Prolonged Dark Time Within A 24-h Light Regime Improves Eggshell Quality by Enhancing Calcium Deposition in Laying Hens

Background: The absorption and transportation of calcium and phosphorus is mainly relied on their corresponding transporters. Eggshell is mainly formed during dark time in one egg cycle. The aim of this study was to determine the effect of different light regime on eggshell quality and the expression of the relevant calcium and phosphorus transporters in laying hens. Seventy two 56-week-old laying hens were randomly divided into two groups and subjected to the following treatments: 16 h light: 8 h dark (control) and 9 h light: 15 h dark (LDP). The expression of phosphorus transporters type IIb Na/Pi co-transporter NaPi-IIa (NPt2a) and NaPi-IIb (NPt2b), calcium transporter calbindin-D28k (CaBP-D28k), and plasma membrane Ca ATPase 1b (PMCA1b) were measured in small intestine, kidney, and eggshell gland. Results: The results showed that the feed intake (P < 0.001) and egg weight (P = 0.05) was decreased by LDP treatment, while laying rate, egg production, and feed eciency were not signicantly inuenced (P > 0.05). Compared to control, eggshell hardness was increased (P < 0.05) by LDP treatment whereas eggshell thickness and eggshell percentage were not signicant changed. Eggshell calcium (Ca) and phosphorus (P) contents were elevated in LDP-hens, compared to control birds. Compared to control birds, serum Ca (P < 0.01) and P levels (P = 0.079) at dark time were increased in LDP-hens while and alkaline phosphatase (ALP) activity was lowered (P < 0.05). The protein expression levels of CaBP-D28k and PMCA1b were not inuenced in duodenum but were decreased at light time in jejunum of LDP hens. In kidney, the expression of CaBP-D28k, PMCA1b and NPt2a were not changed by LDP treatment. In eggshell gland, however, the expression of CaBP-D28k and osteopontin (OPN) were relative higher in LDP hens compared to control birds, whereas the PMCA1b expression was not altered. Conclusions: The result indicates that the increased circulating Ca and P concentrations in dark time are favorable for the deposition of calcium and phosphorus in eggshell. The result offers an alternative strategy for the laying hen with a worse eggshell problem.


Introduction
Eggshell quality plays an important role in the production e ciency of commercial egg industry and the eggshell problems cause signi cant economic losses. The sustain of eggshell quality is essential for the persistency in lay of the long-life layer, which will be capable of producing 500 eggs in a laying cycle of 100 weeks [1]. The unsynchronous increase of calcium (Ca) deposition with eggshell weight is one of the underlying reason of poor shell quality as laying hen ages [2,3].
It is crucial to provide calcium for the high metabolic calcium demand during eggshell mineralization. It is assumed that the continuous supply of calcium from digestive tract and bone mobilization could satisfy the requirement of eggshell formation. However, the limitation of hen access to feed during dark period [4] and bone loss with age [5,6] make the effective calcium supply a problem in the laying period.
Photoperiod is generally considered as the primary cue to stimulate reproduction of laying hen [7]. The light source [8], spectrum [9,10], intensity [11], and the uniformity of light distribution [12] all have an in uence on the laying performance. Altered photoperiod has an in uence on eggshell quality. Eggshell quality is improved by increasing the photoperiod to 24 hours [13]. The midnight lighting programmes provide a means of supporting egg shell quality of older laying hens during the summer months without a signi cant reduction in egg production [14]. In contrast, an increase of 10% shell weight in hens under 27-h photoperiod (14 h light, 13 h dark) is ascribed to the increased time spent in the shell gland [15]. The underlying mechanism remains unclear.
Plasma membrane calcium ATPase (PMCA) participates in the translocation of calcium, mediates Ca 2+ extrusion from cells [21,22,23]. The plasma membrane Ca ATPase 1b (PMCA1b) is the primary isomer expressed in small intestine, kidney, and uterus of birds [24,25,26,27,28]. The NaPi-IIb (NPt2b) cotransporter is the major phosphorus (P) transporter in small intestine that primary expressed at the brush-border membranes of epithelium [29]. Recently, our result indicates that duodenum has the highest expression level of NPt2b and is in uenced by dietary phosphorus level [30]. Hence, we hypothesized that photoperiod take an effect on eggshell formation with the involvement of changed gene expression of the genes.
In this study, 56-weeks-old hens were employed to investigate the effect of prolonged dark period in 24-h cycle. The laying performance, eggshell quality, and the expression of P and Ca transporters in small intestine, kidney, and shell gland in laying hens were investigated.

