Our hypotheses that BMD, BMC and mRNA or protein expression levels of ALP, OC, OPG and BMP-2 in bone might be new sensitive criteria to evaluate dietary Ca requirements of broilers fed a corn-soybean meal diet, and dietary Ca requirements of broilers from 1 to 21 d of age might be different from the current NRC Ca requirement (1.00%) have been partially supported by the results of the current study. The current study indicated that tibia and middle toe BMD, serum and tibia ALP activities, and tibia ALP protein level were new sensitive criteria to evaluate dietary Ca requirements of broilers, and the Ca requirement would be about 0.6% to obtain the best growth rate and 1.00% to meet all of the Ca metabolisms and bone development of broilers fed a conventional corn-soybean meal diet from 1 to 21 days of age. These findings could better characterize requirements and meet the growth, bone development and Ca metabolic functions of broilers.
In earlier studies, growth performance was often used to assess Ca requirements of broilers [6, 13]. In order to maximize the growth performance of broilers, using diets with 0.6% Ca becomes more widespread . As birds possess specific Ca appetite , they get adapted to low Ca diets by increasing absorption and utilization efficiency, which decreases excretion of the restricted nutrients  and elevates plasma 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) concentration and duodenal calbindin concentrations [32, 33]. The current study showed that the increase of dietary Ca level from 0.70 to 1.18% had a negative impact on growth rate of broilers from 1 to 21 days. Similar results were reported by Rama Rao et al. , indicating that the increase of dietary Ca level depressed the weight gain and feed intake at a 0.3% dietary NPP level. The possible reason for the negative effect of high-Ca diet on broiler’s growth rate might be due to that relatively high Ca reduced P availability , leading to the formation of extremely insoluble Ca-phytate complexes and severe P deficiency. In addition, higher dietary Ca could increase intestinal pH, which reduced the absorptivity of minerals . Driver et al. (2005) found that dietary Ca requirement of male broilers from 1 to 16 days was 0.49 ± 0.11% based on body weight gain . Additionally, some researchers recommended relatively lower levels of dietary Ca (0.6 to 0.65%) for broilers according to weight gain and feed intake [36, 37], which is similar to the results from the present study. However, Valable et al. (2017) and Wilkinson et al. (2014b) reported that the reduction of dietary Ca did not affect the growth performance of broilers [38, 39]. Different breeds and growth phases of broilers might account for the discrepancy of the above results.
The Ca and P contents in serum and bone have been considered as good parameters to reflect the nutritional status of Ca in broilers [40, 41]. However, the current study showed that dietary Ca level had no effect on serum and tibia Ca, P content and Ca to P ratio, indicating that these parameters were not suitable to estimate the Ca requirements. Some researchers reported similar results [42, 43]. Hurwitz et al. (1987) explained that modern broilers had good ability to keep serum Ca and P contents in narrow range regardless of the varying dietary Ca levels mainly because of the oscillations regulation system, in which parathyroid hormone, calcitonin and 1,25(OH)2D3 play an important role . In addition, the Ca content in middle toe ash increased linearly with increasing dietary Ca levels, indicating that middle toe ash Ca is not a useful marker for the assessment of Ca requirement of broilers.
Bone characteristics, such as bone ash percentage and breaking strength, have been traditional criteria to evaluate bone mineralization in broilers . The NRC Ca recommendations for broilers were mainly based on maximizing bone ash. According to the results from the current study, the Ca requirements were 0.93% and 0.88% based on tibia ash percentage and breaking strength, respectively. And these two criteria increased quadratically as dietary Ca increased, which is in line with the results reported by Bar et al. (2003) . However, some previous studies (Yoshida and Hoshii, 1982; Hurwitz et al., 1995) showed that the Ca requirement (1.3%) based on bone ash contents of 21-d-old broilers was higher than the current NRC Ca requirement (1.00%) [6, 45], which might be due to the high dietary P levels (0.7 ~ 0.75%) in these studies. Shafey (1993) reported that increasing dietary Ca enhanced the breaking strength of cockerel tibia . However, Onyango et al. (2003) did not observe the same phenomenon, and thought that the high variability in breaking strength contributed to the lack of statistical significance . Actually, several factors can affect breaking strength, such as cross head speed, handling of bones before testing and measurement technique [15, 47, 48]. Some researchers determined BMC and BMD of broilers using DEXA [49, 50]. The DEXA technology provided a rapid and noninvasive advantage measurement of bone mineralization compared with traditional measurement. Yan et al. (2005) found that BMC and BMD were good indicators for Ca nutrition estimation . Onyango et al. (2003) and Valable et al. (2017) reported that BMC and BMD showed a linear and quadratic increase with the increase of dietary Ca level [15, 38], which is similar to the results from the present experiment. The optimal Ca level was estimated to be 0.93% and 0.91% based upon tibia and middle toe BMD, which was close to that (0.93%) estimated by the tibia ash percentage. Onyango et al. (2003) showed that there was a high correlation between bone ash and BMC or BMD . The above results indicate that the requirement for maximizing bone development was greater than that for the best growth performance of broilers, which was further confirmed by Bar et al. (2003) . It might be viable to reduce the dietary Ca level in order to maximize the growth performance of broilers, however, it should not be at the expense of bone health.
