Multilevel sensor for monitoring external and internal environment of eggs.

Although it is well known that incubation environment has a great influence on embryogenesis and post-hatching performance of birds, not much is known about how external thermal, sound and light stimuli are isolated by eggshells and perceived by embryos. In this context, this study aimed to develop, calibrate and evaluate a multilevel sensor for integrated monitoring of the external (incubator) and internal environment of eggs. The variables of interest for the external environment were air temperature and relative humidity. For the internal environment, shell temperature, internal temperature, luminosity and sound pressure level were considered. The sensor was developed with an ATmega328 microcontroller, in open-source prototyping, using electronic components which are compatible with the egg's physical structure. Calibrations were carried out in a controlled environment, comparing the multilevel sensor with commercial equipment, obtaining coefficients of determination of R 2 > 0.90 for all variables studied. The multilevel sensor was also validated, simulating a commercial incubation situation and comparing eggs with 2 shell colors (white and brown) and internal volume (intact and empty). Validation results showed that white-shelled eggs insulate less external light (P < 0.001) and full eggs presented higher internal temperatures, greater light and lower sound pressure levels compared to empty eggs (P < 0.001). The multilevel sensor developed here is an innovative proposal for monitoring, simultaneously and in real time, different variables of interest in the commercial incubation environment.

High albumen height by expression of GALNT9 and thin eggshell by decreased Ca2+ transportation caused high hatchability in Huainan partridge chicken.

Hatchability could be quite different among individuals of indigenous chicken breed which might be affected by the egg quality. In this study, hatchability was individually recorded among 800 forty-wk-old Huainan partridge chickens. The chickens were then divided into high and low hatchability groups (HH and LH group) with 50 birds in each group. Egg quality was further determined in the 2 groups. Eight birds from each group were selected for slaughtering and tissue, responsible for egg formation, collection for structure observation by staining and candidate gene expression by transcriptome analysis. The hatchability in HH was 100% and 61.18% in LH. The eggshell thickness and shell strength were significantly lower, while the albumen height and Haugh unit were significantly higher in HH group than those in LH group (P < 0.05). The magnum weight and index, and the expression of polypeptide N-acetylgalactosaminyltransferase 9 (GALNT9), which responsible for thick albumen synthesis, in HH group were also significantly higher than that of LH group (P < 0.05). Compared with the LH group, there were 702 differentially expressed genes (DEGs) in HH group, of which 402 were up-regulated and 300 were down-regulated. Candidate genes of calbindin 1 (CALB1) and solute carrier family 26 member 9 (SLC26A9), which regulate calcium signaling pathway so as to affect Ca2+ transportation, exhibited significant high and low expression, respectively, in HH group compared to those in LH group (P < 0.05). Therefore, indigenous chicken with high expression of GALNT9 in magnum to form thick albumen to provide more protein for embryo, while high CALB1 and low expression of SLC26A9 to decrease Ca2+ transportation so as to form a thinner eggshell and provide better gas exchange during embryo development.

Genomic insights into shank and eggshell color in Italian local chickens.

Eggshell and shank color in poultry is an intriguing topic of research due to the roles in selection, breed recognition, and environmental adaptation. This study delves into the genomics foundations of shank and eggshell pigmentation in Italian local chickens through genome-wide association studies analysis to uncover the mechanisms governing these phenotypes. To this purpose, 483 animals from 20 local breeds (n = 466) and 2 commercial lines (n = 17) were considered and evaluated for shank and eggshell color. All animals were genotyped using the Affymetrix Axiom 600 K Chicken Genotyping Array. As regards shank color, the most interesting locus was detected on chromosome Z, close to the TYRP1 gene, known to play a key role in avian pigmentation. Additionally, several novel loci and genes associated with shank pigmentation, skin pigmentation, UV protection, and melanocyte regulation were identified (e.g., MTAP, CDKN2A, CDKN2B). In eggshell, fewer significant loci were identified, including SLC7A11 and MITF on chromosomes 4 and 12, respectively, associated with melanocyte processes and pigment synthesis. This comprehensive study shed light on the genetic architecture underlying shank and eggshell color in Italian native chicken breeds, contributing to a better understanding of this phenomenon which plays a role in breed identification and conservation, and has ecological and economic implications.