Materials And Methods
All procedures used in this study were approved by the Animal Care Committee of Shandong Agricultural University and were carried out in accordance with the guidelines for experimental animals of the Ministry of Science and Technology (Beijing, China).

Animals
Seventy two 56-week-old ISA hens with similar body weight and laying rate were obtained. The hens were randomly divided into two groups of 36 hens and reared in two environmental controlled houses with the same rearing facilities. The laying hens were cage reared with two hens per cage (45 cm * 35 cm * 35 cm).Three cages were served as one replicate. The two groups of hens were reared in one of the two following light regimes: 16-h light and 8-h dark (16 L:8 D, control group) or 9-h light and 15-h dark (9 L:15 D, long-dark period treatment, LDP). The experiment lasted for 8 weeks. During the whole experimental period, the hens had free access to feed and water.
Feed intake, egg number, and egg weight were recorded daily, and laying rate and egg production were calculated. The egg quality was measured at 2, 4, 6, and 8 weeks after treatment. All the eggs were collected for three consecutive days every twice weeks and the eggshell hardness, percentage of eggshell, yolk weight, albumin height, Haugh unit, eggshell thickness (mean from the equator and two ends), shape index, and egg yolk color were measured.
At the end of the experiment, sixteen hens were randomly selected from each treatment and sampled at the midpoint of light and dark periods respectively, 13:00 pm and 01:00 am in control group and 9:30 am and 21:30 pm in 9 L:15 D group (Fig. 1). After a blood sample was obtained from a wing vein, the hens were sacri ced by exsanguination after cervical dislocation [31,32]. The duodenum, jejunum, ileum, kidney, and shell gland mucosa were sampled. Tissue samples were immediately snap-frozen in liquid nitrogen and stored at -80 • C for further analysis. Serum was separated by centrifugation at 1,500 g for 15 min and stored at -20 • C until analysis.

Egg quality measurement
Shell thickness was determined at the sharp, blunt ends, and equator by an eggshell Thickness Meter (EFG-0503, ROBOTMATION, Japan), and took the average of the three data. Egg shape index was calculated by long diameter/short diameter. Eggshell breaking strength was determined based upon the vertical axis measured by an eggshell strength tester (EFG-0503, ROBOTMATION, Japan). After breakout, yolk was separated and weighed. Relative weight of yolk was calculated against egg weight. The albumen height, yolk color, and Haugh unit were detected by the multifunctional egg detector (EMT-5200, JapanRobotmation). Eggshells were weighed after dried. Relative weight of eggshells was calculated against egg weight.

Determination of calcium and phosphorus in eggshell
Determination of calcium content in feed and eggshell with potassium permanganate titration [33,34].
Serum calcium, phosphorus, and alkaline phosphatase analysis Serum Ca and P levels and alkaline phosphatase (ALP) activity were measured with commercial kits (Sichuan Mike Biotechnology Co., Ltd., China) by automatic biochemistry analyzer (7020 Clinical Analyzer: Hitachi High-Tech GLOBAL) [35].

