Tibial dyschondroplasia (TD) is an extremely common skeletal abnormality associated with rapid growth rate in broiler chickens (Leach and Lilburn, 1992). It leads to enormous economic losses worldwide and to animal welfare problems (Pines et al., 2005). Tibial dyschondroplasia is characterized by the formation of a lesion composed of non-vascularized, non-mineralized cartilage that extends from the epiphyseal growth plate into the metaphysic (Leach and Nesheim, 1965). It has been reported that TD is influenced by several factors, including genetics and nutrition (Waldenstedt, 2006). Nutrition plays a major role in the development and maintenance of bone structure, such as calcium, phosphorus, and vitamin D (Fleming, 2008). Vitamin D₃ (cholecalciferol) has been widely used as a feed additive to improve calcium and phosphorus metabolism and bone development in poultry (Baker et al., 1998). It has also been demonstrated that supplementation of vitamin D metabolites could efficiently prevent TD in broiler chickens (Edwards, 1990; Roberson and Edwards, 1996; Whitehead et al., 2004).

Conversion of vitamin D₃ to 25-hydroxycholecalciferol (25(OH)D₃) is catalyzed by 25-hydroxylase in livers. 25(OH)D₃ is further converted to 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) by 1α-hydroxylase in kidney. 1,25(OH)₂D₃ is the biologically active form of vitamin D in vivo (Henry et al., 1979; Henry et al., 1985). It has been shown that 25(OH)D₃ significantly improved body weight and feed efficiency of broiler chickens compared with those of the birds fed vitamin D₃ (Yarger et al., 1995; Fritts and Waldroup, 2003; Koreleski and Swiatkiewicz, 2005). Recently, we also demonstrated that broilers fed 1α-hydroxycholecalciferol (1α(OH)D₃) had higher body weight gain, feed intake, and tibia breaking strength compared with birds fed 25(OH)D₃ at 42 days of age (Han et al., 2016). However, no significant effects were found on body weight or feed efficiency in broiler chickens after supplementation of diet with 1,25(OH)₂D₃ (Roberson and Edwards, 1996).

Vitamin D₃ is involved in the regulation of levels of minerals such as calcium and phosphorous. Several mechanisms have been proposed to elucidate the vitamin D₃ metabolism and mechanisms of calcium transport in the small intestine (Norman et al., 1982; Kumar, 1990). However, little is known about the transcriptional level of intestinal calcium homeostasis-related genes in response to different vitamin D₃ metabolites in broiler chickens. Therefore, the present study was conducted to investigate the effects of different vitamin D metabolites including 1α(OH)D₃, 25(OH)D₃, and 1,25(OH)₂D₃ on intestinal calcium homeostasis-related gene expression in broiler chickens.

All experiments were performed in accordance with approved guidelines. The animal protocol was approved by local Institutional Animal Care and Use Committee (IACUC case No. 104-14). The concentration of vitamin D metabolites described earlier (Yarger et al., 1995; Roberson and Edwards, 1996; Chou et al., 2009; Han et al., 2009) was used in the present study. For examination of growth performance, mineral levels, and breaking strength, forty-eight one-day-old broilers (Avian) were randomly allocated to four different treatment groups (n = 12 per group) that were fed: basal diet (control), basal diet plus 5 μg/kg of 1α(OH)D₃, basal diet plus 69 μg/kg of 25(OH)D₃, and basal diet plus 5 μg/kg of 1,25(OH)₂D₃. The basal diets were formulated based on National Research Council recommendations (NRC, 1994) (Table 1). Water and feed was provided ad libitum over the entire experimental period. Total individual body weight, feed intake, and feed conversion ratio were recorded every week. At the end of the experiment, broilers were sacrificed by cervical dislocation. The blood was collected and centrifuged to harvest serum for biochemistry. The left tibia was removed for determination of mineral deposition and breaking strength measurement. For examination of calcium homeostasis-related gene expression, sixteen one-day-old broilers (Avian) were randomly allocated to four different treatment groups (n = 4 per group): basal diet (control), basal diet plus 5 μg/kg of 1α(OH)D₃, basal diet plus 69 μg/kg of 25(OH)D₃, and basal diet plus 5 μg/kg of 1,25(OH)₂D₃. After 0, 6, and 12 h of feeding, the duodenum was excised for RNA extraction.

Tissue RNA was extracted from the duodenum of broilers using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and resuspended in diethyl pyrocarbonate-treated water. Reverse transcription was performed with a Transcriptor Reverse Transcriptase kit (Roche Applied Science, Indianapolis, IN, USA). Quantitative reverse transcriptase-polymerase chain reaction (PCR) for each gene (Table 2) was performed using Miniopticon Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Boston, MA, USA). Polymerase chain reaction was performed by 40 cycles at 95 °C for 30 s, 58-60 °C for 60 s, and 72 °C for 30s. Beta-actin mRNA was determined as the internal control gene. The mRNA expression of each gene was normalized to its β-actin mRNA expression in the same sample. Threshold cycle (Ct) values were obtained and relative gene expression was calculated using the formula: (1/2)Ct target genes-Ct β-actin.

