Phosphorus (P) is an essential nutrient for animal growth and development that has a crucial role in skeletal development and bone mineralization (1). However, more than two-thirds of the P in plant-based raw materials is phytate P. Monogastric animals, including poultry, cannot use bound P in phytate efficiently because of insufficient levels of endogenous phytase (2). Therefore, inorganic phosphates (Pi) such as mono-dicalcium phosphate (MDCP) and dicalcium phosphate (DCP) are commonly used in diets to support animal health and performance (3). However, P digestibility by poultry varies with different sources of Pi (3). Sauvant et al. (4) reported actual ileal P digestibility of MDCP and DCP produced from rock phosphate was 69.3 and 60.2%, respectively. According to Bikker et al. (5), actual ileal phosphate digestibility of DCP was 59.0%, whereas that of MDCP reached 70.7%. An et al. (6) reported apparent ileal digestibility of P in MDCP and DCP by poultry was 86.0 and 76.2%, respectively. Previous studies indicate that actual phosphate digestibility was lower for DCP from rock phosphates than for MDCP. Differences in Pi digestibility are attributed to phosphate characteristics as well as animal age, Ca:P ratio, and application of phytase (7, 8). Therefore, in-depth exploration of different mineral sources of P is required to adjust dietary requirements and ensure highly efficient production while reducing pressure on the environment.

In addition, phytase has been widely used in poultry diets to reduce Pi supplementation. Phytase is one of the most popular environmentally friendly feed additives of the 21st century. Supplementing with large amounts of phytase can further improve P utilization (9). Keshavarz (10) reported that supplementation with 300 FTU/kg phytase effectively restores laying performance when dietary NPP content is <0.25% at laying hens from 18 to 51 weeks of age. Wang et al. (11) indicated that laying performance could be maintained ifphytase at 360 FTU/kg was supplemented into a basal diet with supplementation of <0.10% NPP. Taheri et al. (12) and Adeola and Cowieson. (13) reported that dietary supplementation with phytase above 1,000 FTU/kg could reduce the amount of Pi, even without extra supplementation. Ren et al. (14) reported that corn–soybean meal-based diets containing 0.12% NPP and 2000 FTU/kg phytase would meet the requirements for egg production in Hy-Line Brown laying hens from 29 to 40 wk. of age. It is necessary to decrease dietary Pi supplementation because phosphate ore resources are limited globally and fecal P contaminates soils (15). Based on our understanding of the current literature, information is scant regarding the formulation of low-P diets for aged laying hens by replacing DCP with MDCP. The objective of this study was to evaluate the effect of replacing DCP with MDCP to formulate low-P diets on laying performance, egg quality, calcium-phosphorus metabolism, and bone metabolism of 69–78-week-old aged laying hens.

The Animal Care and Use Committee of Shandong Agriculture University (protocol code SDAUA-2021-019) approved this study.

Jing et al. (23) reported that reducing dietary NPP up to 0.15% is adequate to maintain health and performance of laying hens. Cheng et al. (24) reported that laying performance of Hy-Line Brown laying hens(29 to 40 weeks of age) was well maintained by diets supplemented with 2,000 FTU/kg phytase and 0.05%–0.15%NPP MDCP Pi (dietary NPP levels of 0.17–0.27%). Jing et al. (15) indicated that dietary NPP levels could be reduced to 0.10% with the inclusion of 1,000 FTU/kg phytase, without negative effects on production performance and health of the Lohmann hens from 22 to 34 weeks of age. In the current study, laying performance and egg quality were not significantly different among dietary treatments, indicating that 0.07%-0.15% NPP MDCP Pi with 1,470 FTU/kg phytase was sufficient to support the laying performance of Hy-Line Brown 69–78-week-old laying hens compared to 0.20% NPP DCP Pi. Because significant differences were not observed in egg weight and feed-to-egg mass ratio in the current trial, use of MDCP Pi could clearly reduce feed costs and environmental pollution by reducing the amount of Pi. However, the feeding trial was conducted in autumn, and the new corn was low in energy, resulting in an average daily hen intake of more than 120 g, which exceeded the standard of Hy-Line Brown-Conventional Systems in 2021. Given that limitation, long-term feeding studies with large numbers of hens are needed to confirm the findings of this study. There are large changes in P intake due to differences in feed intake.

Tibia quality is a crucial indicator of P nutrition in poultry (25), and inadequate or excess dietary P can cause bone disease (26, 27). Cheng et al. (24) reported that tibia P and Ca storage and mobilization were well maintained when diets were supplemented with 0.05%–0.15% NPP MDCP Pi (dietary NPP levels of 0.17–0.27%). Ren et al. (14) found that 0.20–0.30% DCP Pi (dietary NPP levels of 0.32–0.42%) did not significantly increase tibia breaking strength, ash content, or Ca and P contents in Hy-Line Brown 29–40-week-old laying hens. In the current study, compared with 0.20% NPP DCP Pi, tibia measurements were not affected in hens fed 0.11% and 0.15% NPP MDCP Pi. The supplementation with 0.11% and 0.15% NPP MDCP Pi (dietary NPP levels of 0.23 and 0.27%) is sufficient for the skeleton health of aged laying hens (69 to 78 weeks of age) with the presence of 1,470 FTU/kg phytase. In modern egg production, laying hens face a number of welfare problems related to the increased prevalence of fractures, especially a continuous high egg production in commercial layer lines have led to increased bone fragility and susceptibility to fracture (28, 29). Bone disease are detrimental to egg production, thereby negatively affecting egg quality and production performance (30). Still, minimal information is available on the effects of MDCP use on skeletal-related welfare problems in laying hens. In this study, the tibia breaking strength was significantly higher in the 0.18 and 0.20% NPP MDCP Pi groups than in the 0.20% NPP DCP Pi group, indicating that MDCP was more beneficial than DCP for tibia quality. Therefore, replacing DCP with MDCP to supplement P can be considered a positive welfare attribute.