Total RNA extraction and Real-Time PCR analyses
The mRNA expression levels of NaPi-IIa (NPt2a), NPt2b, CaBP-D28k, PMCA1b were measured by real-time PCR. Total RNA was extracted from the duodenum, jejunum, ileum, kidney, and eggshell gland using TRIzol reagent (TransGen Biotech, China). The primers used in this study were designed using Primer 5.0 software and synthesized by Sangon Bioteach (Shanghai, China, Table 1). Total RNA concentration was then quanti ed by measuring the absorbance at 260 nm in a photometer (Eppendorf Germany). Ratios of absorbance (260/280 nm) were between 1.75 and 2.01 for all preparations. Next, reverse transcription was performed using total RNA (1 µg) for first-strand cDNA synthesis with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). The cDNA was ampli ed in a 20 µL PCR reaction system containing 0.2 µM of each speci c primer (Sangon, China) and of the SYBR Green master mix (Roche, Germany) according to the manufacturer's instructions. Real-time PCR was performed at the ABI QuantStudio 7500 PCR machine (Applied Biosystems; Thermo, United States) [34,36]. The mRNA values were normalized with the expression of chicken b-actin mRNA. The analyses of the relative gene expression data were performed by the 2 -ΔΔCT method [37,38,39]. All of the samples were run in duplicate, and the primers were designed to span an intron to avoid genomic DNA contamination. Therefore, all gene transcription results are reported as the n-fold difference relative to the calibrator. The speci city of the ampli cation product was veri ed by the standard curve and dissolution curve.

Western blot analysis
The tissue samples were homogenized in 1 mL of lysis buffer (Beyotime, China). After centrifuged at 12000 g for 10 min at 4 °C to remove cellular debris, the supernatant was collected and then quanti ed for protein by the method of BCA protein assay kit (Beyotime, China) according to the manufacturer's protocol. An equal amount of proteins were separated by 10% SDS polyacrylamide gels (Bio-Rad, Richmond, 246 CA) and the proteins were transferred onto polyvinylidene fluoride membrane (Millipore, United States) at 200 mA for 2 h in a Tris-glycine buffer with 20% anhydrous ethanol at 4 °C. Then membranes were blocked with western blocking buffer (Beyotime, China) for 1 h at room temperature. The membranes were incubated with speci c primary antibodies at 4 °C with gentle shaking overnight.
Western blots were developed and quanti ed using BioSpectrum 810 with VisionWorks LS 7.1 software (UVP LLC) [36, 38]. The band intensity was normalized to the β-actin or β-tubulin band in the same sample.

Statistical analyses
Prior to analysis, all data were examined for the homogeneity and normal distribution plots of variances among the treatments by using UNIVARIATE procedure. The main effect of photoperiod treatment was evaluated by a one-way ANOVA and a two-way ANOVA with the Statistical Analysis Systems statistical software package (Version 8e, SAS Institute, Cary, NC, USA). P < 0.05 was considered statistically signi cant.

Laying performance
Compared with control, feed intake (P < 0.001) and egg weight (P < 0.05) were decreased by LDP treatment, while the laying rate and feed to egg ratio were not signi cantly in uenced by treatment (P > 0.05; Table 2, Fig. 2).

Egg quality
Compared with the control group, eggshell hardness was signi cantly increased by LDP treatment (P < 0.05; Table 3, Fig. 3a). In contrast, eggshell thickness, egg shape index, albumen height, yolk color, Haugh units, percentage of yolk and eggshell were not changed by treatment (P > 0.05; Table 3).

Ca and P contents in eggshell
Compared with the control group, eggshell Ca (P < 0.05) and P (P < 0.01) contents were increased by LDP treatment (Fig. 3d, e). The LDP-hens consumed less Ca and P compared to control (P < 0.01; Fig. 3f, g). In contrast, the Ca excretion via eggshell of was not changed (P > 0.05) while P excretion was increased signi cantly (P < 0.01; Fig. 3i).

