TY - JOUR
AU - Liesegang, A.
AB - ABSTRACT The purpose of this study was to investigate whether diets differing in Ca concentration would have an influence on vitamin D (VitD) receptor (VDR) and calbindin D9k (Calb9k) immunoreactivities in the gastrointestinal tract of growing goats. In addition, the effect of a single VitD injection was studied, to clarify whether exogenous VitD would further increase the active Ca absorption mechanisms. The hypothesis of the study was that reduced Ca intake would lead to greater active Ca absorption, and with that, to greater amounts of VDR and Calb9k immunoreactivities. The normal Ca kid group (according to age requirements) received 2.5 to 6 g of Ca/d, whereas the lesser Ca kid group (less than requirements) received 1.5 to 4 g of Ca/d from wk 6 (weaning) to 15 (slaughter). In addition, 5 and 6 goat kids, respectively, of each group (normal Ca kid group, lesser Ca kid group), were injected with VitD (0.05 mg of cholecalciferol/kg of BW) in wk 14 of life. Blood samples were taken in wk 14 and 15. Calcium and VitD (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D) concentrations were determined in serum. Immediately after slaughter, the duodenum (DD) and rumen (RU) were mounted in conventional Ussing chambers. Unidirectional flux rates of Ca across gastrointestinal tissues were measured. Additionally, tissue specimens of the gastrointestinal tract were collected, and formaldehyde-fixed paraffin sections were used for VDR and Calb9k immunohistochemistry. In all kid groups, a net absorption in the RU and a net secretion of Ca in the DD were observed. Immunoreactions of VDR were greatest in the duodenal mucosa, whereas Calb9k immunoreactions were observed in the forestomach and intestinal tissues. The greatest expression was observed in the duodenal surface epithelium. Additionally, in the VitD-injected groups, an immunoreaction occurred in the jejunal superficial and basal glands and the ileal superficial epithelium. In contrast, the other groups showed no Calb9k immunoreactions at these sites. In conclusion, there is clear evidence for the RU as a main site for Ca absorption. The results of this study also indicate that VDR and Calb9k are highly expressed in the duodenal mucosa. The active absorption may not have such an important role in the DD because active transport was also evident in the RU. However, Calb9k expression seems to be stimulated by VitD administration. INTRODUCTION In growing animals, Ca is essential for normal growth, maintenance of bones and teeth, and the prevention of metabolic bone diseases. The Ca balance is controlled by 3 organ systems: the gastrointestinal tract, bone, and kidneys (Bronner, 1997). Normocalcemia is maintained through the actions of calcitonin, parathyroid hormone, and 1,25-dihydroxyvitamin D (1,25VitD), the active form of vitamin D (VitD; Russel, 2001). In nonruminant animals, intestinal Ca absorption is assumed to be achieved by 2 mechanisms: an active transcellular transport and a passive paracellular pathway (Bronner et al., 1986; Bronner 1998). Active Ca transport is mainly regulated by 1,25VitD via transcriptional activation of genes; 1,25VitD binds to its classical nuclear receptor (Erben et al., 2002), the VitD receptor (VDR). The transcellular Ca absorption in the intestine is divided into 3 steps: first, passive entry of ionized Ca into enterocytes via transient receptor potential vanilloid channel type 6 (TRPV6; Peng et al., 1999; Wilkens et al., 2009); second, cytosolic transfer of Ca bound to calbindin D9k (Calb9k), a VitD-dependent Ca-binding protein (Bronner, 1987,1991; Feher et al., 1992); and third, the extrusion of Ca across the basolateral membrane, where Ca pumps, predominantly plasma membrane Ca-ATPase and sodium Ca exchanger, transport Ca into the blood (van Abel et al., 2003; Wasserman, 2004). These transporters [TRPV6, Calb9k, plasma membrane Ca-ATPase (PMCA1b), and sodium Ca exchanger] are considered to be essential for transcellular Ca absorption. Additionally, in ruminants, Ca is absorbed in the rumen (RU; Breves and Schröder, 2005). It is not known how the ruminal Ca absorption influences Ca homeostasis (Martens, 2005). Different studies have reported different results on the permeability of Ca across the ruminal epithelium (Pfeffer et al., 1970; Dillon and Scott, 1979; Schröder et al., 1995, 2001). Although it has clearly been shown that in sheep these Ca flux rates in the RU must be active (Schröder et al., 2001), it is still not clear how this transport is realized on a molecular basis. Moreover, the absorption of Ca from the intestines may not be identical, even between ruminant species. Liesegang et al. (2004,2006,2008) showed that dairy goats and dairy cows have different Ca and bone metabolism characteristics compared with milk sheep. Growing goats had a greater bone turnover than growing sheep (Liesegang and Risteli, 2005). Thus, the mechanisms in relation to Ca in ruminants are not completely understood, especially the active Ca absorption mechanisms in the different parts of the gastrointestinal tract. Generally, the organism can adapt to different stages of reproduction and growth, but the mechanisms of adaptation to changing Ca needs are currently unknown. The goal of the present study was to test the hypothesis that VDR and Calb9k concentrations vary in the gastrointestinal tract, depending on Ca intake. Another hypothesis was that different Ca feeding regimen plus a VitD injection would be factors that would influence Ca absorption. It was of special interest to see whether changes occurred according to Ca absorption in the RU. MATERIALS AND METHODS All procedures in the present experiment were approved by the Cantonal Veterinary Office of Zurich. The guidelines used were according to the 2009 animal welfare law of Switzerland. Animals and Experimental Design The trial included 22 goat kids (Saanen breed). Animals were housed in pens bedded with sawdust. During feeding, the animals were maintained in single pens and allowed to stay in groups the rest of the time. After weaning (6 wk postnatal) goat kids were fed individually for 9 wk. The lesser Ca kid group (Ca-lk) received 1.5 to 4 g of Ca/d and the normal Ca kid group (Ca-nk) received 2.5 to 6 g of Ca/d from wk 6 to 15 of life, respectively (Figure 1). All animals received a diet that was balanced in energy and protein. The diet consisted of hay and concentrate (Table 1). The diet contained 1,500 IU of VitD/kg of DM, in accordance with recommendations of the Swiss Federal Research Station for Animal Production (RAP, 1999). Additionally, the animals had free access to water and a salt block (NaCl, Agrosal, Heilbronn, Germany). Seven days before slaughter (wk 14 postnatal), 5 or 6 goat kids from each group (Ca-lk and Ca-nk) were injected once intramuscularly with VitD (cholecalciferol, 0.05 mg/kg of BW intramuscularly, vitamin D3 “S” ad us. vet., Streuli, Uznach, Switzerland). The groups were then defined as Ca-lkVitD and Ca-nkVitD. Blood samples to describe the VitD status [1,25VitD and 25-hydroxyvitamin D (25VitD)] were taken before VitD injection (wk 14) and at wk 15. During the