Considerable speculation exists concerning the mechanism by which 1-hydroxylated cholecalciferol compounds improve dietary phytate-phosphorus utilization in chickens. Responses to feeding hydroxylated cholecalciferol compounds [1,25-(OH)₂ D₃ and 1-OH D₃⁴ in low phosphorus (P), phytate-containing corn-soybean meal diets have been dramatic. Reductions in the incidence of tibial dyschondroplasia and improved phytate-P utilization are the main benefits of these compounds (Biehl et al. 1995, Edwards 1993, Mitchell and Edwards 1996, Rennie and Whitehead 1996, Rennie et al. 1993), but dramatic improvements in the utilization of zinc and manganese occur as well (Biehl et al. 1995, Roberson and Edwards 1994). Surprisingly, utilization of iron and copper is not improved when corn-soybean meal diets are fortified with 1-OH D₃ or 1,25-(OH)₂ D₃, respectively (Aoyagi and Baker 1995, Biehl 1997).

There is a paucity of evidence indicating how hydroxylated cholecalciferol compounds may be acting in the body to improve phytate-P utilization. There is good evidence that 1,25-(OH)₂ D₃ stimulates gut absorption of P in chicks (Wasserman and Taylor 1973) and rats (Tanaka and DeLuca 1974). The primary mechanism of action for 1-hydroxylated cholecalciferol compounds would seem to involve their action on dietary phytate, because phytate-bound P, Zn and Mn are released when 1-hydroxylated cholecalciferol compounds are added to chick diets (Biehl et al. 1995, Edwards 1993). We have speculated that orally fed 1-OH D₃ is absorbed from the intestine and then transported to the liver, where it is hydroxylated at the 25-position to become 1,25-(OH)₂ D₃. This active metabolite of cholecalciferol may be transported back to the gastrointestinal (GI) tract via the bile, where it can activate or stimulate intestinal or microbial production of phytase in the GI tract. This in turn would cause increased release of phytate-bound minerals. By direct feeding of 1-OH D₃, the necessary hydroxylation step in the kidney is avoided, i.e., only the liver is needed to hydroxylate 1-OH D₃ at the 25 position to 1,25-(OH)₂ D₃.

The presence of intestinal phytase in chicks is supported by the findings of Davies et al. (1970) and Iqbal et al. (1994). The activity of intestinal phytase and alkaline phosphatase has been reported to increase when elevated levels of cholecalciferol are fed to chicks (Davies et al. 1970, Pileggi et al. 1955). Data from our laboratory have suggested, however, that surfeit levels of cholecalciferol are much less effective than low levels of 1,25-(OH)₂ D₃ or 1-OH D₃ in increasing phytate-P utilization (Biehl 1997). Because of the potency of these hydroxylated cholecalciferol compounds, they could potentially enhance intestinal phytase activity to a greater extent than cholecalciferol itself, which would explain the greater magnitude of response compared with high levels of cholecalciferol.

Our objectives herein were to determine whether either 1,25-(OH)₂ D₃ or 1-OH D₃, given orally to chicks or added to mucosal incubation tubes, would increase the specific activity of mucosal phytase. Also, to address the question of GI microbial phytase stimulation, 1-OH D₃ efficacy was evaluated in cecectomized chicks fed P-deficient corn-soybean meal diets. Cecectomy is known to remove most of the microbial activity from the avian GI tract (McNab 1973).

MATERIALS AND METHODS

After thawing for a short period of time, each mucosal sample (~400 mg wet weight) was homogenized in 45 mL cold sucrose (50 mmol/L)-Tris HCl (2 mmol/L) buffer (pH 7.1) containing 100 mg/L Tween 20. Homogenization was performed with a Sorvall tube homogenizer for ~20 strokes. Between tissue homogenates, the tube and homogenizer were washed with 70% ethanol and then rinsed with deionized water. The tissue homogenates were centrifuged at 8000 × g for 3 min and the supernatant was then separated from the pellet. The pellet was discarded. An aliquot of the supernatant was taken for protein determination by the microprotein method of Bradford (1976).