Serum parameters
In control hens, the serum Ca and P concentrations were higher in light time (P < 0.05) compared to that in dark time, whereas there was no difference (P > 0.05) between light and dark time in LDP treatment ( Fig. 4a, b). Moreover, serum Ca level in dark time was higher in LDP treatment (P < 0.05) compared with control (Fig. 4a). The ALP activity had no detectable difference (P > 0.05) in light and dark time in both control and LDP treatments. Compared to control, the hens in LDP treatment had lower ALP activity during dark time (P < 0.05; Fig. 4c).

The expression of calcium transporters
In light time, the mRNA expression level of CaBP-D28k in the eggshell gland was lower (P < 0.05) in both treatments, compared to that in the dark time (Fig. 5a). In contrast, there was no difference in the protein level of CaBP-D28k (P < 0.05) between light and dark time (Fig. 5b). In light time, however, the protein level of CaBP-D28k was higher in control than that in LDP treatment (P < 0.05). Neither the mRNA nor the protein level of PMCA1b was altered by photoperiod treatment (P > 0.05; Fig. 5d-f).
In the control group, the mRNA expression level of CaBP-D28k was higher in dark period compared to the light period in duodenum and jejunum (P < 0.05) but not in ileum (P > 0.05; Fig. 6, 7, 8). In LDP treatment, however, there was no difference in the mRNA expression level of CaBP-D28k between light and dark period (P > 0.05). The mRNA level of PMCA1b was increased during dark period in the duodenum of control hens compared with the light period (P < 0.05), whereas no detectable in uence (P > 0.05) was observed in jejunum and ileum of control hens. In LDP treatment, the expression of PMCA1b had no difference between light and dark time (P > 0.05). In duodenum and jejunum, the expression of NPT2b was not changed by light and dark cycle or photoperiod treatment (P > 0.05). In ileum, however, the PMCA1b expression was decreased (P < 0.05) during dark time in LDP treatment compared to that in the light or dark time of control (Fig. 8).
In kidney, the light and dark period had no signi cant (P > 0.05) in uence on the mRNA level of CaBP-D28k and PMCA1b and the protein level of CaBP-D28k and PMCA1b (Fig. 9).

The expression of phosphorus transporters
In control group, there was no difference (P > 0.05) in the mRNA level of NPt2a in the kidney between light and dark time (Fig. 10a). In contrast, the NPt2a mRNA level was higher (P < 0.05) in light time than that in dark time. The mRNA expression level of NPt2b in the duodenum and jejunum was not affected by light treatment (P > 0.05; Fig. 10b, c). However, in the LDP group the mRNA expression level of NPt2b in ileum was signi cantly upregulated in light time, compared with dark time (P < 0.05; Fig. 10d). The mRNA expression level of NPt2b in control group was signi cant higher than LDP group at dark time in ileum (P < 0.05).

Discussion
In the present study, the result indicated that prolonged dark time in a 24 h light-dark cycle decreases feed intake, hen-day egg production, and egg weight but has no unfavorable in uence on feed e ciency. Longer dark time increases Ca and P contents of eggshell and improves eggshell strength. The result suggests that the elevated serum Ca and P concentrations at dark time are involved in the improved eggshell quality. The result highlights an effective method to ameliorate eggshell quality at late-phase of laying period.

