British Poultry Science Volume 46, Number 1 (February 2005), pp. 87–96 Effects of dietary boron supplementation on some biochemical parameters, peripheral blood lymphocytes, splenic plasma cells and bone characteristics of broiler chicks given diets with adequate or inadequate cholecalciferol (vitamin D3) content F. KURTOĞLU1, V. KURTOĞLU2 , I_. ÇELI_K3, T. KEÇECI_4 AND M. NI_ZAMLIOĞLU1 1 Department of Biochemistry, 2Department of Animal Nutrition and Nutritional Diseases, Department of Histology and Embryology and 4Department of Physiology, University of Selçuk, Faculty of Veterinary Medicine, Kampüs, Konya, Turkey 3 Abstract 1. The effects of 5 and 25 mg/kg boron supplementation of diets with inadequate (6.25 mg/kg) or adequate (50 mg/kg) cholecalciferol (vitamin D3) content on some biochemical parameters, tibia characteristics, peripheral blood lymphocyte and splenic plasma cell counts of broilers were investigated. 2. Supplementation of the diet with boron affected plasma concentrations of boron, iron, copper and zinc and also tibia boron, zinc and calcium concentrations but did not have any effect on tibia iron or copper concentrations or tibia ash and tibia weight values. 3. Boron supplementation caused significant increases in splenic plasma cell count but decreased the proximal and distal tibia growth plate widths. There was no effect of boron supplementation on peripheral blood alpha-naphthyl acetate esterase (ANAE) content. Whole blood haematocrit and haemoglobin counts were significantly increased by boron supplementation but there were no effects on leucocyte ratios such as eosinophil, basophil, monocyte, lymphocyte and thrombocyte. 4. In general, the findings of the present study support the hypothesis that boron has an important biological role that affects the mineral metabolism of animals by influencing both biochemical and haematological mechanisms. INTRODUCTION Although boron has been considered as an essential element for higher plants since the 1920s, its importance in human and animal nutrition has only been recognised recently. Most of the current interest in boron in animal nutrition began in 1981 with the finding that physiological amounts of boron (3 mg/kg diet) added to diets low in boron (<0.3 mg/kg diet) stimulated growth in vitamin-D3 deficient chicks (Hunt and Nielsen, 1981). Results presented by Kurtoğlu et al. (2001) indicated that boron had an influence on plasma calcium content and alkaline phosphatase (AP) activity especially at low dietary concentrations of D3 (6.25 mg/kg). The influence of vitamin D3 on cartilage and bone mineralisation is mediated, in part, through its role as a regulator of energy substrate utilisation (Hunt et al., 1994); therefore, calcification is an energy intensive process. There is considerable evidence that dietary boron alleviates perturbations in mineral metabolism that are characteristic of vitamin D3 deficiency (Hunt and Nielsen, 1981; Hunt et al., 1983; Hunt, 1994; Kurtoğlu et al., 2001). Three particular mechanisms involving boron demonstrate the boron—vitamin D3 interaction: boron compensates for perturbations in energy—substrate utilisation; boron enhances the macromineral content of normal bone; dietary boron, independently of vitamin D, enhances some indices of growth cartilage maturation (Hunt et al., 1994). Elliot and Edwards (1990) used a factorial arrangement of treatments involving the addition of calcium, cholecalciferol and boron (3 mg/kg) to purified diets for broilers. They reported that boron supplementation tended to increase bone Correspondence to: Associate Professor Dr. Firuze Kurtoğlu, Department of Biochemistry, University of Selçuk, Faculty of Veterinary Medicine, 42075 Kampüs, Konya, Turkey. E-mail: kurtoglu@selcuk.edu.tr Accepted for publication 13th September 2004. ISSN 0007–1668(print)/ISSN 1466–1799(online) ß 2005 British Poultry Science Ltd DOI: 10.1080/00071660400024001 88 F. KURTOĞLU ET AL. ash and that there was a significant interaction between the effects of boron and cholecalciferol on weight gain. In the poultry industry, processing of spent hens often results in many broken bones, especially in cage-maintained birds. Bone breaking strength and