To measure phytase activity, the supernatant (2 mL) was combined with 100 µL of magnesium chloride solution (40 mmol/L) and 100 µL of zinc chloride solution (4 mmol/L). An antibiotic mixture was added to provide 130,000 units of streptomycin and 2 × 10⁶ units of penicillin per liter of substrate. This was done to minimize microbial phytase activity (Akin and Benner 1988). A 30 mmol/L sodium phytate solution (100 µL) was then added to the experimental tubes, and deionized water was added to the blanks. Reagent blanks consisting of buffer with or without sodium phytate together with antibiotics were included. Tubes were then incubated at 37°C in a water-bath for 2 h, after which an aliquot (250-500 µL) was taken for determination of inorganic phosphate (Pi). Urea solution (360 g/L) was added (1 mL) to the tubes to prevent protein precipitation, and then a malachite green reagent (4 mL) was added to finalize the mixture. The malachite green reagent contained 5 mmol/L HCl and 34 mmol/L ammonium molybdate together with malachite green (340 mg/L). After allowing 10 min for color development, the samples were read spectrophotometrically at 640 nm.

The concentrations of Pi in the incubates and blanks were derived from the standard curve, which was generated using graded doses of Pi (1-5 mmol/L Pi) from KH₂PO₄. The phytase activity was calculated as follows: phytase activity [nmol Pi/(mg protein·min)] = [(Tp  T)  (Bp  B)]/(protein concentration in mg/L × time in min), where Tp = Pi in tubes containing tissue + phytate, T = Pi in tubes containing tissue + water (i.e., tissue blank), Bp = Pi in tubes containing buffer + phytate, and B = Pi in reagent blank (i.e., buffer + water).

Table 1. Composition of phosphorus-deficient and cholecalciferol-adequate corn-soybean meal diet1 [View Table]

The basal diet was designed to be superadequate in cholecalciferol but markedly deficient in bioavailable P, containing only an estimated 0.14 g/100 g nonphytate P and 0.10 g/100 g bioavailable P (NRC 1994). The Ca level was set at 0.63 g/100 g, below the NRC (1994) Ca requirement, but resulting in a Ca:available P ratio of over 6:1. Our previous work had shown that a Ca level higher than 0.63 g/100 g in a diet with only 0.10 g/100 g available P would result in anorexia (Biehl et al. 1995). The experimental design of Assay 1 consisted of the following three dietary treatments: the P-deficient negative control diet, the negative control diet plus 20 µg/kg 1-OH D₃ and a positive control diet with adequate Ca and P levels according to NRC (1994). At assay termination, chicks and feed were weighed to determine performance data, after which the chicks were killed by CO₂ suffocation; right tibiae and duodenal mucosal samples were immediately taken for determination of bone ash (Chung and Baker 1990) and phytase activity as described previously. Bone and mucosa samples were pooled by replicate for purposes of analysis.

Equal portions of mucosa were also taken from each replicate group within Assay 1 treatments 1 and 2 (negative control and negative control + 1-OH D₃) for in vitro studies of the effect of added 1-OH D₃ or 1,25-(OH)₂ D₃ (in incubation tubes) on Pi release from sodium phytate. The two pooled mucosal samples were homogenized and centrifuged as described previously. Each supernatant was then diluted with sucrose buffer to produce four incremental levels of mucosal protein (containing phytase) per treatment sample. Each of these (eight) samples was divided into the following five subsamples: one containing no additive, one containing 0.1 µg of 1,25-(OH)₂ D₃, one containing 1.2µg of 1,25-(OH)₂ D₃, one containing 0.1 µg of 1-OH D₃ and one containing 2.0 µg of 1-OH D₃. Thus, a total of 40 tubes were incubated with sodium phytate for 2 h as described previously. After the incubation, aliquots were taken for Pi determination.