Prolonged dark time has a disadvantage effect on laying performance
Chicken is sensitive to the change of photoperiod. Many studies have been conducted to investigate the effect of changed light-dark period, light source, light intensity, and intermittent lighting [11,40,41]. In the present study, the prolonged dark period in 24-h cycle had a negative in uence on the laying rate, egg weight, and egg production. However, the decreased laying performance seemed to be a result of reduced feed intake in LDP-hens, as the egg to feed ratio was not signi cantly in uenced by photoperiod treatment. In broiler under 16 L:8 D cycle, the feed intake in the dark is almost negligible and take account of less than 3% of total feed consumption [42]. In laying hen subjected to an asymmetric pattern of 0.25 L:0.75 D for 16 h followed by 8 D, the reduction in feed intake is ascribed to reduced total activity time [43]. Hence, the result suggests that the reduced feed intake in LDP hens is a result of shorter light time and in turn shorter forage time.
Prolonged dark period improves eggshell quality by elevating Ca level during dark period During the eight weeks experiment period, the egg quality was evaluated every two weeks. The elevated eggshell strength in LDP-hens was in accordance with the egg broken rate, which had a 36.5% decrease in LDP treatment (Control, 2.60% vs. LDP, 1.65%). Hence, the result indicated that LDP could improve eggshell quality. Eggshell strength and thickness were improved in high Ca consumption group [44]. The inconsistent response of eggshell thickness and eggshell strength is in line with the previous works [45,46]. The breaking strength and shell thickness are respectively negatively and positively in uenced by increasing dietary phosphorus and calcium contents [47]. Except of the shell thickness, the calcium content in eggshell also contributes to eggshell hardness. Although the eggshell thickness is not necessary to correlate with its calcium content [48], the calcium concentration in eggshell decreases with age in laying hens [49]. In the present study, the calcium and phosphorus concentrations were all increased in the eggs from LDP-hens, suggesting that the improved eggshell strength is associated with the elevated Ca and P contents in the eggshell.
The elevated plasma Ca and Pi levels are associated with the improved eggshell strength [46]. The serum Ca exhibits a circadian rhythm which varied with the changed light: dark cycle in laying hens [50]. In the present study, the blood samples were obtained at the intermittent points of light-and dark-period, respectively. The relative higher serum Ca and P levels in light period compared to the dark period indicated that there is a circadian rhythm in the blood Ca and P levels. This result was in line with the previous results [51,52]. Parsons and Combs (1981) [51] reported that blood Ca 2+ concentration varies with the egg position in the oviduct, reaches the maximum level following oviposition and drops until an uncalci ed egg entered the shell gland. The daily change of blood P, however, has different reports. The blood P follows a pattern opposite that of Ca [51] or shows a similar trend as blood Ca [52]. Except of the daily egg cycle, blood Ca 2+ and P levels are related to the light-dark cycle or feed intake. In 4-month-old pullets, serum Ca is lower during daylight hours while is higher during the dark period [51], indicating daylight exposure and/or feed intake take an in uence on blood Ca. In LDP-hens of the present study, however, the circadian rhythm in the blood Ca and P levels disappeared and the serum Ca and P were kept high levels in both light-and dark-period, implying that a more su cient supply of blood Ca and P during the dark hours. It is speculated that the increase of blood Ca 2+ is a result of additional release of bone Ca during the night hours [53]. In contrary, the relative lower activity of ALP at dark time in LDP hens indicated the reduced Ca mobilization from bone during dark time. Similarly, the Ca level in pullets, without the interruption of eggshell formation, is signi cantly elevated during the dark period, dropping to low levels during daylight hours when feeding [51]. The underlying mechanism needs to be investigated further. Collectively, the result suggests that the prolonged dark time is bene cial for the eggshell formation and calcium deposition during night and the increased circulating Ca and P levels during dark time should be at least partially responsible for the elevated eggshell Ca and P contents in LDP treatment.
OPN, a component of the organic matrix of eggshell, play an important role in egg shell calci cation. OPN is mainly secreted by the epithelial cells of the eggshell gland lumen [54,55]. In the present study, the expression level of OPN was higher at dark time compared to light time, in line with the work by Pines and Knopov (1995)[56], who reported that the expression of osteopontin in eggshell gland showed circadian rhythm and the peak value of osteopontin expression appeared in dark period. When the egg enters the uterus, the pressure on the uterine wall is the key factor causing the upregulation of OPN expression [57].
The relative higher expression levels of OPN at both light (4.87 folds) and dark (1.28 folds) periods compared to control suggests that upregulated OPN expression is involved in the enhanced eggshell strength.