whole trial, blood samples were collected to measure Ca concentrations in serum. Figure 1. View largeDownload slide Calcium intake of goat kids (mean ± SE) in grams per day during the 9-wk feeding trial (n = 11 for each group). Different letters (a, b) indicate differences between Ca-nk and Ca-lk (P ≤ 0.05). Shaded bars = kids fed normal (recommended) amounts of Ca; unshaded bars = kids fed less than normal amounts of Ca. An asterisk (*) indicates significant (P < 0.05) differences between the time point before to the time point after; the heavy black line indicates daily Ca requirements: 5 g/d until wk 12 postnatal, and then 4.5 g/d (RAP, 1999). Figure 1. View largeDownload slide Calcium intake of goat kids (mean ± SE) in grams per day during the 9-wk feeding trial (n = 11 for each group). Different letters (a, b) indicate differences between Ca-nk and Ca-lk (P ≤ 0.05). Shaded bars = kids fed normal (recommended) amounts of Ca; unshaded bars = kids fed less than normal amounts of Ca. An asterisk (*) indicates significant (P < 0.05) differences between the time point before to the time point after; the heavy black line indicates daily Ca requirements: 5 g/d until wk 12 postnatal, and then 4.5 g/d (RAP, 1999). Table 1. Daily allowance (kg of original substance) and nutrient intake (daily intake in grams or megajoules in DM) of the experimental diets1 Item  Ca-nk, 6pn to 8pn  Ca-lk, 6pn to 8pn  Ca-nk, 9pn to 15pn  Ca-lk, 9pn to 15pn  Ingredient, daily allowance           Hay2  0.4  0.4  0.5  0.5   Concentrate  0.43  0.44  0.43  0.44  Nutrient intake               DM  0.7  0.7  0.8  0.8   NEl,5 MJ  4  4.1  4.4  4.5   Absorbable protein  58  58  64  65   Ca  5.4  2.9  5.8  3.3   P  2.5  2.5  2.8  2.7   Mg  2.1  2.1  2.3  2.3  Item  Ca-nk, 6pn to 8pn  Ca-lk, 6pn to 8pn  Ca-nk, 9pn to 15pn  Ca-lk, 9pn to 15pn  Ingredient, daily allowance           Hay2  0.4  0.4  0.5  0.5   Concentrate  0.43  0.44  0.43  0.44  Nutrient intake               DM  0.7  0.7  0.8  0.8   NEl,5 MJ  4  4.1  4.4  4.5   Absorbable protein  58  58  64  65   Ca  5.4  2.9  5.8  3.3   P  2.5  2.5  2.8  2.7   Mg  2.1  2.1  2.3  2.3  1During the 9-wk-feeding trial (n = 11 for each group; nk = normal Ca concentration; lk = lesser Ca concentration); pn = weeks postnatal. 2Hay (barn dried, balanced in grasses and leguminoses): 4.5 MJ of NEl/kg of DM, 76 g of absorbable protein/kg of DM, 4.3 g of Ca/kg of DM, 3.1 g of P/kg of DM. 3Concentrate: 7 MJ of NEl/kg of DM, 88 g of absorbable protein/kg of DM, 9.8 g of Ca/kg of DM, 3.5 g of P/kg of DM. 4Concentrate: 7.1 MJ of NEl/kg of DM, 89 g of absorbable protein/kg of DM, 3.5 g of Ca/kg of DM, 3.5 g of P/kg of DM (Kliba 2705b1, 2704b2, Provimi Kliba AG, Kaiseraugst, Switzerland). 5Calculated according to RAP (1999). View Large Table 1. Daily allowance (kg of original substance) and nutrient intake (daily intake in grams or megajoules in DM) of the experimental diets1 Item  Ca-nk, 6pn to 8pn  Ca-lk, 6pn to 8pn  Ca-nk, 9pn to 15pn  Ca-lk, 9pn to 15pn  Ingredient, daily allowance           Hay2  0.4  0.4  0.5  0.5   Concentrate  0.43  0.44  0.43  0.44  Nutrient intake               DM  0.7  0.7  0.8  0.8   NEl,5 MJ  4  4.1  4.4  4.5   Absorbable protein  58  58  64  65   Ca  5.4  2.9  5.8  3.3   P  2.5  2.5  2.8  2.7   Mg  2.1  2.1  2.3  2.3  Item  Ca-nk, 6pn to 8pn  Ca-lk, 6pn to 8pn  Ca-nk, 9pn to 15pn  Ca-lk, 9pn to 15pn  Ingredient, daily allowance           Hay2  0.4  0.4  0.5  0.5   Concentrate  0.43  0.44  0.43  0.44  Nutrient intake               DM  0.7  0.7  0.8  0.8   NEl,5 MJ  4  4.1  4.4  4.5   Absorbable protein  58  58  64  65   Ca  5.4  2.9  5.8  3.3   P  2.5  2.5  2.8  2.7   Mg  2.1  2.1  2.3  2.3  1During the 9-wk-feeding trial (n = 11 for each group; nk = normal Ca concentration; lk = lesser Ca concentration); pn = weeks postnatal. 2Hay (barn dried, balanced in grasses and leguminoses): 4.5 MJ of NEl/kg of DM, 76 g of absorbable protein/kg of DM, 4.3 g of Ca/kg of DM, 3.1 g of P/kg of DM. 3Concentrate: 7 MJ of NEl/kg of DM, 88 g of absorbable protein/kg of DM, 9.8 g of Ca/kg of DM, 3.5 g of P/kg of DM. 4Concentrate: 7.1 MJ of NEl/kg of DM, 89 g of absorbable protein/kg of DM, 3.5 g of Ca/kg of DM, 3.5 g of P/kg of DM (Kliba 2705b1, 2704b2, Provimi Kliba AG, Kaiseraugst, Switzerland). 5Calculated according to RAP (1999). View Large Collection of Blood Samples Blood samples were collected from the external jugular vein (Vacutainer, 9 or 6 mL, without additives, Vacuette Greiner Bio-One Vacuette, St. Gallen, Switzerland). Blood was centrifuged (1,580 × g for 10 min at 20°C) within 30 min of collection. Two tubes of serum were stored at −20°C and 1 tube was stored at −80°C until analyses were performed. Analysis of Serum Samples Serum was analyzed for Ca and 1,25VitD as described previously (Liesegang et al., 2000; Liesegang and Risteli, 2005). Serum 25VitD measurements were performed using a kit from Chromsystems Instruments and Chemicals GmbH (Munich, Germany), which allowed the chromatographic determination of 25VitD using a simple isocratic HPLC system with a UV detector (HP-1100, Agilent Technologies, Palo Alto, CA). The intra- and interassay CV were 4.4 and 5.6%, respectively (Pérez-Llamas et al., 2008). Tissue Sampling and Processing Within 10 min after slaughter, pieces of RU and duodenum (DD) were collected for the Ussing chamber analyses, and within 15 min, 5-cm-long tubular pieces of DD, jejunum (JJ), ileum (IL), cecum (CC), and colon (CO) and pieces of RU, reticulum (RET), omasum (OMA), and abomasum (ABO) were collected and fixed in neutral buffered 4% formaldehyde solution for 26 h. The piece of the DD was taken directly after the pancreatic duct entrance and the JJ, IL, and CC probes were taken from the central part of these segments. The segment of the CO was taken 40 cm cranial to the anus. The pieces from the RU, RET, and OMA were taken from the base, and finally, a piece of the ABO was taken in the middle of the greater curvature. When necessary, gastrointestinal samples were washed with physiological saline. After fixation, all gastrointestinal probes were cut into pieces 1 cm in length and rinsed in tap water for 24 h. This procedure was followed by dehydration in graded ethanol (70, 80, 96, and 100%), xylene, and paraffin (60°C) and finally embedding in paraffin (Histowax, Leica Microsystems AG, Glattbrugg, Switzerland). Transverse sections of 5 µm in thickness were cut, mounted on Superfrost Plus adhesive slides (Menzel-Gläser, Braunschweig, Germany), and dried at 60°C overnight. Sections were stained with hematoxylin and eosin (Romeis, 1989) to exclude animals possibly exhibiting pathological changes in the intestine. Ussing Chamber Technique Tissue specimens of the DD and RU were mounted in a modified Ussing chamber (Ussing and Zehran, 1951) and bathed with a volume of 3.5 mL of buffer solution on both sides of the intestinal wall. The Parson buffer solution contained (in mmol/L): NaCl, 107; KCl, 4.5; NaHCO3, 25; Na2HPO4, 1.8; NaH2PO4, 0.2; CaCl2, 1.25; MgSO4, 1; and glucose, 12.2; and was gassed with 5% CO2 in 95% O2 and kept at 37°C; pH was adjusted to 7.4. The epithelium was continuously short-circuited by an automatic