Table 2. Phosphorus released when sodium phytate is incubated in vitro with increasing concentrations of mucosal tissue from chicks fed phosphorus-deficient diets with or without 1 -hydroxycholecalciferol (1-OH D3 ) (Assay 1)1,2 [View Table]

In chick Assay 2, chicks were fed the same 23% crude protein corn-soybean meal diet as in Assay 1 during the first 13 d posthatching. At d 8, 9 and 10, surgery was performed on a group of 60 chicks each day. The birds were deprived of feed for 12 h before and 12 h after surgery. Also, chicks were administered 0.4 mg ceftiofur sodium (Naxcel, Upjohn-Pharmacia, Kalamazoo, MI) subcutaneously the day before, the day of, and the day after surgery to reduce the chance of infection. On the day of surgery, chicks were anesthetized with a 50:50 mixture of a ketamine/xylazine combination, which was given intramuscularly at a dosage of 17.5 mg ketamine/kg body weight and 3.5 mg xylazine/kg body weight. Once anesthetized, some chicks were sham operated by making a small incision through the skin and facia, locating the ceca in the GI cavity, removing them from the cavity, and then placing them back into the cavity and sewing the skin back together. The cecectomized chicks were treated in the same fashion except that the ceca were tied off and removed from the body before closing the incision. Surgery for each chick took ~15-20 min. On d 14 posthatching after a period of recovery, quadruplicate groups of three chicks from each surgery group were weighed and randomly assigned to one of three dietary treatments. Experimental diets were fed until d 27 posthatching. The dietary treatments consisted of the basal P-deficient diet (Table 1), the basal diet plus 1470 units/kg phytase,⁵ and the basal diet plus 20 µg/kg 1-OH D₃. At assay termination, chicks and feed were weighed, chicks were killed by CO₂ suffocation, and right tibiae were removed for determination of bone ash (Chung and Baker 1990).

RESULTS

Table 3. Phosphorus released when sodium phytate is incubated in vitro with chick mucosal tissue in tubes with or without hydroxylated cholecalciferol compounds [1,25-(OH)2D3 and 1-OHD3] (Assay 1)1 [View Table]

Table 4. Performance, bone ash, and intestinal phytase activity of chicks fed phosphorus-deficient and cholecalciferol-adequate corn-soybean meal diets (Assay 1)1,2 [View Table]

Table 5. Evaluation of supplemental phytase and 1 -hydroxylcholecalciferol (1-OH D3) in sham-operated and cecectomized chicks fed phosphorus-deficient and cholecalciferol-adequate corn-soybean meal diets (Assay 2)1 [View Table]

DISCUSSION

It is possible that the marked efficacy of 1-OH D₃ in improving phytate-P utilization is not related to stimulation of either phytase-protein synthesis or phytase specific activity in the intestinal mucosa. Instead, 1-OH D₃, after conversion to 1,25-(OH)₂ D₃, may function to markedly increase Ca absorption and perhaps also Ca removal from diet-based phytate. This could make the phytate complex more accessible to phytase attack. It is well established that high Ca diets and high Ca:P ratios cause enhanced phytate binding of P (Biehl and Baker 1997, Biehl et al. 1995, Sebastian et al. 1996) and of trace elements such as Zn (Bafundo et al. 1984, Fordyce et al. 1987, O'Dell et al. 1964). This may explain why 1,25-(OH)₂ D₃ so markedly increases Zn release from phytate (Biehl et al. 1995). Removal of Ca from the intestinal lumen may assist in making both phytate-bound P and Pi more bioavailable. The resulting marked improvement in P absorption probably explains why dietary Mn utilization is so dramatically improved (Biehl et al. 1995). Thus, excess Pi in the gut lumen is strongly antagonistic to Mn absorption (Wedekind et al. 1991a and 1991b).

