens, while the mRNA level of Alb was increased. The relative expression of Calb1 mRNA tended to increase in the treated group. The expression of calbindin protein in the cytoplasm of glandular cells of the shell gland was defined by immunohistochemical method. Very strong signals of calbindin were observed in the treated group. The supplementation of the laying hens' diet with dried artichoke (C. scolymus L.) led to a significant increase of Ca content in blood that was reflected in the changes of expression of the eggshell gland genes involved in the mineralization of eggshell.

Expression of follistatin is associated with egg formation in the oviduct of laying hens.

The objective of this study was to examine the expression profiles of follistatin (FST) and its associated molecules (MSTN, INHA, INHBB, INHBA, ACVR2A, and ACVR2B) in the oviduct of laying hens at 3 hr and 20 hr post-ovulation (p.o., n = 5; 35 weeks old), molting (n = 5; 60 weeks old), and non-laying (n = 4; 35-60 weeks old) hens and also to localize the FST by using immunohistochemistry assay. Expression of FST was significantly higher (p < .05), and MSTN was lower in the uterus of laying hens around 15-20 hr p.o. (during eggshell formation), however, their expressions in the magnum remain unchanged across different physiological stages of hens. FST was mainly expressed in the luminal and glandular epithelium of the uterine tissues, and their expression intensity was highest in laying hens during the eggshell mineralization. There was a relatively increased expression of INHA in the magnum of laying hens around 3 hr p.o. as compared to non-laying and molting hens. At the same time (3 hr p.o.), there was a significant (p < .05) decrease in the expression of the INHBB, ACVR2A, and ACV2B. These results indicate that follistatin may regulate the differentiation of uterine luminal and glandular epithelium during eggshell biomineralization.

Effects of Dietary Zinc on Performance, Zinc Transporters Expression, and Immune Response of Aged Laying Hens.

This study was to investigate the effects of dietary zinc (Zn) supplementation on performance, zinc transporter gene expression, and immune function in aged laying hens. In experiment 1, twenty 31-week-old hens (young) and twenty 60-week-old hens (old) with the same genetic background were fed with the same diet for 4 weeks. In experiment 2, a basal diet supplemented with zinc sulfate (ZnS) and zinc glycine chelate (ZnG) at 30, 60, 90, and 120 mg Zn/kg to constitute nine experimental diets. Eight hundred and ten 60-week-old layers were distributed in a completely randomized experimental design with 9 treatments, 6 replicates of 15 birds each, and birds were fed for 10 weeks. In experiment 1, results showed that zinc and metallothionein (MT) concentration in the shell gland of old hens was significantly lower than young layers (P < 0.05). Zinc transporters ZnT1, 4, 5, 6, and 7 messenger RNA (mRNA) abundance in old layers were significantly lower versus the young (P < 0.05). In experiment 2, results indicated that dietary zinc supplementation did not significantly affect the laying rate, average feed intake, egg weight, feed conversion efficiency, broken egg rate, or mortality (P > 0.05). Supplemental ZnG significantly improved eggshell breaking strength than ZnS, with a higher alkaline phosphatase (ALP) activity and more abundant ZnT4 expression in shell gland versus ZnS (P < 0.05). ZnG supplementation at 90 mg Zn/kg affected the duodenal mucus by significantly increasing ZnT1, 6, 7, ZIP13, and MT-4 mRNA level (P < 0.05). Zinc level significantly increased bovine serum albumin (BSA) antibody concentration on 14 day after BSA injection (P < 0.05). Supplementation of ZnG improved eggshell quality of aged layers by upgrading zinc transporter expression in the shell gland and intestine also enhanced humoral immunity.

Evidences in duck (Anas platyrhynchos) by transcriptome data for supporting the biliverdin was mainly synthesized by shell gland.