voltage-clamp device (Aachen Microclamp, AC Copy Datentechnik, Aachen, Germany) with correction for solution resistance. Tissue conductance was measured by recording the voltage resulting from bipolar current pulses (±100 mA) applied across the tissue at 1-min intervals and was calculated according to Ohm's law. The values for tissue conductance and the continuously applied short-circuit current were recorded every minute. Ten minutes after mounting the tissues in the chambers, 10 µL of 45Ca2+ was added to the mucosal side (to measure mucosal-to-serosal Ca fluxes) or the serosal side (to measure serosal-to-mucosal fluxes) of the intestinal wall (labeled side). After an additional 60 min, to allow isotope flux rates to reach a steady state and the short-circuit current to stabilize, unidirectional ion flux rates were determined in sequential 20-min periods. From the measured unidirectional net flux rates (Jms = flux rate from mucosa to serosa; Jsm = flux rate from serosa to mucosa), net ion flux rates (Jnet) were calculated [Jnet = Jms − Jsm; nmol/(cm2/h)] from the mean unidirectional flux rates. Immunohistochemistry for VDR The method used was described previously (Liesegang et al., 2008). A biotinylated rat monoclonal antibody (9A7γ, Neo-Markers, P. H. Stehelin & Cie AG, Basel, Switzerland) was used to label VDR in the gastrointestinal sections. This antibody was used at a 1:200 dilution. Negative controls were performed using Tris-buffered saline instead of primary antibody and positive controls using duodenal cross-sections of pigs (cross-reactivity reported with identical results; Milde et al., 1989; Schröder et al., 2001). In every tissue specimen, nuclear staining intensities (SI) of 500 cells were recorded from the following cell types: basal glandular cells (BG), intermediate glandular cells (IG), superficial glandular cells (SG) and surface epithelial cells (SEP). The SI of the nuclei were scored as negative = 0, very weak = 0.5, weak = 1, intermediate = 2, or strong = 3, correlating to the absence of brown (i.e., blue counterstaining only), light brown, brown, or dark brown staining, respectively (i.e., SI 0 to SI 3). The semiquantitative evaluation of histochemical reactions for VDR was performed as described previously; that is, an immunoreactive score (IRS) was calculated for the colored intensity using the following formula (Boos et al., 2007; Liesegang et al., 2008):  VDR-IRS = 0 × n(SI0) + 0.25 × n(SI0,5) + 1 × n(SI1) + 4 × n(SI2) + 9 × n(SI3), where n is the number of cells. Immunohistochemistry for Calb9k The method for quantifying Calb9k was performed using an immunohistochemical protocol. A Calb9k polyclonal rabbit antibody was used for the immunohistochemistry (Swant, Bellinzona, Switzerland). All subsequent steps were carried out at room temperature. Negative controls were performed using Tris-buffered saline instead of primary antibody, and positive controls were performed using intestinal cross-sections of pigs. For microscopic analysis of the slides, a light microscope (Leica DMLB, Leica Microsystems AG) was used. After histomorphological examination, the slides were assessed with a computerized digital camera (Colorview 12, Leica Microsystems AG) analyzing the mean color intensity (i.e., grayscale values) of cells or tissues. The software used was analySIS Pro (Soft Imaging System GmbH, Münster, Germany). This procedure was always performed under standardized conditions, eliminating primarily errors normally occurring during visual grading of histochemical color intensities. However, this did not eliminate interassay variations, which are caused by the use of slides processed histochemically in several succeeding batches (Grube, 2004; Taylor and Levenson, 2006). Digitalized pictures were taken; white balancing was performed (i.e., measurement in a tissue-free district of the section, thus representing 100% transmission); randomly selected regions of interest were defined, containing immunopositive structures such as SEP, SG, and BG; and color intensity was measured. The mean color intensity (i.e., grayscale value) of a selected structure within a region of interest was measured using 256 different color levels, whereby zero represented black and 255 represented white. This meant that larger grayscale values corresponded to smaller immunoreaction intensities. After assessing 5 tissue areas within the tissue for each immunopositive structure per slide, a mean value per structure and animal was calculated. Transmission (T) values were transformed to extinction (E), which was proportional to dye concentrations at the level of the sections (E = lg1/T). This value was used for further statistical analysis. Statistical Analyses The results are presented as mean ± SEM. A multivariate ANOVA for repeated measures was performed, with group as a cofactor included in the model to test the difference in the time-dependent patterns between groups. The factor “cell type” was included in the model for the VDR-IRS (i.e., location of the cells within the mucosa). To avoid false conclusions resulting from a violation of the assumption of compound symmetry, a Huynh-Feldt correction was performed. Furthermore, the difference between groups was tested with the Mann-Whitney U test (nonparametric) to limit the influence of extreme values. The level of significance was set at α = 0.05 for all tests. All statistical analyses were performed by using SYSTAT for Windows (SPSS Inc., Chicago, IL). RESULTS Ca and VitD Concentrations in Serum Mean serum Ca concentrations were similar (2.9 ± 0.7 mmol/L; P > 0.05) in both groups over the whole trial (reference value in goats: 2.2 to 2.7 mmol/L; Tschuor et al., 2008). However, differences were seen in VitD concentrations in the serum: in the 14th wk, the 25VitD concentrations were 30.7 ± 1.3 ng/mL in the Ca-nk group, 30.2 ± 4 ng/mL in the Ca-lk group, 31.7 ± 1.8 ng/mL in the Ca-nkVitD group, and 32.5 ± 1.3 ng/mL in the Ca-lkVitD group. In the 15th wk, 1 wk after the VitD injection, the mean 25VitD concentrations showed an increase (P < 0.001) in the Ca-nkVitD group (38.7 ± 1.2 ng/mL) and the Ca-lkVitD group (46.7 ± 3.3 ng/mL) compared with the Ca-nk group (29.9 ± 1.8 ng/mL) and the Ca-lk group (27.7 ± 2.2 ng/mL). The 1,25VitD concentrations in serum showed no difference within the groups (14th wk: 158 ± 28 pmol/L; 15th wk: 162 ± 16 pmol/L; P = 0.078 group effect; P = 0.734 time effect). Ca Flux Rates Mucosal-to-serosal Ca flux rates (Jms) exceeded the respective flux rates in the opposite direction (Jsm) in the RU. This resulted in net Ca flux rates (Jnet = Jms − Jsm) ranging between +6 and +7 nmol/(cm2/h) in RU. In the Ca-nk group, the Jms and Jsm flux rates were greater (P = 0.190) compared with the Ca-lk group in RU, but no group effect was indentified. In the DD of the kids without a VitD injection, net Ca flux rates ranged between −9 and −5 nmol/(cm2/h), and in kids with a VitD injection, mean flux rates were approximately +0.5 nmol/(cm2/h). The net flux rates were not different between the groups in the RU (P = 0.927) and DD (P = 0.190; Table 2). The net flux rates were positive in the RU of all groups, indicating