Ca, P, and related hormones (PTH, CT and 1,25-(OH)₂D₃) in the serum are crucial indicators for phosphate–calcium metabolism. Jing et al. (23) reported that the concentrations of plasma P were generally reduced in hens on low-P diets. In this study, there were no significant differences in serum Ca and P in hens from different groups. The differences in findings could be related to different testing environments and sources of phytase (31). In addition, serum P are controlled by a variety of factors, and regulation is not limited to Pi intake alone but also to bone resorption and renal reabsorption (32). Meanwhile, the data from the current study showed that hens fed 0.07 and 0.11% NPP MDCP Pi had higher serum levels of PTH than those in all other groups. PTH, secreted from cells of the parathyroid glands, maintains the serum P levels at a normal level by acting directly or indirectly on the intestine, bone, and kidneys (33, 34).

The intake of P plays an important role in bone health, especially during egg laying. The effects of replacing DCP with MDCP and reducing NPP supplementation levels on several serum bone turnover markers indicating bone health were evaluated in this study. A biochemical marker of bone turnover secreted by osteoclasts, TRACP-5b reflects the status of bone resorption, and its blood concentration is used to detect the total amount of bone resorption (25). Obrant et al. (35) reported that serum TRACP-5b is significantly elevated in women with persistent fractures. CTX-I is a special sequence fragments released by osteoclasts from the degradation of type I collagen during bone resorption (36). Therefore, CTX-I reflects the level of bone destruction and is a specific indicator of bone resorption (37). OPG is an irreducible soluble receptor secreted by osteoblasts, which is considered as a protective factor against bone loss (38, 39). OPG could regulate the differentiation of osteocytes and block the formation of osteoclasts (1). Khosla et al. (40) reported a compensatory increase in OPG inhibition of abnormal bone resorption in postnatal rats due to lactation, which disturbed Ca and P metabolism in vivo. The results showed that the serum levels of OPG in the 0.07% NPP MDCP Pi group increased significantly, indicating that the speed of bone resorption was accelerated, which resulted in abnormal bone loss (41). This conclusion is consistent with the lowest tibia Ca and P contents in the 0.07% NPP MDCP group. Aged hens may be more sensitive to P deficiency than young chicks (42). In the current study, the serum levels of TRACP-5b and CTX-I in the 0.11% and 0.15% NPP MDCP Pi hens were significantly lower than those in 0.18 and 0.20% NPP MDCP Pi and 0.20% NPP DCP Pi hens. Low-P intake caused hens to mobilize bone resorption to maintain the body’s calcium-phosphorus metabolism balance.

In addition to the above biochemical indices, the related mRNA expression of a P transporter was also examined. The type II sodium/phosphate cotransporter (NaPi-II) group primarily includes cotransporters NaPi-IIa, NaPi-IIb, and NaPi-IIc, with NaPi-IIb mainly in the small intestine and NaPi-IIa and NaPi-IIc mediating renal P reabsorption (43). The P-transport capacity of the kidney is the most important factor in the maintenance of P homeostasis in hens (44). In a kidney, NaPi-IIa is an electrogenic transporter that preferentially combines with P, whereas NaPi-IIc is an electroneutral transporter that does not easily combine with P. Dietary P deficiency induces upregulation of renal NaPi-IIa mRNA expression in poultry (45). Katsumata et al. (27) reported a significant decrease in renal NaPi-IIa mRNA expression levels after feeding a high-P diet to rats. Li et al. (46) reported that laying hens fed 0.15% available phosphoru (AP) had higher NaPi-IIa mRNA expression levels than those fed 0.41–0.82% AP. In the current study, the renal NaPi-IIa mRNA expression level in the 0.07%–0.15% NPP hens was higher than that in 0.18–0.20% NPP hens, which indicated that the low-P diet increased renal P reabsorption. Because there are no antibodies to NaPi-IIa, protein expression of NaPi-IIa in the kidney was not measured in the current study. Therefore, the effect of a low-P diet on phosphorus balance in laying hens remains to be elucidated.

Renal P reabsorption and bone resorption were both involved in adapting to a low-P diet. In conclusion, NPP levels can be reduced to 0.11% (dietary NPP level of 0.23%) when MDCP is used to supplement NPP in diets instead of DCP without negatively affecting laying performance and skeletal health of aged hens.

The nutritional value of different P sources should be reconsidered in laying hens, especially in the application of phytase. There is great potential to reduce feed costs and decrease phosphorus excretion by reducing the amount of inorganic phosphates without affecting skeletal health and performance of aged hens.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by The Animal Care and Use Committee of Shandong Agriculture University (protocol code SDAUA-2021-019).

WY and SJ received funding. YR, TZ, NJ, and YL conceptualized and designed the study. YR, TZ, LL, ZZ, and KZ conducted animal experiments, chemical analyses, and analyzed the data. YR and TZ wrote the original. NJ, SJ, and YL reviewed and revised the draft. All authors contributed to the article and approved the submitted version.

This research was supported by the Major Innovative Projects of Shandong Province (2019JZZY020609). WY and SJ received funding.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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