A total of four incubators (NK-hatching; Beili Incubation Equipment Co. Ltd., Sichuan, China) used in the experiment were blacked out with shade cloth. Two were outfitted with monochromatic green LED strips (ND-BIS-2D-D10W, Nodark Biolight Technology Co., Ltd., Wuxi, China) (λ = 520 to 525 nm) while the other two were left with no light source. Fertile eggs were obtained from White Leghorn layers (Shaver strain) of 50 week of age. The pure lines chickens were sourced from the University of Guelph and housed in the experimental farm of Institute of Animal Science of Chinese Academy of Agricultural Sciences. A total of 600 eggs of normal size with an average weight of 62.5±3.0 g were selected and stored for no longer than seven days at 15℃ and 70%-75% relative humidity. Eggs were randomly allocated to two groups, with two incubators (replicates) per group, and 150 eggs per incubator. Eggs of one group were incubated in monochromatic green light-fitted incubators with a light:dark schedule of 12L:12D and light intensity of 200 lx during the first 18 days of incubation, namely 12L:12D group. Eggs in the control group were incubated in complete darkness and named 0L:24D group. The temperature and humidity were calibrated by a standard thermometer and hygrometer for the four incubators before incubation and monitored every 2 h during the whole incubation period. The incubation was maintained at a temperature of 37.8 ± 0.10℃, and a relative humidity around 60% until day 18. From day 19, eggs were transferred to hatching baskets and a temperature of 37.2 ± 0.10℃ and relative humidity of 70% was set for the hatcher. At the end of embryonic days 17 (ED17), ED18, ED19, ED20, and ED21, three embryos or chicks were randomly selected and weighted from each replicate (incubator). The hearts, livers, and pectoral muscles were isolated and weighed to track the embryo and organ development. The weight was calculated using the average weight of each replicate for the statistical analysis.
Hatching time and hatching performance
From d 19 post incubation, all eggs were transferred to the same hatcher (Haijiang Hatching Equipment Co., Ltd., Beijing, China) in constant darkness until hatching. The number of hatched chicks was counted every 2 h from 468 h to 512 h post incubation to monitor the hatching time for each egg and assigned to the nearest time of hatch. Average hatching time, time to 90% hatch, peak hatching period, and hatch window were recorded in each replicate. Average hatching time was calculated as the mean duration from egg setting to the emergence of chicks for all hatched chicks in a given batch. Time to 90% hatch was the hatching time that 90% of the chicks hatched. Peak hatching period was defined as the duration that 30% to 70% chicks hatched . Hatch window was defined as the time interval from the emergence of the first chick to the last chick [18, 19]. Hatchability was calculated as a percentage of fertile egg number. Every hatched chick was assessed for chick quality within 4-6 h after hatching according to a standardized method of scoring in a scale of 100 .
Histology of pineal gland
To explore the possible mechanism that monochromatic green light affects hatching time, another hatching trial was performed using the same incubation conditions and light treatments. Pineal glands were extracted from six birds from both 12L:12D and 0L:24D groups on d 10, 12, 14, 16, and 18 post incubation, and fixed in 10% neutral buffered formalin. On the 7th day after fixation, all samples were dehydrated in a graded series of alcohols, embedded in paraffin, and dissected in 4 μm thick. These sections were stained with Harris haematoxylin and eosin for histomorphology observation using a Zeiss Axioskop (Carl Zeiss, Thornwood, NY) equipped with a QICam digital camera and NIS Elements software (Nikon Instruments, Melville, NY) was used for light microscopy. The pineal follicle related indicators, including wall thickness (WT), follicle (FL) area, and lumen (LM) area were measured using Digimizer Image Analysis software (Ostend, Belgium).
Expression of clock-related and AANAT genes in the pineal gland
The expression of clock-related genes (BMAL1, CRY1 and CLOCK) and the melatonin rate-limiting enzyme related gene (AANAT) in the pineal gland was determined using real-time quantitative RT-PCR and cosinor analysis. Pineal glands were collected every 4 h over a 28 h period from d 17 to d 18 after incubation (zeitgeber time (ZT) 2, 6, 10, 14, 18, 22, and 26, where ZT0 is the time when the light is turned on, ZT12 is the time when the light is turned off). Pineal glands removed during dark period were obtained from birds euthanized and decapitated under dim red light. Six pineal glands were collected at each time point each group, and the sampling time of 0L:24D group was synchronized with those of 12L:12D group. Two pineal glands were pooled and the total RNA of three pooled samples each group were extracted with TRIzol reagents following the manufacturer’s protocol (Tiangen Biotech, Beijing, China). cDNA was generated by reverse transcription using 1 μg total RNA in a total of 20 μL reaction volume following the instruction of the manufacturer (TaKaRa, Shiga, Japan). Primer 6.0 was used to design primer sequences for the target genes (Additional file 1: Table S1). Real-time PCR amplification was performed using 1.5 μL cDNA solution per 10 μL reaction volume with SYBR® FAST qPCR Kit Master Mix (Kapa Biosystems, Wilmington, USA). The relative expression of target genes was calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control by the 2-ΔΔC(T) method . Samples were run in triplicate.
Serum concentrations of melatonin (MT) and IGF-1
Serum concentrations of MT and IGF-1 on d 19 post incubation were analyzed. Circadian time (CT) is a standard marker of time that is based upon the oscillation or rhythm in constant darkness. CT0 and CT12 correspond to the beginning time of the subjective day and night respectively; CT26/2 is the time point of CT2 on the second day. The blood samples of twelve embryos were collected from allantois vein after the birds were euthanized by cervical dislocation every 4 h from CT2 to CT26/2. The sampling time of embryos was synchronized in the treatment groups. Serum was decanted after 2 h of collection at room temperature. Serum of four embryos were pooled and three pools each group were sent for detection of MT and IGF-1 concentration using a commercial ELISA kits (Horabio Biotechnology Co. Ltd, Shanghai, China).
The data of related characteristics of hatching time, hatching performance, pineal follicle, serum hormones, and embryo growth were analyzed using T-test (SAS 9.2, SAS Institute Inc., Cary, NC). The percentage was arcsine-transformed before analysis. Significance of difference was set at P < 0.05. The rhythm of genes was analysed by GraphPad Prism software using the cosinor formula y=c +a cos [2 (t- ø)/24], where c, a and ø denote the mesor, amplitude and phase of the cosine wave, t is time in hours, and R2 is the fitting degree. Statistically significant differences in gene rhythms were indicated by P < 0.05 and calculated using the website https://www.danielsoper.com/statcalc/calculator.aspx?id=15 .