Ca absorption, whereas the net flux rates in the DD were negative or weakly positive, respectively, indicating a net secretion or only sparse absorption. No difference between the groups was observed (P > 0.05). Table 2. Electrical properties, unidirectional and net flux rates1,2 [nmol/(cm2/h); means ± SE3] Item  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Rumen               ms flux  14.18 ± 2.38  11.06 ± 2.85  9.21 ± 1.89  8.25 ± 2.95   sm flux  6.78 ± 2.68  3.74 ± 1.12  2.95 ± 0.81  2.62 ± 1.12   Net flux  6.92 ± 2.23  7.31 ± 1.97  6.26 ± 1.43  5.64 ± 1.92  Duodenum               ms flux  30.85 ± 3.84  15.14 ± 2.43  21.35 ± 3.33  22.31 ± 2.13   sm flux  32.66 ± 5.42  19.17 ± 2.12  30.29 ± 3.69  25.87 ± 4.70   Net flux  −3.54 ± 4.17  0.32 ± 1.98  −8.94 ± 3.18  0.49 ± 4.41  Item  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Rumen               ms flux  14.18 ± 2.38  11.06 ± 2.85  9.21 ± 1.89  8.25 ± 2.95   sm flux  6.78 ± 2.68  3.74 ± 1.12  2.95 ± 0.81  2.62 ± 1.12   Net flux  6.92 ± 2.23  7.31 ± 1.97  6.26 ± 1.43  5.64 ± 1.92  Duodenum               ms flux  30.85 ± 3.84  15.14 ± 2.43  21.35 ± 3.33  22.31 ± 2.13   sm flux  32.66 ± 5.42  19.17 ± 2.12  30.29 ± 3.69  25.87 ± 4.70   Net flux  −3.54 ± 4.17  0.32 ± 1.98  −8.94 ± 3.18  0.49 ± 4.41  1Jnet = Jms − Jsm, where sm = serosal to mucosal and ms = mucosal to serosal. 2nk = normal Ca concentration, n = 5; nkVitD = nk and vitamin D injection, n = 6; lk = lesser Ca concentration, n = 6; lkVitD = lk and vitamin D injection, n = 5. 3No group effect, P > 0.05. View Large Table 2. Electrical properties, unidirectional and net flux rates1,2 [nmol/(cm2/h); means ± SE3] Item  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Rumen               ms flux  14.18 ± 2.38  11.06 ± 2.85  9.21 ± 1.89  8.25 ± 2.95   sm flux  6.78 ± 2.68  3.74 ± 1.12  2.95 ± 0.81  2.62 ± 1.12   Net flux  6.92 ± 2.23  7.31 ± 1.97  6.26 ± 1.43  5.64 ± 1.92  Duodenum               ms flux  30.85 ± 3.84  15.14 ± 2.43  21.35 ± 3.33  22.31 ± 2.13   sm flux  32.66 ± 5.42  19.17 ± 2.12  30.29 ± 3.69  25.87 ± 4.70   Net flux  −3.54 ± 4.17  0.32 ± 1.98  −8.94 ± 3.18  0.49 ± 4.41  Item  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Rumen               ms flux  14.18 ± 2.38  11.06 ± 2.85  9.21 ± 1.89  8.25 ± 2.95   sm flux  6.78 ± 2.68  3.74 ± 1.12  2.95 ± 0.81  2.62 ± 1.12   Net flux  6.92 ± 2.23  7.31 ± 1.97  6.26 ± 1.43  5.64 ± 1.92  Duodenum               ms flux  30.85 ± 3.84  15.14 ± 2.43  21.35 ± 3.33  22.31 ± 2.13   sm flux  32.66 ± 5.42  19.17 ± 2.12  30.29 ± 3.69  25.87 ± 4.70   Net flux  −3.54 ± 4.17  0.32 ± 1.98  −8.94 ± 3.18  0.49 ± 4.41  1Jnet = Jms − Jsm, where sm = serosal to mucosal and ms = mucosal to serosal. 2nk = normal Ca concentration, n = 5; nkVitD = nk and vitamin D injection, n = 6; lk = lesser Ca concentration, n = 6; lkVitD = lk and vitamin D injection, n = 5. 3No group effect, P > 0.05. View Large VDR Vitamin D receptor immunoreactivities were exposed as brown staining in the nuclei in all segments of the intestinal tissues (DD, JJ, IL, CC, and CO; Figures 2 and 3). Goblet cells were always devoid of any specific immunoreaction and were thus not considered in the present study. This brown coloration was of varying degrees and was thus indicative of more or less increased VDR concentrations within all other cell types present within the epithelium of crypts (BG, IG, SG) or intestinal luminal SEP. Nuclear immunostaining was present in all segments of the intestine, whereby immunoreactivities were greatest in the DD compared with the more distal intestinal segments. The IRS of the Ca-nk group did not differ from data obtained from the Ca-lk group, nor did the VitD injection show any effect on the immunoreactivities of VDR (P = 0.454). The intestinal VDR-IRS of BG decreased from larger quantities in the DD and JJ to smaller quantities in the IL, CC, and CO. Mean VDR-IRS [(BG + IG + SG + SEP)/4] differed between all segments (DD > JJ > CO > IL > CC; P ≤ 0.05; Table 3). Differences between the cell types (BG, IG, and SG/crypt epithelial cells and SEP) within an intestinal segment were for the DD (BG, IG > SG > SEP; P ≤ 0.05), JJ, and IL (BG, IG > SG > SEP; P ≤ 0.05), and CC, CO (IG > BG, SG > SEP; P ≤ 0.05). No VDR immunoreactivity was observed in the forestomach tissues of goat kids. Figure 2. View largeDownload slide Vitamin D receptor (VDR) immunohistochemistry in the duodenum (DD; A, a) and jejunum (JJ; B, b) of goat kids. The VDR immunoreaction is demonstrated as brown staining and contrasts well with the blue counterstaining of the nuclei. DD (A) and JJ (B) exhibit a clear gradient in VDR immunostaining. Strong reactions are visible in basal glandular cells (BG), intermediate glandular cells (IG), superficial glandular cells (SG), and surface epithelial cells (SEP) and demonstrate a stepwise reduced immunostaining, respectively. Small inlets (a, b) show the BG marked at a greater magnification to underline the decreasing VDR immunoreaction along the length of the small intestine and the cytoplasmic staining in DD and JJ. MM = lamina muscularis mucosae; V = intestinal villi; N = nucleus. Color version available in the online PDF. Figure 2. View largeDownload slide Vitamin D receptor (VDR) immunohistochemistry in the duodenum (DD; A, a) and jejunum (JJ; B, b) of goat kids. The VDR immunoreaction is demonstrated as brown staining and contrasts well with the blue counterstaining of the nuclei. DD (A) and JJ (B) exhibit a clear gradient in VDR immunostaining. Strong reactions are visible in basal glandular cells (BG), intermediate glandular cells (IG), superficial glandular cells (SG), and surface epithelial cells (SEP) and demonstrate a stepwise reduced immunostaining, respectively. Small inlets (a, b) show the BG marked at a greater magnification to underline the decreasing VDR immunoreaction along the length of the small intestine and the cytoplasmic staining in DD and JJ. MM = lamina muscularis mucosae; V = intestinal villi; N = nucleus. Color version available in the online PDF. Figure 3. View largeDownload slide Vitamin D receptor (VDR) immunohistochemistry in the ileum (A, a), cecum (B, b), and colon (C, c) of goat kids. The ileum, cecum, and colon generally exhibit weaker immunoreactions than the duodenum and jejunum. Goblet cells (G; marked only in panel c) are always devoid of VDR. SEP = superficial epithelium; SG = superficial glandular cells; IG = intermediate glandular cells; BG = basal glandular cells; MM = lamina muscularis mucosae; PP = Peyer's plaques; M = tunica muscularis; V = intestinal villi; N = nucleus. Color version available in the online PDF. Figure 3. View largeDownload slide Vitamin D receptor (VDR) immunohistochemistry in the ileum (A, a), cecum (B, b), and colon (C, c) of goat kids. The ileum, cecum, and colon generally exhibit weaker immunoreactions than the duodenum and jejunum. Goblet cells (G; marked only in panel c) are always devoid of VDR. SEP = superficial epithelium; SG = superficial glandular cells; IG = intermediate glandular cells; BG = basal glandular cells; MM = lamina muscularis mucosae; PP = Peyer's plaques; M = tunica muscularis; V = intestinal