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 influence on feed efficiency. 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 influence 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 significantly influenced 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 influenced 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 Ca2+ concentration varies with the egg position in the oviduct, reaches the maximum level following oviposition and drops until an uncalcified 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 Ca2+ 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 influence 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 sufficient supply of blood Ca and P during the dark hours. It is speculated that the increase of blood Ca2+ 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 significantly 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 beneficial 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 calcification. 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 Ca2+ 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, 4- and 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 influenced 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.