The left tibias were collected, cleaned of adhering tissue, weighed, dried to constant weight at 105 °C, defatted, and then ashed in a muffle furnace at 600 °C. Ash was dissolved in concentrated HCl for mineral determination. Calcium and phosphorus content was measured by atomic absorption spectrophotometry (AOAC, 1995a,b). Tibia breaking strength (breaking force divided by bone weight) was measured using TA.XT plus Texture Analyser (Mason Technology, Dublin, Ireland) with the use of a probe (HDP/3PB). Tibia was placed at the central position with 10 cm clearance, allowing the comparison of breaking strength values.

Data were subjected to one-way ANOVA using the General Linear Model procedure of SAS statistical package (Statistical Analysis System, version 9.2) for completely randomized designs. Duncan's new multiple range test was used to evaluate differences between means (SAS Institute, 2008). P-values ≤ 0.05 were considered statistically significant.

To examine the effect of vitamin D₃ metabolites on growth performance and bone quality, broilers were fed different vitamin D₃ metabolites (1α(OH)D₃, 25(OH)D₃, and 1,25(OH)₂D₃) for three weeks. However, there were no statistically confirmed differences between the growth performance and vitamin D₃ metabolite supplementation during the entire feeding period (Table 3). Although the changes in tibia calcium and phosphorus content were not statistically significant, a trend of increased tibia calcium and phosphorus content was observed with the supplementation of vitamin D₃ metabolites (Table 4). Dietary supplementation of vitamin D₃ metabolites had no significant effect on serum calcium and serum phosphorus content (Table 4). Although they did not reach statistical significance, vitamin D₃ metabolites caused a similar trend in increasing bone breaking strength in broiler chickens after feeding for three weeks (Table 4). These results indicate that supplementation of vitamin D₃ metabolites did not significantly improve growth performance and bone quality of broiler chickens.

To examine the effect of different vitamin D₃ metabolites on calcium homeostasis-related gene expression in the duodenum, broiler chickens were fed 5 μg/kg of 1α(OH)D₃, 69 μg/kg of 25(OH)D₃, and 5 μg/kg of 1,25(OH)₂D₃ for 0, 6, and 12 h. The level of calbindin mRNA was rapidly increased at 6 h after feeding broiler chickens additional amounts of vitamin D₃ metabolites (Figure 1A). Among vitamin D₃ metabolites, 1,25(OH)₂D₃ appeared to be more efficient than 1α(OH)D₃ and 25(OH)D₃ at regulating calbindin mRNA expression at 6 and 12 h (Figure 1A). The level of β-glucuronidase mRNA was induced at 6 h after feeding 1,25(OH)₂D₃ compared with other vitamin D₃ metabolites (Figure 1B). After 12 h of feeding, 1α(OH)D₃ and 25(OH)D₃ also promoted β-glucuronidase mRNA expression and 1,25(OH)₂D₃ increased the β-glucuronidase mRNA even further (Figure 1B). Similarly, 1,25(OH)₂D₃ rapidly induced the level of transient receptor potential cation channel, subfamily V, member 6 (TRPV6) mRNA at 6 h (Figure 1C). After 12 h of feeding, all vitamin D₃ metabolites exhibited increased TRPV6 mRNA expression (Figure 1C). The level of type IIb sodium-dependent phosphate (Na/Pi IIb) cotransporter mRNA was rapidly increased at 6 and 12 h after feeding broiler chickens additional amounts of vitamin D₃ metabolites (Figure 2A). The level of 24-hydroxylase mRNA was reduced at 12 h after feeding 25(OH)D₃ compared with the control diet (Figure 2B). However, none of the vitamin D₃ metabolites changed the vitamin D receptor (VDR) mRNA levels in the duodenum of broilers (Figure 2C). Taken together, these results indicate that vitamin D₃ metabolites were able to rapidly modulate calcium homeostasis-related gene expression in the duodenum and 1,25(OH)₂D₃ was the most effective vitamin D₃ metabolite in regulating calcium homeostasis-associated genes.

In this study, we demonstrated that vitamin D₃ metabolites could rapidly regulate calcium homeostasis-related gene expression in the duodenum from 6-12 h after feeding, although the growth performance and bone quality of broiler chickens at 21 days of age were not significantly changed. Among these vitamin D₃ metabolites, 1,25(OH)₂D₃ was the most effective in modulating calcium homeostasis-associated gene expression.