villi; N = nucleus. Color version available in the online PDF. Table 3. Vitamin D receptor immunoreactive scores (mean ± SE) of the various cell types within the epithelial layers of the different segments of intestines of goat kids1 Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  DD BG  1,190 ± 209  1,439 ± 133  1,432 ± 193  1,576 ± 229  1,412 ± 92a,x  DD IG  1,102 ± 202  1,395 ± 132  1,335 ± 209  1,407 ± 243  1,315 ± 95a,x  DD SG  316 ± 120  288 ± 66  243 ± 54  263 ± 77  276 ± 37a,y  DD SEP  59 ± 9  138 ± 36  94 ± 30  110 ± 47  102 ± 17a,y  Mean DD              776 ± 53a  JJ BG  831 ± 159  797 ± 175  1,288 ± 213  1,086 ± 301  1,004 ± 109a,x  JJ IG  878 ± 214  589 ± 141  1,144 ± 216  1,017 ± 308  903 ± 112b,x  JJ SG  201 ± 78  128 ± 12  315 ± 86  248 ± 76  223 ± 35b,y  JJ SEP  52 ± 16  87 ± 26  172 ± 54  60 ± 21  96 ± 19b,z  Mean JJ              557 ± 65b  IL BG  143 ± 30  224 ± 70  237 ± 55  260 ± 116  221 ± 35b,x  IL IG  137 ± 21  242 ± 40  306 ± 63  263 ± 90  245 ± 30b,x  IL SG  62 ± 13  118 ± 14  97 ± 22  88 ± 23  94 ± 10b,x  IL SEP  33 ± 8  104 ± 29  36 ± 8  91 ± 53  68 ± 16b,z  Mean IL              157 ± 20c  CC BG  72 ± 22  51 ± 23  159 ± 59  89 ± 28  94 ± 20c,x  CC IG  219 ± 44  138 ± 39  355 ± 87  212 ± 65  232 ± 34c,y  CC SG  83 ± 10  126 ± 15  130 ± 52  102 ± 18  112 ± 15a,x  CC SEP  17 ± 11  39 ± 15  43 ± 29  38 ± 17  35 ± 9b,z  Mean CC              118 ± 18d  CO BG  93 ± 33  191 ± 84  269 ± 91  170 ± 38  185 ± 36b,x  CO IG  252 ± 54  603 ± 171  582 ± 152  394 ± 63  470 ± 68b,y  CO SG  105 ± 12  243 ± 82  160 ± 22  144 ± 41  166 ± 26a,x  CO SEP  17 ± 11  30 ± 11  27 ± 12  5 ± 2  20 ± 5c,z  Mean CO              210 ± 30e  Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  DD BG  1,190 ± 209  1,439 ± 133  1,432 ± 193  1,576 ± 229  1,412 ± 92a,x  DD IG  1,102 ± 202  1,395 ± 132  1,335 ± 209  1,407 ± 243  1,315 ± 95a,x  DD SG  316 ± 120  288 ± 66  243 ± 54  263 ± 77  276 ± 37a,y  DD SEP  59 ± 9  138 ± 36  94 ± 30  110 ± 47  102 ± 17a,y  Mean DD              776 ± 53a  JJ BG  831 ± 159  797 ± 175  1,288 ± 213  1,086 ± 301  1,004 ± 109a,x  JJ IG  878 ± 214  589 ± 141  1,144 ± 216  1,017 ± 308  903 ± 112b,x  JJ SG  201 ± 78  128 ± 12  315 ± 86  248 ± 76  223 ± 35b,y  JJ SEP  52 ± 16  87 ± 26  172 ± 54  60 ± 21  96 ± 19b,z  Mean JJ              557 ± 65b  IL BG  143 ± 30  224 ± 70  237 ± 55  260 ± 116  221 ± 35b,x  IL IG  137 ± 21  242 ± 40  306 ± 63  263 ± 90  245 ± 30b,x  IL SG  62 ± 13  118 ± 14  97 ± 22  88 ± 23  94 ± 10b,x  IL SEP  33 ± 8  104 ± 29  36 ± 8  91 ± 53  68 ± 16b,z  Mean IL              157 ± 20c  CC BG  72 ± 22  51 ± 23  159 ± 59  89 ± 28  94 ± 20c,x  CC IG  219 ± 44  138 ± 39  355 ± 87  212 ± 65  232 ± 34c,y  CC SG  83 ± 10  126 ± 15  130 ± 52  102 ± 18  112 ± 15a,x  CC SEP  17 ± 11  39 ± 15  43 ± 29  38 ± 17  35 ± 9b,z  Mean CC              118 ± 18d  CO BG  93 ± 33  191 ± 84  269 ± 91  170 ± 38  185 ± 36b,x  CO IG  252 ± 54  603 ± 171  582 ± 152  394 ± 63  470 ± 68b,y  CO SG  105 ± 12  243 ± 82  160 ± 22  144 ± 41  166 ± 26a,x  CO SEP  17 ± 11  30 ± 11  27 ± 12  5 ± 2  20 ± 5c,z  Mean CO              210 ± 30e  a–eMean values with different letters within a column (i.e., between the different intestinal segments) differ (P ≤ 0.05). x–zMean values with different letters within a column (i.e., between the different epithelia) differ (P ≤ 0.05); no significant group effect was observed (P > 0.05). 1nk = normal Ca concentration, n = 5; nkVitD = nk and vitamin D injection, n = 6; lk = lesser Ca concentration, n = 6; lkVitD = lk and vitamin D injection, n = 5; BG, IG, and SG = basal, intermediate, and superficial glandular cells; SEP = surface epithelium; DD = duodenum; JJ = jejunum; IL = ileum; CC = cecum; CO = colon. View Large Table 3. Vitamin D receptor immunoreactive scores (mean ± SE) of the various cell types within the epithelial layers of the different segments of intestines of goat kids1 Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  DD BG  1,190 ± 209  1,439 ± 133  1,432 ± 193  1,576 ± 229  1,412 ± 92a,x  DD IG  1,102 ± 202  1,395 ± 132  1,335 ± 209  1,407 ± 243  1,315 ± 95a,x  DD SG  316 ± 120  288 ± 66  243 ± 54  263 ± 77  276 ± 37a,y  DD SEP  59 ± 9  138 ± 36  94 ± 30  110 ± 47  102 ± 17a,y  Mean DD              776 ± 53a  JJ BG  831 ± 159  797 ± 175  1,288 ± 213  1,086 ± 301  1,004 ± 109a,x  JJ IG  878 ± 214  589 ± 141  1,144 ± 216  1,017 ± 308  903 ± 112b,x  JJ SG  201 ± 78  128 ± 12  315 ± 86  248 ± 76  223 ± 35b,y  JJ SEP  52 ± 16  87 ± 26  172 ± 54  60 ± 21  96 ± 19b,z  Mean JJ              557 ± 65b  IL BG  143 ± 30  224 ± 70  237 ± 55  260 ± 116  221 ± 35b,x  IL IG  137 ± 21  242 ± 40  306 ± 63  263 ± 90  245 ± 30b,x  IL SG  62 ± 13  118 ± 14  97 ± 22  88 ± 23  94 ± 10b,x  IL SEP  33 ± 8  104 ± 29  36 ± 8  91 ± 53  68 ± 16b,z  Mean IL              157 ± 20c  CC BG  72 ± 22  51 ± 23  159 ± 59  89 ± 28  94 ± 20c,x  CC IG  219 ± 44  138 ± 39  355 ± 87  212 ± 65  232 ± 34c,y  CC SG  83 ± 10  126 ± 15  130 ± 52  102 ± 18  112 ± 15a,x  CC SEP  17 ± 11  39 ± 15  43 ± 29  38 ± 17  35 ± 9b,z  Mean CC              118 ± 18d  CO BG  93 ± 33  191 ± 84  269 ± 91  170 ± 38  185 ± 36b,x  CO IG  252 ± 54  603 ± 171  582 ± 152  394 ± 63  470 ± 68b,y  CO SG  105 ± 12  243 ± 82  160 ± 22  144 ± 41  166 ± 26a,x  CO SEP  17 ± 11  30 ± 11  27 ± 12  5 ± 2  20 ± 5c,z  Mean CO              210 ± 30e  Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  DD BG  1,190 ± 209  1,439 ± 133  1,432 ± 193  1,576 ± 229  1,412 ± 92a,x  DD IG  1,102 ± 202  1,395 ± 132  1,335 ± 209  1,407 ± 243  1,315 ± 95a,x  DD SG  316 ± 120  288 ± 66  243 ± 54  263 ± 77  276 ± 37a,y  DD SEP  59 ± 9  138 ± 36  94 ± 30  110 ± 47  102 ± 17a,y  Mean DD              776 ± 53a  JJ BG  831 ± 159  797 ± 175  1,288 ± 213  1,086 ± 301  1,004 ± 109a,x  JJ IG  878 ± 214  589 ± 141  1,144 ± 216  1,017 ± 308  903 ± 112b,x  JJ SG  201 ± 78  128 ± 12  315 ± 86  248 ± 76  223 ± 35b,y  JJ SEP  52 ± 16  87 ± 26  172 ± 54  60 ± 21  96 ± 19b,z  Mean JJ              557 ± 65b  IL BG  143 ± 30  224 ± 70  237 ± 55  260 ± 116  221 ± 35b,x  IL IG  137 ± 21  242 ± 40  306 ± 63  263 ± 90  245 ± 30b,x  IL SG  62 ± 13  118 ± 14  97 ± 22  88 ± 23  94 ± 10b,x  IL SEP  33 ± 8  104 ± 29  36 ± 8  91 ± 53  68 ± 16b,z  Mean IL              157 ± 20c  CC BG  72 ± 22  51 ± 23  159 ± 59  89 ± 28  94 ± 20c,x  CC IG  219 ± 44  138 ± 39  355 ± 87  212 ± 65  232 ± 34c,y  CC SG  83 ± 10  126 ± 15  130 ± 52  102 ± 18  112 ± 15a,x  CC SEP  17 ± 11  39 ± 15  43 ± 29  38 ± 17  35 ± 9b,z  Mean CC              118 ± 18d  CO BG  93 ± 33  191 ± 84  269 ± 91  170 ± 38  185 ± 36b,x  CO IG  252 ± 54  603 ± 171  582 ± 152  394 ± 63  470 ± 68b,y  CO SG  105 ± 12  243 ± 82  160 ± 22  144 ± 41  166 ± 26a,x  CO SEP  17 ± 11  30 ± 11  27 ± 12  5 ± 2  20 ± 5c,z  Mean CO              210 ± 30e  a–eMean values with different letters within a column (i.e., between the different intestinal segments) differ (P ≤ 0.05). x–zMean values with different letters within a column (i.e., between the different epithelia) differ (P ≤ 0.05); no significant group effect was observed (P > 0.05). 