bone ash are often used as criteria for assessing the value of various dietary supplements, cage designs and bird density in preventing bone breakage. It has been assumed that rapid growth in broilers is a major cause of tibial dyschondroplasia, because restricted feeding reduces the incidence of this disorder (Roberson et al., 1993). For this reason, the National Research Council (NRC, 1984) suggested that trace mineral supplements to chemically defined diets should contain at least 2 mg/kg boron, although the boron requirement for different categories of poultry has not been determined (Hunt, 1989; Nielsen and Shuler, 1992; Rossi et al., 1993). In laying hens, a method of improving mineral balance in order to increase the bone strength of spent laying hens could benefit the poultry industry (Wilson and Ruszler, 1996; Kurtoğlu et al., 2002). Morphological examination of the tibia of chicks indicated that an interaction between boron and cholecalciferol affected bone formation. When dietary cholecalciferol was low, rachitic long bones were found in 17 of 21 boron-deprived chicks, but only 9 of 22 boronsupplemented chicks exhibited rachitic long bones; moreover, the lack of calcification generally was more severe in the boron-deprived chicks (Hunt and Nielsen, 1981). Boron deprivation was found to exacerbate gross bone abnormalities in chicks caused by a diet deficient, but not completely lacking, in cholecalciferol (Hunt and Nielsen, 1981; Hunt et al., 1994). At the microscopic level, boron deprivation exacerbated the distortion of marrow sprouts caused by cholecalciferol deprivation and delayed the initiation of cartilage calcification (Hunt, 1989). Boron deprivation also decreased chondrocyte density in the proliferating zones of the growth plate in cholecalciferol deficient chicks (Hunt et al., 1994). It has also been found that in ovo injections of boron reduced the normal height of the growth plate of chicks hatched from cholecalciferol-deficient eggs (King et al., 1991). It seems that boron may also affect blood cell counts and composition because blood cell formation and maturation are influenced by changes in cell membrane or kidney function or in calcium metabolism (Nielsen et al., 1991). For example, erythropoietin, a hormone synthesised in the kidney, plays a key role in the maturation of red blood cell precursors in bone marrow (Fisher, 1983). Prior studies of boron effects on blood cell variables were made in human subjects (Nielsen et al., 1987, 1990; Nielsen, 1989) but there was no data on blood cell variables affected by boron supplementation in chicks or other animal species. The growth plate, located between the epiphysis and metaphysis of the long bones, is responsible for elongation of these bones (Olsson, 1982; Brighton, 1985). These long bones have two growth plates (Olsson, 1982). Tibia and femur are the longest bones of the body, have the highest growth rate and are also bear the most weight. Therefore, growth plates of these long bones give the most valuable information about the status of skeletal development of poultry. Tibial growth plate histology has often been examined in experimental studies and in the diagnosis of skeletal disturbances (Farquharson et al., 1993; Germiller and Goldstein, 1997). Enzyme histochemical studies have widely been used to evaluate the functional development and maturation of the immune system (Coskun et al., 1998; Celik et al., 2000a, b; Sur and Çelik, 2003). A lymphocyte lysosomal enzyme (Knowles and Holck , 1978), alpha-naphthyl acetate esterase (ANAE), has been demonstrated in mature, immunocompetent circulating Tlymphocytes of many animal species. ANAE positivity has widely been used in the chicken (Pruthi et al., 1987; Maiti et al., 1990). This enzyme is assumed to be responsible for the cytotoxic effects of T-lymphocytes and the phagocytic activity of monocytes (Mueller et al., 1975). Knowles and Holck (1978) have reported that the ANAE positivity of peripheral blood T-lymphocytes is represented by 3 to 5 reddishbrown granules localised adjacent to the cell membrane. Lymphocytes without ANAE reaction product specific granules are considered to be an ANAE-negative B-lymphocytes. The