Alkaline phosphatase is a ubiquitous enzyme that catalyzes the hydrolysis of phosphate monoesters , and can be used as a general indicator of skeletal development . The increase of ALP activity is usually associated with inadequate supply of Ca or P and poor bone mineralization [53, 54]. Xia et al. (2015) found that serum ALP activity was sensitive to the dietary Ca, and can be used to evaluate the dietary Ca requirements for laying Longyan shelducks . The current study showed that both serum and tibia ALP activities changed quadratically as dietary Ca increased, and the Ca requirements to obtain the minimum ALP activities in serum and tibia were 1.00% and 0.80%, respectively. Similarly, Hurwitz and Griminger (1961) reported that serum ALP activity decreased as dietary Ca increased, reaching a minimum in the range of probable Ca requirement, and could be used as an indicator to evaluate Ca adequacy in growing chicks . In the present study, the tibia ALP protein expression level increased quadratically as dietary Ca increased, and the Ca requirement obtained from this indicator was 0.90%, while the tibia ALP mRNA expression level was not affected by dietary Ca. Mehra et al. (2003) explained that changes in protein levels were not directly related to changes in mRNA levels, because of the complexity of transcription, translation and posttranslational modification .
Bone metabolism includes bone formation and resorption. Markers of bone formation and resorption act as key determinants in the regulation of bone mass [17, 58]. Osteocalcin is the most abundant non-collagenous protein of bone matrix and plays an important role in the bone formation and Ca metabolism . Osteoprotegerin is a secreted protein involved in the regulation of bone resorption [58, 60]. The decrease of OPG mRNA expression in the tibia led to the osteoporosis in broilers . Jiang et al. (2013) reported that the increase of dietary Ca level enhanced OPG mRNA and OC protein expression levels in the keel bone of hens . However, in the present study, dietary Ca levels did not affect OPG mRNA and OC protein expression levels in the tibia of broilers. The disparity may mainly result from the different animal species and bone samples. The BMP-2, a potent osteogenic differentiation factor, is essential for osteoblast differentiation and bone formation . Jia et al. (2015) found that the mRNA and protein levels of BMP-2 increased with increasing Ca2+ concentrations in the primary renal tubular epithelial cells . A study with piglets demonstrated that the serum BMP-2 concentration exhibited a trend from rise to decline with increasing dietary Ca levels . Similar tendency was observed for the tibia BMP-2 mRNA levels as dietary Ca levels increased in the present study. In addition, the results from the present study showed that as dietary Ca levels increased, the tibia OC and BMP-2 mRNA levels changed linearly, but not quadratically, indicating that these parameters are not suitable to estimate the requirements of Ca in broilers. We also found that the mRNA levels of OC and BMP-2 in the tibia varied, while their protein levels did not change as dietary Ca levels increased, implying that the transcriptional change would often precede the translational change [60, 65].
The present study showed that the Ca requirement was 0.59% based on ADG. Similarly, Sebastian et al. (1996) found that the optimum body weight, feed intake, and feed efficiency were obtained at 0.60% dietary Ca . We also found that the Ca requirements of broilers ranged from 0.8 to 1.0% based on tibia and middle toe characteristics, serum or tibia ALP activity, and tibia ALP protein expression level. In order to meet all of Ca metabolisms and bone development functions, dietary Ca requirement would be 1.0% for broilers fed a conventional corn–soybean meal diet from 1 to 21 d of age, which is in line with the current NRC Ca requirement. Earlier reports suggested that the Ca requirement should be higher for skeletal development than for optimal growth [9, 13]. Our findings also suggest that the current NRC (1994) recommendation for Ca (1.0%) would be adequate for the optimum bone development but excessive for the best growth rate.