The blue-shelled egg not only plays a key role in helping birds to avoid predation as a result of crypsis and mimetism, but it also provides eggshell strength and filters solar radiation; moreover, it has an important economic trait for poultry. However, the source of biliverdin for blue-shelled egg remains unsolved in ducks. The current study detected the biliverdin content and localization of heme oxygenase 1 (HMOX1) in duck shell gland; moreover, RNA-seq analysis was performed in the shell gland of blue-shelled and white-shelled ducks. Results indicated that biliverdin is a primary pigment for blue-shelled egg in ducks, and the HMOX1 protein showed high expression in ciliated epithelial cells of shell gland between blue-shelled and white-shelled ducks. In the pathway of biliverdin synthesis, only 5-aminolevulinate synthase 1 expression level was significantly upregulated in blue-shelled ducks, and nuclear factor, erythroid 2 like 1 and period circadian clock 2 may be the essential elements in biliverdin synthesis of duck shell gland. Furthermore, some of the transporter genes, such as activator-Like and solute carrier family 13 member 5, may be involved in the formation of blue egg in duck. Results of the current study suggested that the biliverdin is most likely synthesized and secreted from epithelial cells of shell gland. In addition, ALAS1 may play a key role in the formation of blue egg in ducks.

Shell-Less Egg Syndrome (SES) Widespread in Western Canadian Layer Operations Is Linked to a Massachusetts (Mass) Type Infectious Bronchitis Virus (IBV) Isolate.

A disease with a sudden drop in egg production and shell-less eggs called, shell-less egg syndrome (SES) has been observed in Western Canada egg layer flocks since 2010. The etiology of this disease is not known. We hypothesize that SES is caused by an infectious bronchitis virus (IBV) strain since it is known that IBV replicates in the shell gland causing various eggshell abnormalities. In this study, we screened egg layer flocks, in the provinces of Alberta (AB) and Saskatchewan (SK), with and without a history of SES for the presence of IBV infection. During 2015⁻2016, a total of 27 egg layer flocks were screened in AB (n = 7) and SK (n = 20). Eighty-one percent of the screened flocks (n = 22) were positive for IBV infection. Thirty of these isolates were successfully characterized using molecular tools targeting the most variable spike (S) 1 gene. IBV isolates from this study clustered into three genotypes based on partial S1 gene variability. The majority of the IBV isolates (70%) were Massachusetts (Mass) type, and the rest were either Connecticut (Conn) type or an uncharacterized genotype with genetic characteristics of Mass and Conn types. Since the majority of the IBV isolates included within the Mass type, we used a Mass type IBV isolate to reproduce SES in specific pathogen free (SPF) white leghorn chickens in lay. Further studies are warranted to investigate whether other IBV isolates can cause SES, to clarify the pathogenesis of SES and to develop a vaccine in order to prevent SES as observed in Western Canadian layer flocks.

Eggshell Biliverdin and Protoporphyrin Pigments in a Songbird: Are They Derived from Erythrocytes, Blood Plasma, or the Shell Gland?

Biliverdin and protoporphyrin pigments are deposited into the eggshell when the developing egg is in the shell gland. However, the site of synthesis of eggshell pigments is still uncertain, although it may influence the possible costs and potential functions of eggshell coloration in avian species. Eggshell pigments may be derived from red blood cells or be produced in other organs and then transferred to the shell gland, or they may be synthesized de novo in the shell gland. We studied in the canary (Serinus canaria) whether eggshell blue-green and brown pigmentations are associated with experimentally elevated anemia, female hematocrit level, immature erythrocyte percentage, and feces and plasma pigment levels during egg laying to find out the possible origin of eggshell pigments. We found no significant effects of hematocrit level or experimentally elevated anemia on intensity of eggshell blue-green and brown pigmentations; therefore, we consider it less likely that eggshell pigments are derived from erythrocytes. In addition, we found no significant associations between female feces biliverdin concentration during egg laying and intensity of eggshell blue-green pigmentation, suggesting that eggshell biliverdin may not originate from the spleen or liver. We found a negative association between plasma and feces protoporphyrin concentrations during egg laying and eggshell brown chroma. This result suggests that an increased production of protoporphyrin in the liver, which could have elevated plasma and feces protoporphyrin concentrations, could inhibit eggshell protoporphyrin pigmentation, probably through affecting enzymatic activities. We suggest that both pigments are produced de novo in the shell gland in the canary, but circulating pigment levels may influence shell gland pigment synthesis, thus connecting the physiological status of the female to eggshell coloration.