objective of this study was to determine the effects of boron supplementation on plasma and tibia mineral concentrations, peripheral blood lymphocyte and splenic plasma cell counts, ANAE positivity of peripheral blood lymphocytes and some biochemical parameters of 1- to 45-d-old broiler chicks given adequate (50 mg/kg) or inadequate (6.25 mg/kg) dietary vitamin D3 (cholecalciferol). MATERIALS AND METHODS Animals and husbandry Five hundred and forty 1-d-old, unvaccinated broiler chicks (Avian) were used. They were weighed individually and divided into 6 main groups of 90 chicks each as shown in Table 1. To limit differences due to position, these groups were each divided into 6 subgroups of 15 chicks each. The chicks were placed in heated houses lit BORON AND CHOLECALCIFEROL by fluorescent sources. Food and water were provided ad libitum. The experimental period lasted until d 45. In the trial, chickens received a basal diet (Table 2) with vitamin premix, boron and cholecalciferol for each group (Table 1) weighted individually and added to diets in a homogeneous form. Orthoboric acid was used as the boron source, since it is a common inorganic form of boron of high purity (99.995%) and is absorbed well from the gastrointestinal tract (Pfeiffer and Jenney, 1950). Diet proximate analyses were performed using the methods of AOAC (1984). Table 1. Boron and vitamin D3 (cholecalciferol) supplementation of diets Group Boron (B) mg/kg food Vitamin D3 mg/kg food 0 0 5 5 25 25 6.25 50.00 6.25 50.00 6.25 50.00 1 2 3 4 5 6 Biochemical measurements Plasma total protein, albumin and cholesterol levels were measured spectrophotometrically (Shimadzu, UV 2100, Kyoto, Japan) using commercial kits (supplied by Randox Laboratories, Crumlin, UK). Plasma iron, copper and zinc concentrations were determined by atomic absorption spectrophotometry (Buck Scientific 200A, East Norwalk, CT 06855, USA). For plasma analysis, blood samples were taken from 12 chicks in each group by cardiac puncture into heparinised (10 IU heparin per ml of blood; Roche, Istanbul, Turkey) tubes on d 45. Blood samples were immediately centrifuged (Megafuge 1.0 R Heraeus Sepatech GmbH, Berlin, Germany) at 1000 g for 15 min at 4
C to obtain plasma. Plasma boron concentrations were determined by ICP (inductive coupled plasma atomic emission spectrometer; AES Varion Vista Model, Sydney, Australia) with a detection limit for boron of 3 mg/l as described by Hunt (1997). Preparation of tibia for bone ash and bone mineral analysis Table 2. Compositions of diets, g/kg Source 89 Days 1 to 28 28 to 45 Maize Wheat Soybean meal Sunflower meal Fish meal Bone meal Full fat soybean meal Vegetable oil Mixed oil Limestone DCP Salt Mineral premix* Vitamin premix** Antioxidant Coccidiostat Methionine Lysine 553.0 — 230.0 25.0 60.0 — 78.0 24.3 — 17.0 6.0 1.5 1.0 2.5 — — 1.7 — 471.5 148.0 120.5 — 30.0 20.0 150.0 — 3.50 7.0 5.7 2.7 2.0 2.5 1.0 1.0 2.3 0.8 Chemical analysis results ME***, MJ/kg Crude protein, g/kg Dry matter, g/kg Crude ash, g/kg Crude cellulose, g/kg Ether extract, g/kg 13.3 230.0 891.3 56.1 36.0 69.6 13.9 190.0 890.2 51.1 30.6 88.9 *Mineral premix. In each kg: Mn 80.000 mg; Fe 35.000 mg; Zn 50.000 mg; Cu 5.000 mg; I 2.000 mg; Co 400 mg; Se 150 mg. **Vitamin premix. In each 2.5 kg: retinol 3600 mg; -tocopherol acetate 30.000 mg; menaqinone 3.000 mg; thiamin 3.000 mg; riboflavin 6.000 mg; pyridoxine 5.000 mg; cyanocobalamin 15 mg; niacin 25.000 mg; biotin 40 mg; carotenoid 8.000 mg; folic acid 1.000 mg; choline cloride 300.000 mg; ascorbic acid 50.000 mg. ***Calculated. At d 45, 10 chicks from each group were killed by cervical dislocation. The left tibias were removed and boiled for 5 minutes. The tibias were excised and all flesh was removed. Proximal cartilages of the tibias were removed. The bones were airdried at room temperature for 48 h in an airconditioned room and then lipids were removed using petroleum ether as a solvent. Each tibia was broken into small pieces, weighed and then ashed at 550