Calcium transport is not altered by lighting program
In laying hens, CaBP-D28k localizes at the intestinal enterocyte cytoplasm, and highly expresses in duodenum and followed by jejunum and ileum [30,58,59]. The ingested calcium is absorbed into the vascular system in duodenum and upper jejunum with the involvement of CaBP-D28k [60]. PMCA mediates Ca 2+ extrusion from cells [21,22,23]. Therefore, we determined the expression of the CaBP-D28k and PMCA1b in the intestinal tract, kidney, and eggshell gland. The relative higher expression level of CaBP-D28k in duodenum and jejunum and PMCA1b in duodenum at dark time was observed in control hens but not in LDP hens. The protein level of CaBP-D28k and PMCA1b, however, had no detectable difference between control and LDP-hens at either day or dark time. Hence, the result suggests that Ca absorption in duodenum is not changed by light regime. In jejunum, the protein levels of CaBP-D28k and PMCA1b were higher in control hen at day time, compared to LDP-hens. The chyme remained in digestive tract at measuring time may be responsible at least for the relative lower expression level of CaBP-D28k and PMCA1b in LDP hens. In the study, the hens were sampled at the intermediate point of dark period, 4and 7.5-h after light out in control and LDP groups respectively. Moreover, the LDP hens consumed less feed (89.9% of control) than control ones.
The protein levels of CaBP-D28k and PMCA1b in the kidney were not changed by photoperiod treatment and light-dark cycle, suggesting that calcium reabsorption in the kidney plays a less important role in the elevated circulating calcium concentration during dark period in LDP hens. In shell gland, however, the unchanged CaBP-D28k and PMCA1b levels in dark time between control and LDP treatments, suggesting that Ca secretion by shell gland is not changed by light regime treatment. Collectively, the result implies that higher circulating Ca concentration at dark time in LDP hens should be responsible for the increased Ca deposition in eggshell and in turn, the improved eggshell quality.
Phosphorus transport is not altered by photoperiod There are two Na/Pi cotransporter families, Na/Pi-II and NaPi-III. NaPi-II has three kinds of homologs, NaPi-IIa (NPt2a), NaPi-IIb (NPt2b), and NaPi-IIc (NPt2c). In small intestine, P absorption is primary adjusted by NPt2b [61, 62, 63, 64, 65] and the expression of gene NPt2b in intestinal tract mainly locates at the duodenum [30,63]. In the present study, the expression of NPt2b was not in uenced by photoperiod in duodenum and jejunum, whereas the NPt2b level was lower in the dark period in ileum, indicating that light regime treatment has no effect on P absorption. As there is no available antibody on NPt2a, the protein level of NPt2a was not determined in the present study. The elevated circulating P (P = 0.079) in LDP hens at dark time remains to be elucidated.
In kidney, NPT2a is the main type of P transporter. The adaptive capacity of kidney in P transport takes the most important role in the maintenance of P homeostasis in laying hens [66]. The relative lower expression level of NPT2a in LDP-hens was in line with the high serum P level. This result was in accordance with the previous work by Li et al. (2018) [30] who reported that laying hens fed a diet with higher available phosphorus had relative lower NPT2a mRNA level.

Conclusions
In conclusion, the laying performance of hens under 9 L:15 D light regime was lowered compared to the hens at 16 L:8 D light regime. Prolonged dark time from 8-h to 15-h improves eggshell strength and eggshell calcium and phosphorus contents. The result indicates that the increased circulating Ca and P concentrations in dark time are favorable for the deposition of calcium and phosphorus in eggshell. The result offers an alternative strategy for the laying hen with a worse eggshell problem. Data were presented as mean ± SEM (n = 6). a−b Means with different superscripts differ signi cantly (P < 0.05) Effects of different light regimes on laying performance of hens. Egg production (a), egg weight (b), feed intake (c), feed e ciency (d), and hen-day egg production (e). Data were presented as means ± SEM (n = 6); * P < 0.05 Effects of different light regimes on serum calcium (a), phosphorus (b), and alkaline phosphatase (c).