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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.

The molecular mechanisms underlying reproductive aging in avian species are poorly understood. Previous studies have shown the importance of mechanistic target of rapamycin (mTOR) signaling pathway in aging. In this study, we investigated the relationship between the mTOR signaling pathway and ovarian aging in the peak phase and late phase of egg production in laying hens. The egg production rate and egg quality were tracked for 5 consecutive weeks in 30-week-old and 70-week-old Dawu Jinfeng hens (N = 30/group). During the peak phase (week 35) and late phase (week 75), antioxidant and immune indicators were detected by enzyme-linked immunosorbent assay, and mTOR signaling-related genes (CLIP-170, GRB10, LIPIN-1, ATG1, 4E-BP1, S6K, PKC, RHO, and SGK1) were detected in the follicles by quantitative reverse transcription-PCR technology. The protein expression of key genes (mTOR, PKC, 4EBP1) was evaluated by Western blot analysis. The egg production rate and the antioxidant indexes superoxide dismutase and glutathione peroxidase and the levels of total antioxidant capacity and immunoglobulins (IgM and IgG) were significantly higher at week 35 than those at week 75 (P

Melatonin is a key regulator of follicle granular cell maturation and ovulation. The mammalian target of rapamycin (mTOR) pathway plays an important role in cell growth regulation. Therefore, our aim was to investigate whether the mTOR signaling pathway is involved in the regulation of melatonin-mediated proliferation and apoptotic mechanisms in granulosa cells. Chicken follicle granular cells were cultured with melatonin (0, 2, 20, or 200 μmol/L) for 48 h. The results showed that melatonin treatment enhanced proliferation and suppressed apoptosis in granular cells at 20 μmol/L and 200 μmol/L (P < 0.05) by upregulation of cyclin D1 (P < 0.01) and Bcl-2 (P < 0.01) and downregulation of P21, caspase-3, Beclin1, and LC3-II (P < 0.01). The effects resulted in the activation of the mTOR signaling pathway by increasing the expression of avTOR, PKC, 4E-BP1, S6K (P < 0.05), p-mTOR, and p-S6K. We added an mTOR activator and inhibitor to the cells and identified the optimal dose (10 μmol/L MHY1485 and 100 nmol/L rapamycin) for subsequent experiments. The combination of 20 μmol/L melatonin and 10 μmol/L MHY1485 significantly enhanced granulosa cell proliferation (P < 0.05), while 100 nmol/L rapamycin significantly inhibited proliferation and enhanced apoptosis (P < 0.05), but this action was reversed in the 20-μmol/L melatonin and 100-nmol/L rapamycin cotreatment groups (P < 0.05). This was confirmed by mRNA and protein expression that was associated with proliferation, apoptosis, and autophagy (P < 0.05). The combination of 20 μmol/L melatonin and 10 μmol/L MHY1485 also activated the mTOR pathway upstream genes PI3K, AKT1, and AKT2 and downstream genes PKC, 4E-BP1, and S6K (P < 0.05), as well as protein expression of p-mTOR and p-S6K. Rapamycin significantly inhibited the mTOR pathway–related genes mRNA levels (P < 0.05). In addition, activation of the mTOR pathway increased melatonin receptor mRNA levels (P < 0.05). In conclusion, these findings demonstrate that melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptor.

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.05) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.05) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.05), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P

Eggshell translucency is a ubiquitous external eggshell quality problem caused by variations of eggshell ultrastructure or shell membrane. In previous studies, researchers have widely investigated this phenomenon with nutritional, environmental, and genetic perspectives in many breeds. However, most studies referring to phenotypic measurement of shell translucency have been performed using a relatively subjective two-, three-, or four-grading methods, which made it impossible to compare distribution of shell translucency among different breeds. In this study, we aimed to explore variations of translucent eggshell spots in different breeds and their distribution in blunt, middle, and sharp ends of eggshell using a relatively objective grayscale recognition method. We selected 45 eggs from each flock of pure lines, commercial strains, and Chinese local breeds (10 flocks, aged 60 to 70 wk), and stored them in a constant environment for 5 d. Then measured eggshell translucency using grayscale recognition method. Indicators of shell translucency included sum of spot areas on the whole eggshell (SUSA), sum area of the whole eggshell (SUSHA), RSS (ratio of SUSA to SUSHA), quantity of spots (QS), average spot area in eggshell (AAES), and diameter of spots in eggshell (DS). As results, in Hy-Line Brown, Brown-Egg Dwarf Layer, and Taihang (pink-shelled) breeds, phenotypic intensity of eggshell translucency was slight; in Rhode Island Red, Jingfen-1, and Dongxiang breeds, phenotypic intensity of eggshell translucency was relatively extensive; and in Jinghong-1, Hy-Line Sonia, White Leghorn, and Taihang (blue-shelled), phenotypic intensity of eggshell translucency was at an intermediate level. In general, the larger the RSS, the larger the QS, AAES, and DS. Of 3 ends for most breeds, eggshell translucency of blunt and sharp ends was usually greater than that of middle ends, and blunt ends seemed to have the most extensive eggshell translucency. Findings from this study could provide a reference for population selection to locate genes regulating shell translucency and to explore the physical structure mechanism for eggshell translucency formation.