1nk = normal Ca concentration, n = 5; nkVitD = nk and vitamin D injection, n = 6; lk = lesser Ca concentration, n = 6; lkVitD = lk and vitamin D injection, n = 5; BG, IG, and SG = basal, intermediate, and superficial glandular cells; SEP = surface epithelium; DD = duodenum; JJ = jejunum; IL = ileum; CC = cecum; CO = colon. View Large Calb9k Calbindin D9k was histochemically detectable and was assessed quantitatively in tissues in the forestomach (i.e., RU, OMA, RET, ABO) and in intestinal tissues (i.e., DD, JJ, and IL; Figures 4 and 5). No immunohistochemical reactions were observed in the large intestine (i.e., CC and CO). In the forestomach, the reaction was always in the SEP, and no difference was detected between the groups. In the intestines, immune reactions were not evident in the intestinal sections. However, reactions were present in all the DD groups and in part of the JJ groups. The DD showed a greater expression than the JJ (P = 0.003). There were differences between groups (P = 0.023) and between different parts of intestinal sections (P < 0.0001). The greatest immunoreactivities were found in the DD SEP. Between the different layers of DD, differences were obtained (P < 0.0001; DD SEP > DD SG > DD BG). This was also true for the JJ: JJ SEP > JJ SG > JJ BG (SEP vs. SG and SEP vs. BG, P = 0.0001; SG vs. BG, P = 0.012). Differences were also observed in the JJ SG (P = 0.007) and the JJ BG (P = 0.010) between the different kid groups. Figure 4. View largeDownload slide Calbindin D9k (Calb9k) immunoreactions in the duodenum (DD, A), jejunum (JJ, B), and ileum (IL, C) of a goat kid without a vitamin D injection. The Calb9k immunoreaction is demonstrated as brown staining without background staining. The DD and JJ (A, B) exhibit a clear gradient in Calb9k immunostaining. A strong reaction is visible in surface epithelial cells (SEP). The superficial glandular cells (SG) and basal glandular cells (BG) demonstrate a stepwise reduced immunostaining, respectively. The IL showed no Calb9k immunoreaction. MM = lamina muscularis mucosae; M = tunica muscularis; V = intestinal villi. Color version available in the online PDF. Figure 4. View largeDownload slide Calbindin D9k (Calb9k) immunoreactions in the duodenum (DD, A), jejunum (JJ, B), and ileum (IL, C) of a goat kid without a vitamin D injection. The Calb9k immunoreaction is demonstrated as brown staining without background staining. The DD and JJ (A, B) exhibit a clear gradient in Calb9k immunostaining. A strong reaction is visible in surface epithelial cells (SEP). The superficial glandular cells (SG) and basal glandular cells (BG) demonstrate a stepwise reduced immunostaining, respectively. The IL showed no Calb9k immunoreaction. MM = lamina muscularis mucosae; M = tunica muscularis; V = intestinal villi. Color version available in the online PDF. Figure 5. View largeDownload slide Calbindin D9k (Calb9k) immunoreactions in the duodenum (DD, A), jejunum (JJ, B) and ileum (IL, C) of a goat kid with vitamin D injection. The Calb9k immunoreaction is demonstrated as brown staining without background staining. The DD, JJ, and IL (A, B, C) exhibit a clear gradient in Calb9k immunostaining. A strong reaction is visible in surface epithelial cells (SEP). Superficial glandular cells (SG) and basal glandular cells (BG), respectively, demonstrate a stepwise reduced immunostaining. Additionally, Calb9k immunoreactions of the rumen (D) and omasum (E), as examples of the forestomachs, are visible. MM = lamina muscularis mucosae; M = tunica muscularis; V = intestinal villi; OL = omasal leave. Color version available in the online PDF. Figure 5. View largeDownload slide Calbindin D9k (Calb9k) immunoreactions in the duodenum (DD, A), jejunum (JJ, B) and ileum (IL, C) of a goat kid with vitamin D injection. The Calb9k immunoreaction is demonstrated as brown staining without background staining. The DD, JJ, and IL (A, B, C) exhibit a clear gradient in Calb9k immunostaining. A strong reaction is visible in surface epithelial cells (SEP). Superficial glandular cells (SG) and basal glandular cells (BG), respectively, demonstrate a stepwise reduced immunostaining. Additionally, Calb9k immunoreactions of the rumen (D) and omasum (E), as examples of the forestomachs, are visible. MM = lamina muscularis mucosae; M = tunica muscularis; V = intestinal villi; OL = omasal leave. Color version available in the online PDF. The Ca-lk group showed differences in the JJ SG and JJ BG compared with the Ca-lk group with the VitD injection (P = 0.037). Different values in the JJ SEP (P = 0.043) of the Ca-nk group were also measured in comparison with the Ca-nk group with the VitD injection. The most important differences were obtained in the JJ SG, JJ BG, and IL SEP because only the groups with a VitD injection showed an immunoreaction (P = 0.013; Table 4). Table 4. Cytoplasmic calbindin D9k extinction values (mean ± SE) of the various cell types within the epithelial layers of the different segments in the gastrointestinal tract of goat kids1 Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  RU SEP  1.4 ± 0.1  1.4 ± 0.1  1.5 ± 0.1  1.6 ± 0.0  1.5 ± 0.0  RET SEP  1.4 ± 0.1  1.5 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  OMA SEP  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  1.4 ± 0.0  1.4 ± 0.0  ABO SEP  1.5 ± 0.1  1.4 ± 0.0  1.6 ± 0.1  1.6 ± 0.0  0.9 ± 0.2  DD SEP  2.6 ± 0.2  3.0 ± 0.2  2.8 ± 0.2  3.0 ± 0.2  2.8 ± 0.1a,x  DD SG  1.7 ± 0.1  2.0 ± 0.1  2.0 ± 0.1  2.0 ± 0.2  1.9 ± 0.1b,y  DD BG  1.4 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.5 ± 0.2  1.3 ± 0.1b,y  JJ SEP  1.5 ± 0.1  3.0 ± 0.2  1.9 ± 0.4  2.5 ± 0.4  1.7 ± 0.3b,x  JJ SG  0  1.8 ± 0.1  0  1.8 ± 0.1  1.8 ± 0.0*  JJ BG  0  1.4 ± 0.0  0  1.6 ± 0.0  1.5 ± 0.0*  IL SEP  0  1.3 ± 0.1  0  1.2 ± 0.0  1.3 ± 0.0*  IL SG  No immunohistochemical reaction in all groups  IL BG  No immunohistochemical reaction in all groups  CC, CO  No immunohistochemical reaction in all groups  Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  RU SEP  1.4 ± 0.1  1.4 ± 0.1  1.5 ± 0.1  1.6 ± 0.0  1.5 ± 0.0  RET SEP  1.4 ± 0.1  1.5 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  OMA SEP  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  1.4 ± 0.0  1.4 ± 0.0  ABO SEP  1.5 ± 0.1  1.4 ± 0.0  1.6 ± 0.1  1.6 ± 0.0  0.9 ± 0.2  DD SEP  2.6 ± 0.2  3.0 ± 0.2  2.8 ± 0.2  3.0 ± 0.2  2.8 ± 0.1a,x  DD SG  1.7 ± 0.1  2.0 ± 0.1  2.0 ± 0.1  2.0 ± 0.2  1.9 ± 0.1b,y  DD BG  1.4 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.5 ± 0.2  1.3 ± 0.1b,y  JJ SEP  1.5 ± 0.1  3.0 ± 0.2  1.9 ± 0.4  2.5 ± 0.4  1.7 ± 0.3b,x  JJ SG  0  1.8 ± 0.1  0  1.8 ± 0.1  1.8 ± 0.0*  JJ BG  0  1.4 ± 0.0  0  1.6 ± 0.0  1.5 ± 0.0*  IL SEP  0  1.3 ± 0.1  0  1.2 ± 0.0  1.3 ± 0.0*  IL SG  No immunohistochemical reaction in all groups  IL BG  No immunohistochemical reaction in all groups  CC, CO  No immunohistochemical reaction in all groups  a,bMean values with different letters within the column (i.e., between the different intestinal segments) differ (P ≤ 0.05). x,yMean values with different letters within the column (i.e., between the different epithelia) differ (P ≤ 0.05). 