C for 8 h (Rossi et al., 1993). The tibial ash samples were weighed and dissolved in 1 ml 5 M HNO3 and 5 ml 5 M HCl. These samples were then filtered, brought up to 25 ml with deionised water and analysed for boron and calcium content using ICP as before. The emission spectrum of each sample was analysed with two replicates at a wavelength of 250 nm. Tibia iron, copper and zinc were determined using an atomic absorption spectrophotometer (Buck Scientific 200A, East Norwalk, CT, USA). Preparation of tibia for morphologic examination The tibias were fixed in 10% formaldehyde, decalcified in 10% (v/v) nitric acid, divided into two equal halves through a midline incision, dehydrated, cleared and embedded in paraffin wax. Serial sections (6 to 7 mm) were taken through the proximal region and stained with Masson’s trichrome and picro-thionine (Culling et al., 1985). The width of the growth plates at medial, mid-point and lateral lines were measured with a linear ocular micrometer. 90 F. KURTOĞLU ET AL. Mean values were calculated and expressed in mm. Determination of peripheral blood leucocyte percentages Cardiac blood samples were taken into heparinised tubes at the end of the experiment. From each sample, 4 smears were prepared and airdried. The smears were fixed in glutaraldehyde— acetone solution, pH 4.8 for 3 min at 10
C. Two of them were stained with May Grünwald— Giemsa (Konuk, 1981) and peripheral blood leuckocyte percentages of the samples were determined by counting 200 leukocytes on each specimen. The results were expressed as percentage (%). The remaining smears were used for ANAE histochemistry (Coskun et al., 1998). Spleen histology and plasma cell counts For routine histological examinations and plasma cell counts, spleen samples were fixed in alcoholic formalin, dehydrated, cleared and immersed in paraffin blocks. Sections of 6 mm thickness were prepared and stained with either Crossmon’s trichrome stain (Bradbury and Gordon, 1992) or methyl green—pyronin (Stevens and Bancroft, 1992). Spleen histology was investigated in trichrome-stained specimens and plasma cells were counted in 10 randomly selected areas of each specimen by using an ocular square micrometer. Mean plasma cell counts were expressed as cell count/unit area (1.44  104 mm2) for each sample. Statistical analysis The biochemical data and the other parameters were assessed using Duncan’s multiple range test (SPSS, 1998). ANAE histochemistry Two smears were used for ANAE demonstration (Coskun et al., 1998). The incubation solution was prepared as follows: 80 ml of 0.067 M phosphate buffer (pH 5.0) and 4.8 ml of hexazotised pararosaniline [2.4 ml of pararosaniline (Sigma, C.I.N. 42500) and 2.4 ml of 4% sodium nitrite in distilled water] and 20 mg of alpha-naphthyl acetate (Sigma, N-8505) in 0.8 ml of acetone were mixed. The final pH of the incubation solution was adjusted to 5.8 with 1 M NaOH. The blood smears were incubated in the freshly prepared incubation solution for 2 h at 37
C. After incubation, the smears were rinsed in distilled water and counter-stained for 10 min with 1% methyl green (Merck, C.I.N. 2585) prepared in 0.1 M acetate buffer (pH 4.2). The slides were dehydrated and mounted with synthetic mounting medium (Entellan, Merck). On each specimen, 200 lymphocytes were evaluated for ANAE positivity and those having 1 to 4 reddish-brown specific granules were considered as ANAE-positive T-lymphocytes. The results were expressed as positivity percentages (%). RESULTS The greatest plasma boron concentrations were found in groups 5 and 6, while the lowest concentrations were determined in groups 1 and 2 (Table 3). Other plasma mineral concentrations were also affected by vitamin D3 and boron additions. The addition of 25 mg/kg boron (groups 5 and 6) caused significant increases in plasma copper concentrations (P < 0.001). Also, 5 mg/kg boron supplementation of the diets inadequate and adequate in vitamin D3 increased the plasma copper concentrations compared with those in group 1. There were similar increases in plasma iron concentrations (P < 0.001). However, the lowest plasma zinc values were obtained from groups 4 and 5 (Table 3). Boron addition (5 and 25 mg/kg) to the diets containing adequate and inadequate vitamin D3 had no effect on plasma total protein and albumin concentrations but cholesterol concentration was Table 3. Effect of boron supplementation on