Whole transcriptome sequencing reveals key genes and ceRNA regulatory networks associated with pimpled eggs in hens.

Eggshell is one of the most important indicators of egg quality, and due to low shell strength, pimple eggs (PE) are more susceptible to breakage, thus causing huge economic losses to the egg industry. At the current time, the molecular mechanisms that regulate the formation of pimple eggs are poorly understood. In this study, uterine tissues of PE-laying hens (n = 8) and normal egg (NE) -laying hens (n = 8) were analyzed by whole transcriptome sequencing, and a total of 619 differentially expressed mRNAs (DE mRNAs), 122 differentially expressed lncRNAs (DE lncRNAs) and 21 differentially expressed miRNAs (DE miRNAs) were obtained. Based on the targeting relationship among DE mRNAs, DE lncRNAs and DE miRNAs, we constructed a competitive endogenous RNA (ceRNA) network including 12 DE miRNAs, 19 DE lncRNAs, and 128 DE mRNAs. Considering the large amount of information contained in the network, we constructed a smaller ceRNA network to better understand the complex mechanisms of pimple egg formation. The smaller ceRNA network network contains 7 DE lncRNAs (LOC107056551, LOC121109367, LOC121108909, LOC121108862, LOC112530033, LOC121113165, LOC107054145), 5 DE miRNAs (gga-miR-6568-3p, gga-miR-31-5p, gga-miR-18b-3p, gga-miR-1759-3p, gga-miR-12240-3p) and 7 DE mRNAs (CABP1, DNAJC5, HCN3, HPCA, IBSP, KCNT1, OTOP3), and these differentially expressed genes may play key regulatory roles in the formation of pimpled eggs in hens. This study provides the overall expression profiles of mRNAs, lncRNAs and miRNAs in the uterine tissues of hens, which provides a theoretical basis for further research on the molecular mechanisms of pimpled egg formation, and has potential applications in improving eggshell quality.

Effects of 28 h ahemeral light cycle on production performance, egg quality, blood parameters, and uterine characteristics of hens during the late laying period.

This study aimed to systematically determined the effect of 28 h ahemeral light cycle on production performance, egg quality, blood parameters, uterine morphological characteristics, and gene expression of hens during the late laying period. At 74 wk, 260 Hy-Line Brown layers were randomly divided into 2 groups of 130 birds each and in duplicates. Both a regular (16L:8D) and an ahemeral light cycle (16L:12D) were provided to the hens. The oviposition pattern in an ahemeral cycle shifted into darkness, with oviposition mostly occurring 3 to 5 h after light out. Production performance was unaffected by light cycle (P > 0.05). Nonetheless, compared to the normal group, the ahemeral group exhibited increased egg weight, eggshell weight, eggshell percentage, yolk percentage, eggshell thickness, and eggshell strength (P < 0.05). There were rhythmic changes in the uterine morphological structure in both cycles, however, the ahemeral group maintained a longer duration and had more uterine folds than the normal group. In the ahemeral cycle, the phases of the CLOCK and PER2 genes were phase-advanced for 3.96 h and 4.54 h compared to the normal cycle. The PHLPP1 gene, which controls clock resetting, exhibited a substantial oscillated rhythm in the ahemeral group (P < 0.05), while the expression of genes presenting biological rhythm, such as CRY2 and FBXL3, was rhythmically oscillated in normal cycle (P < 0.05). The ITPR2 gene, which regulates intracellular Ca2+ transport, displayed a significant oscillated rhythm in ahemeral alone (P < 0.05), while the CA2 gene, which presents biomineralization, rhythmically oscillated in both cycles (P < 0.05). The ahemeral cycle caused 2.5 h phase delays in the CA2 gene compared to the normal cycle. In conclusion, the 28 h ahemeral light cycle preserved the high condition of the uterine folds and changed the uterine rhythms of CLOCK, PER2, ITPR2, and CA2 gene expression to improve ion transport and uterine biomineralization.

Eggshell translucency: its relationship with specific gravity and eggshell color and its influence on broiler egg weight loss, hatchability, and embryonic mortalities.