1nk = normal Ca concentration, n = 5; nkVitD = nk and vitamin D injection, n = 6; lk = lesser Ca concentration, n = 6; lkVitD = lk and vitamin D injection, n = 5; RU = rumen; RET = reticulum; OMA = omasum; ABO = abomasum; DD = duodenum; JJ = jejunum; IL = ileum; CC = cecum; CO = colon; SEP = surface epithelium; SG = superficial glandular cells; BG = basal glandular cells. *Group effect (P ≤ 0.05). View Large Table 4. Cytoplasmic calbindin D9k extinction values (mean ± SE) of the various cell types within the epithelial layers of the different segments in the gastrointestinal tract of goat kids1 Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  RU SEP  1.4 ± 0.1  1.4 ± 0.1  1.5 ± 0.1  1.6 ± 0.0  1.5 ± 0.0  RET SEP  1.4 ± 0.1  1.5 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  OMA SEP  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  1.4 ± 0.0  1.4 ± 0.0  ABO SEP  1.5 ± 0.1  1.4 ± 0.0  1.6 ± 0.1  1.6 ± 0.0  0.9 ± 0.2  DD SEP  2.6 ± 0.2  3.0 ± 0.2  2.8 ± 0.2  3.0 ± 0.2  2.8 ± 0.1a,x  DD SG  1.7 ± 0.1  2.0 ± 0.1  2.0 ± 0.1  2.0 ± 0.2  1.9 ± 0.1b,y  DD BG  1.4 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.5 ± 0.2  1.3 ± 0.1b,y  JJ SEP  1.5 ± 0.1  3.0 ± 0.2  1.9 ± 0.4  2.5 ± 0.4  1.7 ± 0.3b,x  JJ SG  0  1.8 ± 0.1  0  1.8 ± 0.1  1.8 ± 0.0*  JJ BG  0  1.4 ± 0.0  0  1.6 ± 0.0  1.5 ± 0.0*  IL SEP  0  1.3 ± 0.1  0  1.2 ± 0.0  1.3 ± 0.0*  IL SG  No immunohistochemical reaction in all groups  IL BG  No immunohistochemical reaction in all groups  CC, CO  No immunohistochemical reaction in all groups  Layer  Ca-nk  Ca-nkVitD  Ca-lk  Ca-lkVitD  Mean  RU SEP  1.4 ± 0.1  1.4 ± 0.1  1.5 ± 0.1  1.6 ± 0.0  1.5 ± 0.0  RET SEP  1.4 ± 0.1  1.5 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  OMA SEP  1.4 ± 0.0  1.5 ± 0.1  1.4 ± 0.0  1.4 ± 0.0  1.4 ± 0.0  ABO SEP  1.5 ± 0.1  1.4 ± 0.0  1.6 ± 0.1  1.6 ± 0.0  0.9 ± 0.2  DD SEP  2.6 ± 0.2  3.0 ± 0.2  2.8 ± 0.2  3.0 ± 0.2  2.8 ± 0.1a,x  DD SG  1.7 ± 0.1  2.0 ± 0.1  2.0 ± 0.1  2.0 ± 0.2  1.9 ± 0.1b,y  DD BG  1.4 ± 0.0  1.4 ± 0.0  1.5 ± 0.1  1.5 ± 0.2  1.3 ± 0.1b,y  JJ SEP  1.5 ± 0.1  3.0 ± 0.2  1.9 ± 0.4  2.5 ± 0.4  1.7 ± 0.3b,x  JJ SG  0  1.8 ± 0.1  0  1.8 ± 0.1  1.8 ± 0.0*  JJ BG  0  1.4 ± 0.0  0  1.6 ± 0.0  1.5 ± 0.0*  IL SEP  0  1.3 ± 0.1  0  1.2 ± 0.0  1.3 ± 0.0*  IL SG  No immunohistochemical reaction in all groups  IL BG  No immunohistochemical reaction in all groups  CC, CO  No immunohistochemical reaction in all groups  a,bMean values with different letters within the column (i.e., between the different intestinal segments) differ (P ≤ 0.05). x,yMean values with different letters within the column (i.e., between the different epithelia) differ (P ≤ 0.05). 1nk = normal Ca concentration, n = 5; nkVitD = nk and vitamin D injection, n = 6; lk = lesser Ca concentration, n = 6; lkVitD = lk and vitamin D injection, n = 5; RU = rumen; RET = reticulum; OMA = omasum; ABO = abomasum; DD = duodenum; JJ = jejunum; IL = ileum; CC = cecum; CO = colon; SEP = surface epithelium; SG = superficial glandular cells; BG = basal glandular cells. *Group effect (P ≤ 0.05). View Large DISCUSSION In all kid groups, a large positive net flux rate (net absorption) for Ca was measured in the RU in the Ussing chambers. It was concluded that the RU is a main site for active Ca transport mechanisms in growing goat kids. Previous studies described Ca net absorption before the DD in single- and multifistulated ruminants (Pfeffer et al., 1970; Dillon and Scott, 1979; Wylie et al., 1985). Höller et al. (1988) and Schröder et al. (1997, 1998) also observed active Ca transport mechanisms in the caprine and ovine RU by the Ussing chamber technique. Furthermore, in the sheep RU, active positive Ca net flux rates were observed, but there was no influence of Ca supply or 1,25VitD (Schröder et al., 2001). In contrast, studies in nonruminant animals have shown that active Ca absorption is stimulated in response to Ca intake and that this effect is mediated by 1,25VitD (Nemere and Norman, 1991; Breves et al., 1995). Less dietary Ca intake led to a stimulation of active Ca absorption by more than 50% in the RU of growing goats, but not in sheep (Schröder et al., 1997). In contrast, the current study demonstrated no increase in Ca net flux rates in dietary Ca-depleted growing goats. In addition, the VitD injection showed no effect on Ca absorption in the RU of goat kids. In the DD, negative net flux rates were measured in groups not injected with VitD. From the present study, there was evidence of decreased Ca absorption in the DD of goat kids with a VitD injection, but this needs to be investigated further. The reason for the single VitD pulse treatment 7 d before slaughter was to simulate a therapy after calving to prevent parturient paresis, which is used in dairy cattle practice 1 wk before calving. Hollis and Conrad (1976) showed a 1-wk lag in the conversion of cholecalciferol to 25(OH)VitD in dairy cattle. Horst and Littledike (1982) recommended administering 10 × 106 IU of VitD approximately 1 wk before the expected date of parturition and to repeat the injections, if necessary. The effectiveness of this latter procedure apparently relies on the transient increase in plasma 1,25VitD that follows the VitD injection. The additional VitD injection should then increase intestinal Ca absorption (Collins and Norman, 1990). In nonruminant species [e.g., pigs (Schröder et al., 1998) and rats (Brommage et al., 1995)], active Ca absorption from the upper small intestines was stimulated by 1,25VitD. Schröder et al. (1997) observed an increase in net Ca absorption independent of Ca supply after VitD injection in the DD of goat kids. It is possible that 1,25VitD changes Ca absorption in the upper small intestine. Schröder et al. (1995) assumed that the duodenal mucosa is an optional target organ of 1,25VitD in goats. However, the mechanisms of Ca absorption in the RU are still unclear. The results of the present immunohistochemical study in growing goats demonstrated that VDR are present in all intestinal segments, as was also shown in earlier studies (Boos et al., 2007; Liesegang et al., 2008; Riner et al., 2008). The observed VDR immunoreactivities were prominent in DD mucosa, with less in the JJ and CO, a further decline in the IL, and the least in the CC. The results indicate that VDR are highly expressed at the site of active, VitD-dependent intestinal Ca absorption. Calcium intake, as well as VitD injections, had no influence on VDR immunoreactions. Interestingly there were no immunohistochemical reactions of VDR in the forestomach, but a clear active net flux rate of Ca was shown in the Ussing chamber. This active transport seems to be independent of VitD but dependent on Calb9k because it is expressed in the forestomach. To the knowledge of the authors, these are the first results presenting the expression of the Ca-binding protein Calb9k in caprine gastrointestinal tissues. Calbindin D9k was detected immunohistochemically in the forestomach and intestinal epithelium of growing goats. The DD and JJ exhibited immunohistochemical staining for Calb9k in all kid groups. Interestingly, goat kids with a VitD injection showed an additional immunohistochemical reaction in the JJ and IL that was not detectable in noninjected animals at these sites. There were 2 differences in immunoreactive Calb9k