The phytase assay system we used (Davies et al. 1970, Iqbal et al. 1994) was based on enzymatic release of Pi from sodium phytate at pH 7.1. We did not test Ca phytate in our assay system, nor did we test either sodium or Ca phytate at a higher or lower pH. It may be possible that Pi release from Ca phytate in mucosal incubation tubes would be affected by 1-OH D₃ feeding. Clearly, one cannot exactly simulate the conditions of the gut when using in vitro tissue preparations, nor can one assume that the phytate complex existing in corn and soybean meal is the same as either sodium or Ca phytate.

It is well established that cholecalciferol itself can improve phytate-P utilization (Davies et al. 1970, Mohammed et al. 1991, Pileggi et al. 1955). Our work with a cholecalciferol-free, low P corn-soybean meal diet showed that chick bone ash would continue to respond to vitamin D-3 levels up to 37.5 µg/kg, over seven times the estimated NRC (1994) requirement for cholecalciferol (Biehl 1997). This same study, however, showed that 1-OH D₃ was five times more biologically active than cholecalciferol itself, and no level of (excess) cholecalciferol was as effective in elevating tibia ash as 20 µg/kg 1-OH D₃. Earlier, DeLuca (1974) and Soares et al. (1978) had come to a similar conclusion regarding the potency of 1-OH D₃. This suggests that any 1,25-(OH)₂ D₃ precursor (i.e., 1-OH D₃, 25-OH D₃ or cholecalciferol itself ) should be expected to have efficacy in vivo for releasing Pi from Ca-containing phytate complexes that exist in cereal grains and oilseed meals.

Our last assay was designed to explore whether the ceca may be involved in the response to feeding hydroxylated vitamin D-3 compounds. The role of the GI microflora in Pi release from phytate is controversial. It has been suggested that gut microflora play no major role in phytate breakdown in rats (Miyazawa et al. 1996). However, several earlier rat studies had suggested that microflora in the GI tract are involved in phytate breakdown (Moore and Veum 1983, Moore et al. 1984, Sandberg and Andersson 1988, Wise and Gilburt 1982). Hydroxylated cholecalciferol compounds could be enhancing Pi release from phytate via both mucosal and microbial phytase. Assuming microbial phytase in the ceca could be releasing some Pi from phytate, the resulting Pi could be pushed back into the gut lumen at the ileal-cecal junction by peristaltic and antiperistaltic action (Hodgkiss 1984). Some Pi absorption could then occur, and it may even be possible that some Pi absorption could occur from the ceca as well. By removing the ceca, we were able to determine their contribution to the efficacy of 1-OH D₃ in releasing Pi from phytate. Regardless of cecectomy or sham operation, the tibia ash response was similar, indicating that the ceca and the cecal microbes have little influence on the 1-OH D₃ response. The tibia ash response when microbial phytase was added to the basal diet of both sham and cecectomized chicks was expected, because exogenous microbial phytase acts in the doudenum to release phytate-P; thus cecectomy should not have interfered with this mechanism.

Another assay in our laboratory attempted to reduce the microbial population with high levels of antibiotics in the diet and then to evaluate the response to 1-OH D₃ when added to a P-deficient corn-soybean meal basal diet (Biehl 1997). The results indicated that bone ash would respond to 1-OH D₃ supplementation in both the presence or absence of antibiotics (Biehl 1997).

In summary, these studies did not lead to a direct conclusion regarding the mechanism responsible for the Pi releasing efficacy of 1-hydroxylated cholecalciferol products. Further research should focus on the reasons why 1-OH D₃ is so markedly efficacious in chickens fed P-deficient diets, but has no efficacy in pigs fed similar diets (Biehl and Baker 1996). Anatomical and physiological differences (e.g., crop, gizzard, duodenal pH or transit time) between pigs and avians should be considered. Another area that deserves attention is the kidney, where 1,25-(OH)₂ D₃ can markedly affect mineral reabsorption.