Eggshell quality is among the most important factors affecting hatchability in broiler breeders, and therefore several methods for its assessment are available in the poultry industry. Among them, eggshell translucency has received special attention in recent years due to its connection with ultrastructural disorganization of the shell layers. However, there is very limited data on the impact of translucency on hatching eggs and on the possible links between this trait and specific gravity (SG) or shell color. Thus, our study investigated associations and interactions between eggshell translucency, SG, and color on incubation parameters of eggs from the same breeding flock (Ross 308AP, 51 wk of age). To this end, light and dark eggs within 5 different SG categories (≥1.065, 1.070, 1.075, 1.080, and ≤1.085) were selected from 15,976 eggs, graded into 3 translucency scores, and later incubated to evaluate egg weight loss, hatchability and embryonic mortalities. In general, translucency scores were evenly distributed within SG categories (χ2 [8, N = 1,138] = 13.67, P = 0.090) and color (χ2 [2, N = 1,138] = 4.93, P = 0.084). No interactions between eggshell translucency and SG or between translucency and color were found for the analyzed variables. An interaction was observed between SG and eggshell color for the variable egg weight loss, where the light-shelled eggs, in most SG categories lost more weight throughout incubation than dark eggs. Eggshell translucency affected egg weight loss, hatchability, and embryonic mortality on 11 to 18 d of incubation, with highly translucent eggs showing the worst results. At the same time, eggs with SG lower than 1.070 displayed the greatest weight loss, lowest hatchability, and highest contamination. We found no influence of eggshell color on weight loss or hatchability, but light-shelled eggs exhibited higher late embryonic mortality. Together, these data suggest that despite its effects on certain hatching parameters, shell translucency bears no relationship to SG or color.

TMT-based quantitative proteomic analysis reveals eggshell matrix protein changes correlated with eggshell quality in Jing Tint 6 laying hens of different ages.

The decline in eggshell quality resulting from aging hens poses a threat to the financial benefits of the egg industry. The deterioration of eggshell quality with age can be attributed to changes in its ultrastructure and chemical composition. Specific matrix proteins in eggshells have a role in controlling crystal growth and regulating structural organization. However, the variations in ultrastructure and organic matrix of eggshells in aging hens remain poorly understood. This study assessed the physical traits, mechanical quality, chemical content, as well as the microstructural and nanostructural properties of eggs from Jing Tint 6 hens at 38, 58, 78, and 108 wk of age. Subsequently, a quantitative proteomic analysis was conducted to identify differences in protein abundance in eggshells between the ages of 38 and 108 wk. The results indicated a notable decline in shell thickness, breaking strength, index, fracture toughness, and stiffness in the 108-wk-age group compared to the other groups (P < 0.05). The ultrastructure variations primarily involved an increased ratio of the mammillary layer and a reduced thickness of the effective layer of eggshell in the 108-wk-age group (P < 0.05). However, no significant differences in eggshell compositions were observed among the various age groups (P > 0.05). Proteomic analysis revealed the identification of 76 differentially expressed proteins (DEPs) in the eggshells of the 38-wk-age group and 108-wk-age group, which comprised proteins associated with biomineralization, calcium ion binding, immunity, as well as protein synthesis and folding. The downregulation of ovocleidin-116, osteopontin, and calcium-ion-related proteins, together with the upregulation of ovalbumin, lysozyme C, and antimicrobial proteins, has the potential to influence the structural organization of the eggshell. Therefore, the deterioration of eggshell quality with age may be attributed to the alterations in ultrastructure and the abundance of matrix proteins.

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.

Comparative analysis of hepatic transcriptomes and metabolomes of Changshun green-shell laying hens based on different green eggshell color intensities.