Expression of arginine vasotocin and estrogen receptor alpha (ERα) in the shell gland altered by the specific phase relations of neural oscillations affects the reproductive physiology of Japanese quail.

In order to study the effect of specific phase relation of neural oscillations on reproductive regulation and the response of AVT (the avian homologue of mammalian AVP) the expression of AVT in the shell gland was monitored in sexually immature quail. In this study 3-week-old female Japanese quail were administered with serotonin precursor, 5-hydroxytryptophan followed by the dopamine precursor, l-dihydroxyphenylalanine at interval of 8h and 12h daily over a period of 13days. At thirty two days post treatment, a significant decrease in gonadal activity was seen in 8h quail although 12h quail exhibited an increase as compared to controls. A significant decrease in plasma estradiol level was noted in 8h quail while 12h exhibited no significant difference compared to controls. To address the relative roles of estrogen mediated action we also investigated estrogen receptor alpha (ER-α) expression and localization in the shell gland by visualizing it through confocal immuno-fluorescence microscopy. Results indicate increased expression of immunoreactive (ir)-AVT (myometrium), ir-ER-α (epithelial cells of endometrial region), along with significant increase in hypothalamic, plasma and shell gland AVT and a rapid increase in egg laying thus maintaining full breeding condition in 12h while low expression of ir-AVT and ir-ER-α was observed in 8h quail along with a significant decrease in hypothalamic, plasma and shell gland AVT with the suppression of gonads thereby stopping the egg-laying behaviour was noted. These findings not only suggest the modulation of gonadal development by changing the specific phase relation of neural oscillations but also demonstrate a parallel relation of AVT and gonadal activity in both conditions. It is concluded that the egg laying performance in response to AVT is regulated by the temporal phase relationship of neurotransmitters, and in part, this effect appears to be estrogen dependent.

Morphological analysis of the oviduct, oviducal gland and isthmus of the blue shark Prionace glauca (Linnaeus, 1758) (Elasmobranchii: Carcharhiniformes)

Oviducal gland present in elasmobranchs is correlated to the organism's reproductive strategy, and its functions are to produce mucus, to form the egg's tertiary envelope and to store sperm. The gland contains four zones: club, papillary,baffle and terminal. The structures of the oviduct, oviducal gland and isthmus of blue shark Prionace glauca were described using macroscopic, light microscopy and scanning electron microscopy techniques. The epithelium of the oviduct and isthmus is folded and is a simple, columnar, ciliated lining epithelium with glandular cells. In the oviducal gland, the lining tissues in the four zones are similar to the oviduct and isthmus lining. The terminal zone shows the presence of sperm in the lumen of the secretory tubules, which remains stored even in the absence of recent copulation. Here, these organs were studied and their connections in an attempt to elucidate the mechanisms of reproduction in the blue shark, showing the three-dimensional aspects, thus adding morphological information important for the understanding of the structure and functioning of these organs of fundamental importance in the life of the majority of elasmobranchs.

A glândula oviducal presente nos elasmobrânquios está correlacionada com a estratégia reprodutiva, cuja função é de produzir o muco, formar o envelope terciário do ovo e armazenar espermatozóides. A glândula contém quatro zonas: club, papilar, baffle e terminal. Foi descrita a estrutura do oviduto, glândula oviducal e istmo do tubarão-azul, Prionace glauca , pelas técnicas macroscópica, microscópica de luz e eletrônica de varredura. Foi observado que no oviduto e istmo o epitélio é pregueado de revestimento simples, colunar, ciliado com células glandulares. Na glândula oviducal o tecido de revestimento é semelhante ao oviduto e istmo nas quatro zonas. Na zona terminal observou-se a presença de espermatozoides no lúmen dos túbulos secretores que permanecem estocados mesmo na ausência de copula recente. Aqui, estudaram-se esses órgãos e suas conexões na tentativa de elucidar os mecanismos da reprodução no tubarão azul, apresentando os aspectos tridimensionais, desta forma agregando informações morfológicas importantes para o entendimento da estrutura e funcionamento desses órgãos de fundamental importância na vida da maioria dos elasmobrânquios.