Effect of LED green light incubation on pecking hormones under normal conditions
Experimental design
In this study total 296 eggs of each Roman pink and Hy-line brown broiler breeders were taken and randomly divided into 2 groups; LED green light incubation (experimental group), and dark light incubation (control group). Each group was further divided into 4 replicates (37 eggs/replicate).
Incubation Process
The experiment adopted an NK-525 microcomputer automatic incubation control box (Shandong Agricultural Science Incubation Equipment Co., Ltd., Shandong Dezhou) for egg incubation, and its size specifications were 1100×1000×900 mm. Two 60 cm long LED green light tubes were fixed in the egg incubator at 15cm above the two sides of the egg tray. To measure the light intensity, 9 points were selected on each egg plate, and a lux meter was used to measure the light intensity of each point in the left, right and horizontal directions. An electromagnetic pulse controller was used to adjust the light intensity of each test point, and the light intensity was controlled at 20 to 50 lux. The light cycle was adjusted to 16-hour light: 8-hour dark (16 L:8 D). The incubation temperature throughout the experiment was 38.0±0.1 °C (1 to 7 days), 37.8± 0.1 °C (7 to 14 days), and 37.6 ± 0.1 °C (14 to 21 days), and the humidity was 60±2%. The egg turning interval was 1.5 hours throughout the experiment, and the egg pan was adjusted at the angle of 45° from 0 to 18 days of incubation. On days 9, 12, and 15, the eggs were examined using a Cool-lite tester to sort out dead embryos and azoospermia from the eggs. On day 18, all eggs were transferred from egg trays to the hatching baskets.
Tissue sample extraction
Embryonic sample extraction
On day 18, four eggs/replicate were randomly chosen from the same positions within egg trays of each experimental group at 6:00, 12:00, 18:00 and 24:00 O-clock to validate the survival rates. Eight eggs/group were randomly broken at the blunt end to take the samples of embryos. Liver tissue (150 to 300 mg weight) was extracted from each embryo, putted into Eppendorf (EP) tubes and stored in a -80°C refrigerator for further analysis.
Detection of pecking hormone from liver samples
The contents of 5-HT, CORT, DA, and TES were detected by ELISA method as given below.
Preparation: Set up standard curve sample wells and experimental sample wells in the microplate. Add 50 μL of different concentration standard samples to the standard curve wells. Set up 1 blank well without any samples (control well). Add 40 μL of sample dilution and 10 μL of the sample to be tested (5x dilution) to the sample detection wells.
Incubation: Add 100 μL of microplate reagent to each well (except blank wells), seal the microplate and incubate at 37°C for 60 minutes.
Washing: Dilute the concentrated washing liquid 20 times with distilled water. Remove the microplate, discard the liquid, and wash each well 5 times with the diluted washing solution.
Color Development: Add 50 μL of color developer A and 50 μL of color developer B to each well (including blank wells). Incubate the microplate at 37°C for 15 minutes, protected from light.
Termination: Add 50 μL of stop solution to each well (including blank wells) to terminate the reaction.
Measurement: Within 15 minutes, measure the OD value of each well at 450 nm wavelength in a microplate reader, using the blank well to zero the measurement.
Effect of LED green light incubation on different hormones during hormonal stress
Experimental design
A total of 704, 52-week-old broiler breeder eggs were selected and weighed 55.4 ± 0.41 g (average ± SEM). The eggs were sterilized and randomly divided into 4 groups of 176 eggs for follow-up trials. The experimental groups were as follows: 100 μL corticosterone solution injection (CI); 100 μL corticosterone + melatonin mixed solution injection (CMI), 100 μL PBS solution injection (PI), and no injection (UI). The eggs in each group were further divided into 2 subgroups, with 88 eggs per subgroup, LED green light-regulated incubation (G) and dark incubation (D). Therefore, all eggs were divided into G+CI, D+CI, G+CMI, D+CMI, G+PI, D+PI (experimental groups) and G+UI and D+UI (control groups). Each subgroup was further divided into 4 replicates with 22 eggs each. The blunt end centers of all eggs underwent with a puncher, followed by a Hamilton injection needle (Hangzhou Cyst Firefly Technology Co., Ltd., Hangzhou) carefully inserted into the blunt end central hole of each egg, penetrating 20 mm (to ensure the accurate injection in to the yolk). Paraffin wax was used to seal the pores of eggs to prevent contamination during incubation. All injections in the experiment were performed by an experimenter to eliminate non-systematic errors.
Corticosterone/melatonin solution preparation
To produce CORT and MLT solution, 100 mg of CORT/MLT dry powder dissolved in 6 ml of absolute ethanol, then 10 ml of sodium bicarbonate buffer was added. Distilled water was used to set the volume to 100 ml to prepare a stock solution having concentration of 1 μg/μL for later use. One ml of mother solution mixed with distilled water to make total volume 100 ml, and a concentration of 1 μg/100 μL was obtained for later use.
Corticosterone + melatonin mixed solution preparation
Prepared a solution by combining 1 ml of CORT mother liquor with 3 ml of MLT mother liquor. Adjusted the volume of 100 ml by using distilled water, resulting a solution containing 1 μg CORT and 3 μg MLT per 100 mL of solution for later use.
Sample preparation and detection of physiological hormone levels
On the first day of hatching (HD1), 2 chicks/replicate in each group were selected at 6:00 AM, 12:00 AM, 18:00 PM and 24:00 AM. Blood sample of 1-1.5 ml was obtained from each chick, mixed with sodium citrate for anti-coagulation, and agitated for 15 minutes. The mixture was then centrifuged at 2000-3000 rpm for 20 minutes, and the supernatant was collected and stored in a -20°C freezer. The ELISA method was used to detect the concentrations of 5-HT and CORT in the blood serum.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA (with nonparametric or mixed methods) was used to compare hormone concentrations. The correlation between pecking hormone levels in egg yolks and eggshell size and color was analyzed using the Pearson correlation coefficient (Pearson). All the data are expressed as the standard error mean ± (SEM±), and P<0.05 was regarded as a significant difference.