The eggshell color of avian species is an important trait that is predominantly determined by the pigments biliverdin and protoporphyrin. Various factors affect eggshell pigment deposition and coloration; however, the underlying mechanisms remain unclear. We analyzed the hepatic transcriptomes and metabolomes of Changshun green-shell hens laying dark green and light green eggs to investigate the potential role of the liver in regulating the intensity of the green eggshell color. In total, 350 differentially expressed genes and 211 differentially altered metabolites were identified. Gene set enrichment analysis revealed that the enriched pathways and Gene Ontology (GO) terms were mainly associated with energy, immunity, and nutrient metabolism. Metabolite set enrichment analysis revealed that the enriched pathways were mainly associated with amino acid, vitamin, bile acid, and lipid metabolism. Moreover, gene-metabolite interaction network analysis revealed 1 subnetwork. Most genes and metabolites in this subnetwork were determined to be related to melanin metabolism and transport. In conclusion, our results suggest that hepatic melanin metabolism and transport are critical for eggshell coloration. Six candidate genes (CDKN2B, DDC, PYCR1, ABCG5, SLC3A1, and P2RX2) and 7 candidate metabolites (serotonin, 5-hydroxyindoleacetic acid, ornithine, acetylcholine, L-tryptophan, D-ornithine, and ADP) were suggested to play important roles in this process. Meanwhile, this study suggests that changes in hepatic energy metabolism, immune status, antioxidation activity, nutrient availability, and bile acid synthesis can impair eggshell coloration.

Estimation of genetic parameters of eggshell translucency and production traits in different genotypes of laying hens.

The translucency of eggshells is a ubiquitous appearance problem caused by moisture translocation and the accumulation of egg contents into the eggshell ultrastructure. Previous studies have mainly investigated the causes of eggshell translucency from nutritional and environmental perspectives. However, little is known of the effect of genetics the causes of eggshell translucency on hen production performance. To evaluate the genetic parameters of eggshell translucency and other production performance indicators, we performed an experiment on 3 pure hen lines: 624 Dwarf Layer-White, 1,612 Rhode Island Red, and 813 Rhode Island Red-White. We collected eggs from each hen over 5 d and measured eggshell translucent level (TL) using the grading method. Additionally we measured indicators of each hen during the laying period, including age at laying of the first egg (AFE), body weight at laying of the first egg (BWFE), weight of the first egg (FEW), body weight at 40 wk (BW40), egg weight at 40 wk (EW40), egg production up to 40 wk of age (EN), and calculated the genetic parameters among the indicators. The results showed that the estimated heritability of TL in the 3 genotypes were 0.30, 0.24, and 0.20, respectively, suggesting a low or moderate level of heritability. We found a positive correlation between TL and AFE, with genetic correlation coefficients 0.19 to 0.41, and negative genetic correlation between TL and EN, with correlation coefficient -0.36 to -0.19. Additionally, we observed positive correlation exists between AFE and FEW, BWFE and FEW, and BW40 and EW40; and negative correlation between AFE and EN in the 3 pure lines. These results enriched the research on heritability of eggshell translucency in different hen breeds and demonstrated moderate or low heritability of the indicator. Furthermore, eggshell translucency was negatively affected by AFE and EN. Our results provide a valuable reference for predicting selection response of eggshell translucency and production performance in brood hens, and locating the genes regulating eggshell translucency.

Effect of phytase and limestone particle size on mineral digestibility, performance, eggshell quality, and bone mineralization in laying hens.

The effect of microbial phytase and limestone particle size (LmPS) was assessed in Lohmann Tradition laying hens from 31 to 35 wk of age. Seventy-two hens were used in a completely randomized trial according to a 2 × 2 factorial arrangement with 2 levels of phytase/basal available P (aP); 0 FTU/kg with 0.30% aP or 300 FTU/kg with 0.15% aP, and 2 limestone particle sizes; fine particles (FL, <0.5 mm) or a mix (MIX) of 75% coarse limestone (CL, 2-4 mm) and 25% FL. Diets contained equivalent levels of Ca (3.5%), phytic P (PP; 0.18%), and aP (0.30%) considering the P equivalency of phytase. Thus, dietary treatments were FL0 and MIX0 without phytase, and FL300 and MIX300 with 300 FTU/kg phytase. Performance were recorded daily and eggshell quality (eggshell weight proportion, weight, thickness, and breaking strength) was measured weekly. At the end of the trial, bone parameters (tibia breaking strength, elasticity, and ash) and the apparent precaecal digestibility (APCD) of P and Ca were determined. No differences were observed between treatments in feed intake, FCR and bone parameters. Addition of MIX increased the eggshell proportion, weight and thickness in groups receiving no phytase (+6.5, +6.9, and +4.5%, respectively) while no effect was observed in groups receiving phytase (Phytase × LmPS, P < 0.05). In hens receiving FL, the APCD of P was lower in diets supplemented with phytase (-14 percentage points; Phytase × LmPS, P < 0.001). A higher phytate disappearance was observed in hens fed diets with phytase in combination with MIX (Phytase × LmPS, P = 0.005). Phytase and MIX together increased the APCD of Ca by 7.3 percentage points (Phytase × LmPS, P < 0.001). In conclusion, addition of CL could limit the formation of Ca-phytate complex thus improving the response of the birds to phytase compared to FL.