It has been reported that feeding broilers 69 μg/kg of 25(OH)D₃ for three weeks did not affect body weight gain, feed conversion ratio, or feed intake (Chou et al., 2009). There were no significant effects on body weight or feed efficiency at 21 days of age in broiler chickens after supplementation of the diet with 1,25(OH)₂D₃ (Roberson and Edwards, 1996). Similarly, our results also confirm the previous reports that no significant effects were observed on growth performance of broiler chickens at 21 days of age after supplementation of the diet with 1α(OH)D₃, 25(OH)D₃, or 1,25(OH)₂D₃. Furthermore, 1α(OH)D₃ did not alter bone breaking strength, tibia calcium, and phosphorus content in broiler chickens at 21 days of age (Han et al., 2009). Consistently, we also found that vitamin D₃ metabolites did not improve bone breaking strength and bone mineral deposition after feeding for three weeks. In contrast, broilers fed 6 μg/kg of 1,25(OH)₂D₃ for three weeks exhibited significantly increased bone ash content (Roberson and Edwards, 1996). The contradiction may be due to the differences between composition of basal diet and chicken strains. Together, these findings demonstrate that vitamin D₃ metabolites do not have a significant effect on growth performance, but mineral deposition and bone breaking strength in broiler chickens may vary after feeding for three weeks.

Calbindin is a vitamin D-induced calcium-binding protein that plays a key role in intestinal intracellular calcium transport. The expression of calbindin in the intestine and kidney has been shown to be regulated by 1,25(OH)₂D₃ in rats and chickens (Brehier and Thomasset, 1990; Hall and Norman, 1990; Ferrari et al., 1992). Supplementation of 1,25(OH)₂D₃ could induce calbindin mRNA expression in the duodenum of 1α-hydroxylase knockout mice (Hoenderop et al., 2004). Here, we further demonstrated that vitamin D₃ metabolites are able to induce intestinal calbindin mRNA expression in vivo. Particularly, the expression of calbindin peaked at 6 h and declined afterwards, 12 h after 1,25(OH)₂D₃ treatment. The intestinal epithelial Ca²⁺ channel, TRPV6, is primarily activated by β-glucuronidase. It has been found that β-glucuronidase secretion from isolated intestinal epithelial cells was remarkably increased by supplementation of 1,25(OH)₂D₃ (Khanal et al., 2008). The intestinal TRPV6 gene expression was significantly increased after incubation for 6 h with 25(OH)D₃ or 1,25(OH)₂D₃ (Balesaria et al., 2009). Supplementation of 1,25(OH)₂D₃ significantly upregulated the TRPV6 mRNA expression in the duodenum of 1α-hydroxylase knockout mice (Hoenderop et al., 2004). Our findings further suggested that vitamin D₃ metabolites, including 1,25(OH)₂D₃, transcriptionally regulate β-glucuronidase and TRPV6 mRNA expression in vivo. The NaPi-IIb cotransporter is primarily expressed in the brush-border membranes of the small intestinal epithelium, where it is considered the major sodium-Pi cotransporter (Hilfiker et al., 1998). Dietary supplementation of 1,25(OH)₂D₃ significantly regulates the expression of NaPi-IIb cotransporter in the intestine of rats (Katai et al., 1999). In broilers, 1α(OH)D₃ also has a similar effect on regulation of intestinal NaPi-IIb cotransporter gene expression (Han et al., 2009). Here, we also demonstrated that vitamin D₃ metabolites could rapidly induce the levels of NaPi-IIb cotransporter mRNA in the intestine of broiler chickens. Several genes associated with calcium homeostasis have been shown to be regulated by VDR, such as calbindin and TRPV6 (Bolt et al., 2005; Meyer et al., 2006). Since dietary 1,25(OH)₂D₃ could be absorbed and bound to intestinal VDR and then regulate its downstream gene transcriptionally, we speculate that intestinal gene expression may be directly regulated by VDR in response to vitamin D₃ metabolites.

The 24-hydroxylase belongs to cytochrome P450-containing enzyme (CYP24) that is important in the regulation of vitamin D metabolism (Minghetti and Norman, 1988). The expression of 24-hydroxylase in the kidney has been demonstrated to be regulated by 1,25(OH)₂D₃ and to initiate the inactivation of 1,25(OH)₂D₃ (Armbrecht et al., 1997). Interestingly, we found that intestinal 24-hydroxylase mRNA expression was reduced by dietary supplementation of 25(OH)D₃ in broiler chickens. However, 1α(OH)D₃ and 1,25(OH)₂D₃ did not cause a significant effect on intestinal 24-hydroxylase mRNA expression. The metabolite 1,25(OH)₂D₃ plays a central role in regulating mineral metabolism through direct interaction with intracellular VDR in intestinal and kidney epithelial cells (Haussler et al., 1998). It has been reported that vitamin D analogs auto-regulate the expression of the VDR gene expression in cultured kidney cells (Costa et al., 1985; Santiso-Mere et al., 1993). In contrast, intestinal VDR gene expression was not affected by 25(OH)D₃ or 1,25(OH)₂D₃ in human cells (Balesaria et al., 2009). Similarly, we also found that vitamin D₃ metabolites did not have an effect on the regulation of VDR gene expression in broiler chickens. How vitamin D₃ metabolites differently regulate intestinal calcium homeostasis-associated gene expression in broiler chickens remains to be investigated in the further studies.

Calbindin, β-glucuronidase, TRPV6, and NaPi-IIb cotransporter gene expression is rapidly responsive to dietary vitamin D₃ metabolites in the duodenum of broilers. Among vitamin D metabolites, 1,25(OH)₂D₃ is the most effective at regulating calcium homeostasis-associated genes in broilers.