plasma mineral concentrations in chicks at d 45 Treatment Plasma Vitamin D3 (mg/kg) Boron (mg/kg) 5 25 6.25 50   þ þ       þ þ þ  þ  þ   þ  þ  þ Boron (mg/ml) Iron (mg/ml) Copper (mg/ml) Zinc (mg/ml) 0.045  0.0017c 0.043  0.0021c 0.174  0.0063b 0.171  0.0078b 0.325  0.0214a 0.318  0.0211a 0.42  0.04c 0.58  0.08c 0.82  0.06b 1.29  0.07a 1.39  0.07a 0.78  0.08b 0.27  0.02e 0.31  0.01de 0.36  0.02c 0.35  0.01cd 0.51  0.06a 0.45  0.01b 1.50  0.03c 1.71  0.04b 1.88  0.06a 1.20  0.06d 1.27  0.03d 1.45  0.04c Values represent the mean  SEM of 6 groups of 12 broiler chicks per treatment. a—e Means within columns with no common superscript are significantly different (P < 0.001), according to Duncan’s multiple range test. 91 BORON AND CHOLECALCIFEROL Table 4. Effect of supplementation on some biochemical parameters in chicks at d 45 Treatment Boron (mg/kg) Plasma Vitamin D3 (mg/kg) 5 25 6.25 50   þ þ       þ þ þ  þ  þ   þ  þ  þ Total protein (g/dl) Albumin (g/dl) Cholesterol (mg/dl) 6.56  0.27 6.87  0.40 6.66  0.39 6.41  0.26 6.13  0.31 6.81  0.28 2.84  0.15 2.94  0.13 3.01  0.21 3.04  0.12 2.98  0.14 3.43  0.15 120.06  8.94ab 106.59  8.57b 140.88  10.85a 136.82  9.48a 142.31  11.23a 138.11  7.31a Values represent the mean  SEM of 6 groups of 12 broiler chicks per treatment. a,b Means within columns with no common superscripts are significantly different (P < 0.05), according to Duncan’s multiple range test. Table 5. Effect of boron supplementation on selected histological variables in chicks at d 45 Treatment Boron (mg/kg) Vitamin D3 (mg/kg) 5 25 6.25 50   þ þ       þ þ þ  þ  þ   þ  þ  þ Peripheral blood lymphocyte ANAE, % Splenic plasma cell counts in unit area* (1.44  1204 mm2) Proximal tribal growth plate width (mm) Distal tribal growth plate width (mm) 42.80  2.92 40.50  3.32 37.00  2.87 33.67  2.76 37.63  2.63 32.33  2.72 78.10  2.60b 83.20  1..27a 83.18  1.20a 74.90  1.73b 86.80  0.53a 85.11  0.96a 2386.07  260.60a 2451.92  286.41a 2746.20  404.65a 1714.02  188.79b 1629.39  101.08b 1698.44  149.46b 822.92  90.81ab 902.08  89.94a 607.14  70.35bc 584.82  69.60c 545.08  72.52c 572.17  58.95c Values represent the mean  SEM of 6 groups of 10 broiler chicks each per treatment. a,c Means within columns with no common superscript are significantly different (P < 0.05). *(P < 0.001), according to Duncan’s multiple range test. affected by boron addition (P < 0.01; Table 4). As shown in Table 5, ANAE-positive lymphocyte percentages in the peripheral blood were not influenced by the addition of boron. However, mean plasma cell counts per unit area of spleen (P < 0.001) and width of both proximal and distal tibial growth plates were significantly (P < 0.05) affected by boron supplementation. Supplementation with boron had no effect on tibia weight or tibia ash (% and g DM), but tibial boron (P < 0.01), zinc and calcium (P < 0.05) concentrations were significantly affected by boron addition (Table 6). Table 7 shows that 25 mg/kg boron supplementation of the adequate and inadequate vitamin D3 diets (groups 5 and 6) significantly (P < 0.05) increased the blood haemoglobin content and haematocrit compared with the other groups but other blood parameters were not affected by any of the boron-supplemented groups, as seen in Table 7. DISCUSSION The findings of the present study support the hypothesis that boron has an important biological role that influences mineral metabolism via biochemical and haematological mechanisms. Moreover, the responses of the chicks to boron deprivation were also modified by the changes in dietary vitamin D3 concentrations, in agreement with data from Hunt (1989). The results of the present study suggest that plasma boron concentration is under homeostatic control, and boron seems to have a regulatory role in mineral metabolism but the mechanism of action is not clear. Plasma and tibia boron concentrations increased in a closely related manner to the dietary boron concentrations. Boron supplementation caused greater plasma iron and copper concentrations independently of dietary vitamin D3 concentration. These findings are in agreement with previous results (Hunt, 1989; Nielsen and Shuler, 1992; Hunt et al., 1994; Seaborn and Nielsen, 