New insights into the relationships between egg maternal components: the interplays between albumen steroid hormones, proteins and eggshell protoporphyrin.

Recent studies have shown that the egg yolk maternal components, which are a mixture of substances that can affect the developing embryo, do not act separately but are interconnected and co-adapted. Surprisingly, no study to date has focused on the associations between maternally derived albumen steroids and albumen and eggshell compounds with pleiotropic effects. Eggshell pigment protoporphyrin (PROTO IX) should provide primary antimicrobial protection for eggs, but as a proven pro-oxidant, it may compromise female fitness. Abundant albumen proteins ovotransferrin (OVOTR) and lysozyme (LSM) have been shown to have antimicrobial, antioxidant, immunoregulatory and growth-regulatory roles. To investigate associations between albumen steroids and OVOTR, LSM and eggshell cuticle PROTO IX, we used chicken eggs with differently pigmented eggshells. We found that albumen steroid hormones were strongly intercorrelated. In addition, we revealed that albumen LSM and testosterone (T) were positively associated, while a negative association was found between albumen LSM and pregnenolone (P5). Eggshell cuticle PROTO IX was negatively associated with the concentration of albumen 17α-hydroxypregnenolone (17-OHP5). Finally, of all the hormones tested, only the concentration of albumen 17-OHP5 correlated negatively with egg volume and varied with eggshell colour and chicken breed. Although experimental evidence for the effect of maternal albumen steroids on avian developing embryo is still scarce, our study is the first to highlight co-variation and potential co-adjustment of maternally derived albumen steroids, proteins and eggshell cuticle pigment suggesting similar allocation mechanisms known for yolk maternal compounds with the potential to influence the avian embryo and offspring phenotype.

Transcriptomic analysis of the liver in aged laying hens with different eggshell strength.

Eggshell is composed of a very ordered and mineralized structure and is important for egg quality. Eggshell strength is particularly important because of its direct association with economic outcomes and egg safety. Various factors related to laying hens and their environment affects eggshell strength. However, the molecular mechanisms of liver functions related to decreased eggshell strength of aged laying hens are largely unknown. Therefore, this study aimed to identify potential factors affecting eggshell strength in aged laying hens at the hepatic transcriptomic level. A total of five hundred 92-wk-old Hy-line Brown laying hens were screened to select those exhibiting the greatest variation in eggshell strength. Based on the final eggshell strength, 12 hens producing eggs with strong eggshell strength (SES) and weak eggshell strength (WES) were finally selected (n = 6) for liver tissue sampling. The RNA-sequencing was performed to identify differentially expressed genes (DEGs) between the 2 groups. We identified a total of 2,084 DEGs, of which 1,358 genes were upregulated and 726 genes were downregulated in the WES group compared with SES group. According to the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, the DEGs indicated the mammalian target of rapamycin signaling pathway, the Janus kinase-signal transducer and activator of transcription pathway, the mitogen­activated protein kinase signaling pathway, and the insulin resistance pathways. Genes related to fatty liver disease were upregulated in WES group compared with SES group. In addition, expression of several genes associated with oxidative stress and bone resorption activity was altered in aged laying hens with different eggshell strength. Overall, these findings contribute to the identification of genes involved in different intensity of eggshell strength, enabling more understanding of the hepatic molecular mechanism underlying in decreased eggshell strength of aged laying hens.

Effects of late incubation temperature and moment of first post-hatch feed access on neonatal broiler development, temperature preference, and stress response.