staining: a proximal-to-distal decrease within the intestinal segments, based mainly on increased Calb9k in the SEP, and SG to BG epithelial cell decreases within the segment. The distribution pattern of the Calb9k protein demonstrated in the present study is in accordance with several reports on Calb9k expression in the duodenal tissue of cows, mice, and goats (Schröder et al., 1998; Yamagishi et al., 2002; van Abel et al., 2003; Yamagishi et al., 2006). Clemens et al. (1988) formerly suggested that the expression of VDR in the chicken intestine may be correlated with the stage of differentiation of the enterocytes. In this study, VDR immunoreactivity was localized mainly in the nuclei of epithelial cells and was more abundant in the crypt than in the villus cells. Calbindin was found predominantly in villus tip cells in the normal rat DD (Taylor et al., 1984; van Corven et al., 1985). Calcium transport was approximately 2-fold greater in the villus tip than in the crypt-cell fraction basolateral membranes, although the affinity for Ca uptake was similar to that in rats (Smith et al., 1985). It may be concluded that Calb9k and ATP-dependent Ca transport are most prominent in absorbing villus cells and that VitD-dependent Ca absorption primarily takes place here as well (van Corven et al., 1985). In the present study, the enterocytes displayed the greatest VDR-IRS in the BG and IG cells and the greatest Calb9k reactivities in the SEP. The SEP have a very short lifetime, and on that account, the receptors in the SEP would not be effective for long periods of time. The SEP that develop from dividing stem cells in the crypts differentiate when they move to the neck of the glands. There, they are integrated into the SEP and are shed after apoptosis. The results of Walters and Weiser (1987) supported the opinion that mature enterocytes have the greatest capacity for transcellular movement of Ca. Furthermore, their findings showed that partially differentiated midvillus cells have the capacity to respond most rapidly to 1,25VitD. The villus tip cells have differentiated to a point at which they are no longer able to synthesize additional proteins for Ca absorption (Walters and Weiser, 1987). Using in situ hybridization, Freeman et al. (1995) found the greatest density of PMCA1b in the crypts and basal cells. Consequently, essential active Ca absorption is shown by coordinated actions of the ligand-active transcription factor VDR and the Ca pump PMCA1b (Freeman et al., 1995). In DD and JJ villus tip cells, TRPV6 has been shown to co-localize with Calb9k (Hoenderop et al., 1999; Peng et al., 2003). Furthermore, Weber et al. (2001) presented a co-localization of TRPV6 and Calb9k, suggesting that apical TRPV6 and intracellular Calb9k are interrelated in a system controlling Ca entry and intracellular Ca concentrations in the intestinal epithelium (Weber et al., 2001). In contrast, Benn et al. (2008) postulated active intestinal Ca absorption in the absence of TRPV6 and Calb9k in mice. Akhter et al. (2007) supported an alternative pathway for intestinal Ca absorption and intracellular Ca diffusion that does not require intestinal calbindin. There is evidence that calbindin possibly may have other roles in the intestine, for example, as a modulator of Ca channel activity, as an intracellular Ca buffer, or both (Benn et al., 2008). These investigations relating to nonruminant animals may also explain a possible mechanism in the RU of ruminants. The assumption of VitD-sensitive Ca absorption is supported by the detection of VDR and Calb9k in the goat small intestine (Schröder et al., 1995, 1997). In sheep, VDR and Calb9k were found in the JJ (Schröder et al., 2001). Additionally, the presence of TRPV5 and TRPV6 in the DD and JJ could be demonstrated with an in situ hybridization technique (Wilkens et al., 2009). In this study, TRPV6 expression was greater in the JJ than in the DD (Wilkens et al., 2009). It seems that the JJ is more important than the DD for active Ca absorption in sheep (Schröder et al., 1997). In contrast, our results and the results of Riner et al. (2008) show that the DD is more important for Ca absorption in goats. The treatment with 1,25VitD had no effect on preintestinal Ca absorption. However, an increase in Calb9k was present on injection of VitD in the small intestine, JJ, and IL. These findings show that intestinal Calb9k transcription was stimulated by the binding of VitD to its receptor and the activation of the complex thereafter. The daily Ca intake seems to be important for the active and passive mechanisms, respectively. Active Ca absorption is dominant at conditions of decreased Ca intake (Pansu et al., 1993), whereas the passive mechanism is more prominent when greater amounts of Ca are present in the feedstuff (Bronner and Pansu, 1999). It has to be considered that, in the present study, the Ca requirements decreased from the second to the third month in growing goats. For this reason, the animals fed the decreased Ca diet were not really deficient compared with the animals that received the diet with a normal Ca content. In addition, it seems that these young animals have a greater ability to recover and cope with decreased amounts of Ca in their diet. This may be seen by the small differences in the VDR and Calb9k immunohistochemistry reactions at 15 wk of age. In the present study, the Ussing chamber results revealed active Ca absorption in the RU, but only Calb9k, and no VDR, was detected by immunohistochemical methods. Also in the DD, there was evidence of active Ca absorption after VitD injection obtained with the Ussing chamber technique. Additionally, the immunohistochemical methods revealed intestinal VDR and Calb9k in goat kids. This study verified the results of Schröder and Breves (2006) showing that the proximal small intestines are major sites for controlled known active Ca absorption in nonruminant animals and ruminants. The mechanisms related to active transport in the rumen are not yet known, as seen in other studies (Schröder et al., 1995), and need to be addressed in further research. There is clear evidence for the RU as a main site for Ca absorption in growing goats, although the exact mechanisms related to this have not yet been clarified. The VDR and Calb9k exist mainly in the intestinal tract of goat kids. The results of this study indicate that VDR are highly expressed at the site of expected maximal intestinal Ca absorption, in the duodenal mucosa. Nuclei of enterocytes stained positive for VDR, with the strongest immunoreactions in the IG and BG. Calbindin D9k revealed the most intensive staining in the SEP in the forestomach and the DD. 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TI - Influence of different calcium supplies and a single vitamin D injection on vitamin D receptor and calbindin D9k immunoreactivities in the gastrointestinal tract of goat kids
JF - Journal of Animal Science
DO - 10.2527/jas.2009-2682
DA - 2010-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/influence-of-different-calcium-supplies-and-a-single-vitamin-d-HTR3YWCLFA
SP - 3598
EP - 3610
VL - 88
IS - 11
DP - DeepDyve
ER -