The blood gas and biochemical reference range established with i-STAT clinical analyzer in avian has become common, however, the reference value for various laying hen lines is limited. Therefore, blood gas and biochemical reference intervals will be established for Lohmann Silver layers in the pre- and post-laying periods. The blood sample was collected at a 4-wk interval. A total of 230 Lohmann Silver layers including 80 pullets (5–17 wk) and 150 laying hens (21–37 wk) were collected for whole blood measurement with the i-STAT clinical analyzer. The CG8+ cartridge provides values of the following 13 parameters: sodium (Na mmol/L), potassium (K mmol/L), ionized calcium (iCa mmol/L), glucose (Glu mg/dL), hematocrit (Hct% Packed Cell Volume [PCV]), pH, partial pressure carbon dioxide (PCO2 mm Hg), partial pressure oxygen (PO2 mm Hg), total concentration carbon dioxide (TCO2 mmol/L), bicarbonate (HCO3 mmol/L), base excess (BE mmol/L), oxygen saturation (sO2%), and hemoglobin (Hb g/dL). The correlation of these parameters and the effect of physiological status were investigated. The reference value interval was established with a reference value advisor for pre-laying and post-laying birds. Correlations were found to be statistically significant, especially between BE and HCO3 and TCO2. Besides, values in Na, iCa, K, Hct, Hb, sO2 differed significantly between the pre- and post-laying periods. Data in this study might serve as important information for facilitating the genetic selection and assessing the health of Lohmann Silver laying hens.

Feather pecking is a typically abnormal behavior that significantly impacts breeding efficiency and animal welfare in the egg production sector. Serotonin (5-HT) is essential for neuronal development and behavioral regulation. This study evaluated the effects of birds' behavioral development (including feather pecking) and changes in serum hormones in chickens followed in ovo injection of 5-HT. On day 11, incubated eggs were injected with 5-HT at 0 (saline control), 5 ug (low) or 15 ug (high) (n = 166 per treatment). The hatched female chicks were raised under similar conditions up to 20 weeks of age (n = 60 per treatment). Birds' behaviors were recorded using a digital video recording system. The time to first vocalize and first move, along with the duration of vocalization and escape attempts during the isolation test, during isolation test were analyzed on day 1, and week 4, 8, 12, 16 and 20. Blood samples were collected followed behavioral tests (n = 5/treatment). The expression of 5-HTR1A genes in the hypothalamus was measured by real-time PCR. Compared to controls, 5-HT administrated pullets had greater body weight (P < 0.05) with an improved feed conversion rate (P < 0.05) as well as higher serum concentrations of norepinephrine (NE) regardless of their doses. In addition, serum dopamine (DA) concentrations were lower in both high- and low-dose pullets at 8 and 12 weeks of age (P < 0.05). Also, a decrease in fearfulness response was observed based on the test to vocalize and duration of vocalization (P < 0.05). Further, this exhibited a lesser frequency of total aggressive behavior compared with the chicks in the control group, especially at 8 weeks of age (P < 0.05), where it is associated with elevated serum 5-HT concentration and upregulated hypothalamic expression of 5-HTR1A (P < 0.05). The changes of these hormone concentrations and gene expressions suggested that 5-HT accumulation in early embryonic stages may alter both the adrenergic and serotonergic systems, which could further regulate the isolation behavior and improve birds' growth performance to a certain extent.