Early life experiences are known to be of great importance for later life. For instance, exposure to stress during early life can increase fearfulness at later age. In broilers, delayed feeding after hatch may cause metabolic stress. Besides, delayed feeding after hatch may affect neonatal broiler development and thermogenesis and consequently preferred ambient temperature. Moreover, these effects of feeding strategy may be dependent on late incubation temperature. To study this, eggs (n = 1,338) from a 54-wk-old Ross broiler breeder flock were incubated at 37.8°C (control) or 36.7°C (lower) eggshell temperature (EST) during late incubation (≥ embryonic d 17). At hatch, two feeding strategies were applied (direct access (early feeding) or 51 to 54 h delayed access (delayed feeding)). Broilers (n = 960) were equally divided over 32 pens and grown for 3 wk. Stress was assessed by determination of corticosterone in blood at 0 h, 48 h, 96 h and d 21 after hatch. Fearfulness was assessed by tonic immobility at d 13. Temperature preference was assessed at d 2 and d 12. Broiler development was determined at 0 h, 48 h, and 96 h after hatch. There was no EST × feeding strategy interaction for any parameter (P ≥ 0.07). Early feeding resulted in a 2.5× lower plasma corticosterone concentration at 48 h (P < 0.01) and a 2.2°C and 2.0°C lower preference temperature for d 2 and d 12 respectively (P = 0.01) compared to delayed feeding. Tonic immobility was not affected. In conclusion, early feeding reduces exposure to stress in the short term and stimulates thermoregulatory ability of broilers in the longer term.

Natural Materials Modified and Applied to the Detection of Drugs In Situ: Modification of Eggshell and Quantification of Oxytetracycline.

This work describes a novel sensing system using eggshells as substrate for the first time, targeting the detection and semiquantitative determination of antibiotics in waters from aquaculture, enabling simple, inexpensive, and in situ drug monitoring. Eggshell was ground and the resulting powder was modified by adsorption of suitable reagents, and it takes a typical colour after contact with the antibiotic. The colour intensity is correlated with the concentration of the antibiotic. This novel approach was applied to oxytetracycline, one of the antibiotics commonly used in aquaculture. The chemical changes on the eggshell powder were evaluated and optimised to produce an intense colour change as a function of the concentration of the antibiotic. The colour changes were evaluated by visual comparison with images taken with a digital camera, applying an appropriate mathematical treatment to the colour coordinates of the HSL system used by Windows. The selectivity of the response was tested against other antibiotic drugs. The materials were also used in the analysis of a spiked environmental water sample. Overall, this work presents a rapid, inexpensive, simple and equipment-free method for screening and discrimination of tetracycline drugs in aquaculture. The method is a green approach by reusing eggshells and decreasing the level of contamination correlated to analytical methods, thus being a promising tool for local, rapid, and cost-effective antibiotic monitoring.

Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Properties, Genetics and Innate Immune Function of the Cuticle in Egg-Laying Species.

Cleidoic eggs possess very efficient and orchestrated systems to protect the embryo from external microbes until hatch. The cuticle is a proteinaceous layer on the shell surface in many bird and some reptile species. An intact cuticle forms a pore plug to occlude respiratory pores and is an effective physical and chemical barrier against microbial penetration. The interior of the egg is assumed to be normally sterile, while the outer eggshell cuticle hosts microbes. The diversity of the eggshell microbiome is derived from both maternal microbiota and those of the nesting environment. The surface characteristics of the egg, outer moisture layer and the presence of antimicrobial molecules composing the cuticle dictate constituents of the microbial communities on the eggshell surface. The avian cuticle affects eggshell wettability, water vapor conductance and regulates ultraviolet reflectance in various ground-nesting species; moreover, its composition, thickness and degree of coverage are dependent on species, hen age, and physiological stressors. Studies in domestic avian species have demonstrated that changes in the cuticle affect the food safety of eggs with respect to the risk of contamination by bacterial pathogens such as Salmonella and Escherichia coli. Moreover, preventing contamination of internal egg components is crucial to optimize hatching success in bird species. In chickens there is moderate heritability (38%) of cuticle deposition with a potential for genetic improvement. However, much less is known about other bird or reptile cuticles. This review synthesizes current knowledge of eggshell cuticle and provides insight into its evolution in the clade reptilia. The origin, composition and regulation of the eggshell microbiome and the potential function of the cuticle as the first barrier of egg defense are discussed in detail. We evaluate how changes in the cuticle affect the food safety of table eggs and vertical transmission of pathogens in the production chain with respect to the risk of contamination. Thus, this review provides insight into the physiological and microbiological characteristics of eggshell cuticle in relation to its protective function (innate immunity) in egg-laying birds and reptiles.