1994). Boron addition at a concentration of 5 mg/kg to adequate and inadequate vitamin D3 diets seems to decrease plasma zinc concentration. Nielsen et al. (1992) found that 3 mg/kg boron supplementation reduced bone zinc concentration from 131 to 96 mg/kg but a decrease was not found in plasma zinc concentration. Zinc is an essential element for normal skeletal growth and development. Thus, significant changes in plasma and bone zinc concentrations may reflect striking changes in bone characteristics (Nielsen et al., 1992). Also, it was reported that substitution of zinc for 92 Table 6. Effect of boron supplementation on selected variables of tibia in chicks at d 45 Treatment Boron* Vitamin D3 (mg/kg) 5 25 6.25 50   þ þ       þ þ þ  þ  þ   þ  þ  þ Iron Zinc (mg/g dry weight) 1.05  0.16c 0.98  0.12c 1.64  0.17ab 1.50  0.27ab 2.11  0.23a 2.04  0.34a 77.56  4.70 80.85  6.46 73.37  6.34 75.77  4.11 75.61  6.45 79.95  6.11 Values represent the mean SEM of 6 group of 10 broiler chicks per treatment. Means within columns with no common superscript are significantly different (P < 0.05). *(P < 0.01), according to Duncan’s multiple range test. DM ¼ dry matter. a—c Copper 1.21  0.26 1.24  0.15 1.25  0.22 1.27  0.19 1.31  0.22 1.40  0.19 135.88  3.10ab 138.07  2.81a 127.56  2.20bc 129.00  2.22bc 124.48  3.29c 125.70  4.08c Calcium Tibia weight Ash (mg/g dry weight) (g DM) (% DM) (g DM) 114.58  3.41c 116.87  2.18bc 125.77  3.47ab 127.77  4.43a 129.06  4.23a 128.17  3.56a 5.62  0.28 5.72  0.24 4.94  0.18 5.47  0.19 6.03  0.35 5.57  0.22 53.43  0.78 53.97  1.97 55.30  0.60 56.36  0.44 54.43  0.34 55.83  0.45 2.99  0.14 3.06  0.08 2.73  0.09 3.08  0.11 3.28  0.19 3.11  0.12 F. KURTOĞLU ET AL. Boron (mg/kg) Tibia Table 7. Effect of boron supplementation on selected whole blood variables of chicks at d 45 Boron (mg/kg) Whole blood Vitamin D3 (mg/kg) 5 25 6.25 50   þ þ       þ þ þ  þ  þ   þ  þ  þ Haemoglobin counts (g/100 ml) Haematocrit counts (%) WBC counts (103/mm3) RBC ratio (106/mm3) Thrombocyte ratio (105/mm3) Lymphocyte ratio (%) Monocyte ratio (%) Eosinophil ratio (%) Heterophil ratio (%) Basophil ratio (%) 10.27  0.60c 10.66  0.66bc 10.52  0.71bc 11.00  0.54bc 12.55  0.80ab 13.43  0.92a 32.50  1.37c 34.10  1.28bc 33.20  1.61bc 34.50  1.96abc 38.10  1.99ab 39.60  2.12a 44.14  1.84 45.60  1.75 43.78  1.69 45.33  2.42 42.98  1.84 44.41  1.95 2.59  0.040 2.62  0.072 2.61  0.062 2.66  0.089 2.80  0.129 2.85  0.141 0.31  0.014 0.33  0.020 0.35  0.019 0.34  0.027 0.32  0.023 0.33  0.036 60.90  2.02 59.40  2.17 60.30  1.62 61.40  2.29 62.80  1.62 59.60  2.08 3.40  0.22 3.60  0.27 3.50  0.31 3.70  0.21 3.40  0.34 3.30  0.21 1.50  0.31 1.60  0.27 1.40  0.22 1.60  0.31 1.70  0.21 1.50  0.22 31.70  1.98 32.80  2.07 32.10  1.72 30.80  2.09 29.70  1.70 33.00  1.84 2.50  0.27 2.60  0.16 2.70  0.21 2.50  0.17 2.40  0.22 2.60  0.22 Values represent the mean SEM of 6 groups of 10 broiler chicks per treatment. Means within columns with no common superscript are significantly different (P < 0.05) according to Duncan’s multiple range test. a—c BORON AND CHOLECALCIFEROL Treatment 93 94 F. KURTOĞLU ET AL. calcium or enhanced zinc binding may explain the close relation between zinc accumulation and calcium depletion in osteoporotic bone (Aitken, 1976). It was concluded that both low plasma and tibia zinc concentrations and conversely greater tibia boron and calcium concentrations of boron-supplemented chicks (Table 6) might be a characteristic of normal bone. Detectable boron concentrations were found in the tibia and plasma even of borondeprived chicks (groups 1 and 2) and tibia boron concentrations were especially increased in vitamin-D3-deficient groups. These findings are in accord with the results of Hunt (1989). No differences in bone weight and ash content of the tibia were found between the 5 and 25 mg/kg boron-supplemented groups. Rossi et al. (1993) reported similar findings in their studies with diets containing 0, 60, 120, 180, 240 and 300 mg/kg boron. Armstrong et al. (2000) also reported that there was no difference in bone ash of pigs fed on diets with 5 and 15 mg boron/kg. Wilson and Ruszler (1998) also failed to find any significant effects of 0, 50, 100, 200 or 