Scoring is a common method to evaluate eggshell translucency, and it mainly depends on the area and the density of translucent spots in eggshells. However, the lack of common scoring criteria and the difficulty of quantitatively measuring spots in eggshells impede effective comparisons between research papers and greatly hinder the progress of research on translucent eggshell. To make measurement of translucent eggshells more objective, we optimized the scoring method and compared it with 2 new methods: grayscale recognition and the colorimeter method. Briefly, a total of 354 eggs from 600, 395-day-old dwarf brown laying hens were collected and classified into 4 score groups according to their degree of translucency. This subjective process was repeated 5 times. Then, we captured the profile side of each egg using a camera and measured spot characteristics using grayscale recognition, which involved measuring the quantity of spots (QS), diameter of each spot (DS), average area of each spot (AAES), sum of spot areas (SUSA), sum of shell area (SUSHA), and ratio of SUSA to SUSHA (RSS) on the eggshell. Furthermore, the L, A, and B values of each egg at the sharp, middle, and blunt ends were separately measured using a colorimeter. As a result, average values of 31.31, 29.78, 29.81, and 9.08% of all eggs were divided into score levels 1, 2, 3, and 4 (from opaque to translucent), which correspond with RSS values of 1.34, 3.23, 6.21, and 11.89%, respectively. By grayscale recognition, QS, DS, AAES, SUSA, SUSHA, and RSS all increased along with increased translucency scores (P < 0.05). Using scoring, an egg with a specific RSS value was more easily assigned to a specific score level (50%) or adjacent score levels (50%). The L, A, and B values of eggshells in score level 1 were significantly different from those of eggshells in levels 3 or 4; however, there were no significant differences between any adjacent score levels. In summary, the present study explored objective reference metrics to measure eggshell translucency.

Background: Eggshell ultrastructure organization, including effective layer thickness, mammillary layer thickness, and average size of mammillary cones, is important for breeding and significantly influences eggshell mechanical properties. Several matrix proteins were known to be important in eggshell formation. However, the proteins and variations that determine eggshell ultrastructure organization are not known. Results: In this study, 17 single-nucleotide polymorphisms of three major genes in a hen population using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Five single-nucleotide polymorphisms with a very low minor allele frequency (< 1%) were excluded from further analysis. The remaining 12 single-nucleotide polymorphisms in Hardy-Weinberg equilibrium were used for analysis of associations with eggshell ultrastructure organization. Associations were found for (i) ovocleidin-116 with effective layer thickness (EFF), mammillary layer thickness (MAM), and average size of mammillary cones (SMAM); (ii) ovalbumin with eggshell thickness (ESH), effective layer thickness, and density of the mammillary cone (DMAM); and (iii) calmodulin1 with density of the mammillary cone. Conclusions: The single-nucleotide polymorphisms identified in the present study may be used as potential markers to improve eggshell quality.

Translucent eggshells negatively affect the appearance of eggs and decrease their economic value. Translocation and accumulation of water from the contents to the shells of eggs are frequent occurrences. Causes of translucent eggshell formation have been investigated, but the primary reason is uncertain. In previous studies, scientists have found that the thickness of the eggshell membrane was significantly different between translucent and opaque eggs. However, there are some conflicts among studies. We performed 2 experiments with 3 breeding flocks of chickens to target the reasons for egg translucence. In experiment 1, eggs of 1,024 Brown-Egg Dwarf Layers (DWL) were used. Approximately 1,600 eggs were collected over 2 consecutive days. They were stored for 3 days, and then 120 translucent and 120 opaque eggs were selected for measurement of egg quality traits and weight loss over several weeks. In experiment 2, we used DWL and White Leghorn pure line (WLL) for assessment of eggshell ultrastructure and membrane traits. We chose 120 translucent and 120 opaque eggs from 3,500 DWL eggs and 125 translucent and 125 opaque eggs from 5,028 WLL eggs. The results are as follows: (1) translucent eggs had greater eggshell strength and lower ultimate failure stress of shell membrane than opaque eggs in both DWL and WLL groups, (2) translucent eggs had thicker shells and thinner shell membranes than opaque eggs in DWL, (3) no significant differences were found in either gas pore or bubble pore traits between translucent and opaque eggs in either line, and (4) no significant differences were detected in internal egg quality or weight loss between translucent and opaque eggs in either line. In summary, the present study suggests that variations in both eggshells and shell membrane structures are implicated in the formation of translucent eggs.