Osteoblasts are considered as a major factor contributing to bone development and mineralization, however, few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  Therefore, in the present study, two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  In experiment 1, osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology, alkaline phosphatase (ALP) and alizarin red staining.  Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4, 8, 12, 16, 20, 24, 28 and 32 after incubation, respectively.  The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped, triangular or polygonal morphology.  More than 95% of the cells were stained blue-black after ALP staining, and mineralized nodules were formed after 4 days of continuous incubation.  in experiment 2, lactate dehydrogenase (LDH) activity stayed at a relatively stabilized level although incubation time affected (P=0.0012) it during the whole culture period.  Additionally, incubation time affected (P≤0.0001) the number and proportion of the area of mineralized nodules.  They increased linearly and quadratically (P<0.04) with the increase of incubation time, and remained at a stabilized level from 24 to 32 days of incubation.  The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models (P<0.0001) of the number and proportion of the area of mineralized nodules, respectively.  These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method, and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization, and could maintain a relatively stabilized vitality from 4 to 32 days of incubation; and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.  Therefore, it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.

The bone morphogenetic protein (BMP) and mitogen-activated protein kinase (MAPK) signaling pathways play an important role in regulation of bone formation and development, however, it remains unclear that the effect of dietary different levels of non-phytate phosphorus (NPP) on these signaling pathways and their correlations with bone phosphorus (P) retention and bone development in broilers.  Therefore, this experiment was conducted to investigate the effect of dietary P supplementation on BMP and MAPK signaling pathways and their correlations with bone P retention and bone development in broilers.  A total of 800 one-day-old Arbor Acres male broilers were randomly allotted to 1 of 5 treatments with 8 replicates in a completely randomized design.  The 5 treatments of dietary NPP levels were 0.15, 0.25, 0.35, 0.45 and 0.55% or 0.15, 0.22, 0.29, 0.36 and 0.43% for broilers from 1 to 21 days of age or 22 to 42 days of age, respectively.  The results showed that extracellular signal-regulated kinase 1 (ERK1) mRNA expression in the tibia of broilers on days 14 and 28, phosphorylated-ERK1 (p-ERK1) on day 14, and BMP2 protein expression on days 28 and 42 decreased linearly (P<0.04), while c-Jun N-terminal kinase 1 (JNK1) mRNA expression on day 42 increased linearly (P<0.02) with the increase of dietary NPP level.  At 14 days of age, total P accumulation in tibia ash (TP_TA), bone mineral concentration (BMC), bone mineral density (BMD), bone breaking strength (BBS) and tibia ash were negatively correlated (r=–0.726 to –0.359, P<0.05) with ERK1 and JNK1 mRNA as well as p-ERK1; tibia alkaline phosphatase (ALP) and bone gal protein (BGP) were positively correlated (r=0.405 to 0.665, P<0.01) with ERK1 mRNA and p-ERK1.  At 28 days of age, TP_TA, BMC, BMD, BBS and tibia ash were negatively correlated (r=–0.518 to –0.370, P<0.05) with ERK1 mRNA and BMP2 protein, while tibia ALP was positively correlated (r=0.382 to 0.648, P<0.05) with them.  The results indicated that TP_TA, BMC, BMD, BBS or tibia ash had negative correlations, while tibia ALP and BGP had positive correlations with ERK1 and JNK1 mRNAs, BMP2 protein and p-ERK1, suggesting that bone P retention and bone development might be regulated by BMP and MAPK signaling pathways in broiler chickens.