Functional connections between bird eggshell stiffness and nest characteristics through risk of egg collision in nests.

Eggs and nests are two critical traits for the ecological success of birds. Their functional interactions, however, remain unclear. Here, we examined the functional connections between egg stiffness and nest attachment, site and structure for 1350 avian species. We revealed high eggshell stiffness for eggs in nests with a pensile attachment, located on non-tree vegetation or having a domed shape, suggesting that birds produce stiffer eggs in response to higher egg-collision risk in unstable or enclosed nests. Interdependence models suggested that the evolution of eggshell stiffness was more likely to be driven by than drive that of nest characters. Our results implied a trade-off between investment in competing for established nesting niches and producing stiff eggs to explore novel niches with high collision risk, possibly mediated by predation or thermoregulation. This study highlights an overlooked connection between nests and eggshells that may have broadened the ecological niches of birds.

Impact of relative humidity on the laying performance, egg quality, and physiological stress responses of laying hens exposed to high ambient temperature.

The present study investigated the effects of relative humidity (RH) on the laying performance, egg quality, and stress indicators of laying hens raised at high ambient temperatures. A total of 180 Hy-Line Brown laying hens (68-wk-old) were randomly allotted to one of the following three RH conditions for 12 h a day (9:00 a.m.-9:00 p.m.) over four weeks: low RH (LRH; 25% RH), moderate RH (MRH; 50% RH), and high RH (HRH; 75% RH); ambient temperature was 30 °C under all treatments. None of the RH treatments affected hen-day egg production, egg weight, or egg mass (P > 0.05). However, feed intake was lower in the HRH group than in the LRH group (P < 0.05). Plasma corticosterone (CORT) concentration on day 21, yolk CORT concentration on day 3, and albumen CORT concentration on day 7 following RH exposure were higher in the HRH group than in the LRH group (P < 0.05). Moreover, plasma HDL-cholesterol concentration on day 14 was higher in the HRH group than in the LRH group (P < 0.05). On days 3 and 14, the Haugh unit decreased (P < 0.05) in the LRH group compared with that in the MRH and HRH groups. The HRH-exposed laying hens showed the lowest (P < 0.05) eggshell thickness on day 14. The absolute weights of eggshell, yolk, and albumen decreased in the HRH group compared with those in the MRH and LRH groups. Overall, high RH lowered feed intake and egg quality except for the Haugh unit, and induced stress response as manifested by elevated plasma, yolk, and albumen CORT concentrations. To our best knowledge, the present study is the first to demonstrate the role of RH in triggering temperature stress responses in laying hens.

phytanoyl-CoA dioxygenase domain-containing protein 1 plays an important role in egg shell formation of silkworm (Bombyx mori).

Yun7Ge is a giant egg mutant found in the silkworm variety Yun7. In comparison with the giant mutant Ge, the eggs of Yun7Ge are larger. The number of laid eggs and hatching rate of Yun7Ge are reduced, which is not conducive to reproduction. In this work, the target gene controlling giant egg trait is located on the Z chromosome and was determined through genetic analysis. Transcriptome results showed that phytanoyl-CoA dioxygenase domain-containing protein 1 (PHYHD1) on the Z chromosome was silenced, and the 25 chorion genes on chromosome 2 were remarkably downregulated. Sequence analysis showed that the 73.5 kb sequence including the PHYHD1 was replaced by a ~3.0 kb sequence. After knocking out the PHYHD1 by using CRISPR/Cas9, the chorion genes were significantly downregulated. Hence, the silencing of PHYHD1 leads to the downregulation of many chorion protein genes, thus directly causing giant eggs.