400 mg/kg boron addition on bone ash content. In contrast, Wilson and Ruszler (1997) found significant differences in bone ash of growing pullets fed on diets supplemented with 50, 100 and 200 mg/kg boron, the highest value for bone ash (34.9%) being obtained in the 50 mg/kg boron group. Similarly, Qin and Klandorf (1991) reported results from three experiments that the addition of 100 mg boron/kg of the diet of broiler breeder hens significantly increased the tibial bone ash. Also, Wilson (1991) reported that the shear-breaking strength increased with bone ash content in laying hens. In the present study, although boron supplementations (5 and 25 mg/kg) were lower than in the other reports (Qin and Klandorf, 1991; Wilson, 1991; Wilson and Ruszler, 1997), the results indicated that bone ash content was not correlated with boron supplementation concentrations. Boron supplementation significantly affected plasma cholesterol concentrations in the present study. Increases in cholesterol concentration with boron addition agree with Hunt (1989) who reported that 3 mg/kg boron supplementation significantly increased plasma cholesterol concentration. However, Seaborn and Nielsen (1994) reported that 500 mg/kg boron added to the food of male rats significantly reduced plasma cholesterol concentrations. In a later report (Armstrong and Spears, 2001), the blood cholesterol concentration of barrows was not affected by 5 or 15 mg/kg boron addition. Since specific lipoproteins might be masked in the measurements of total plasma lipids, there is a need to measure the specific lipoproteins such as HDL and LDL cholesterol in these metabolic changes in chicks. In the boron-supplemented groups with adequate vitamin D3, both proximal and distal tibial growth plates narrowed significantly. Although this might mean that the growth rates of the animals were limited, it might also show that the animals had healthy bones. There are only a few studies reporting a close relationship between boron and blood parameters such as haematocrit (HCT) and haemoglobin (Hb) content and counts for leucocytes, erythrocytes and monocytes. In the present study, it was found that Hb and HCT amounts significantly increased in the 25 mg/kg boronsupplemented group. However, the elevation of plasma iron concentration was much greater than the increase in plasma Hb in all groups. Mean corpuscular volume (MCV) (data not shown) may be a better indicator than Hb or HCT values for plasma iron concentrations. Leucocyte counts, red blood cell counts, thrombocyte counts and their peripheral blood percentages were not affected by any level of boron supplementation. Increases in HCT and Hb values for the 25 mg/kg group may be related to elevated plasma iron content (expect from group 6) and copper concentrations obtained from the 5 and 25 mg/kg boron supplementation groups. However, it can be concluded that high Hb values are not necessarily beneficial in fast growing broiler chicks and may in fact be symptomatic of cardiopvascular stress. Also, increased resistance to blood flow through the lung can cause pulmonary hypertension in broilers (Crespo and Shivaprasad, 2003). Hunt (1989) reported that 3 mg/kg boron addition to cholecalciferol-deficient chicks had no effect on HCT or Hb values. However, Seaborn and Nielsen (1994) found that HCT and Hb concentrations of male rats were significantly depressed by high dietary additions (500 mg/kg) of boron. Armstrong et al. (2001) showed that 5 mg/kg boron supplementation caused a significant decrease in inflammatory response against intra-dermal injections of phytohaemagglutinin (PHA). Similarly, in a later study, Armstrong and Spears (2003) found that pigs fed long term with 5 mg/kg developed a decreased inflammatory response following PHA injection. Interestingly, TNF- and IFN- production by monocytes increased in the boron-supplemented group. Nevertheless, boron did not affect the mitogenic response of lymphocytes to mitogenic stimulation or the humoral immune response against a sheep red blood cell suspension. Although the results of others (Armstrong et al., 2001; Armstrong and Spears, 2003) did not explain the reduction in localised inflammatory response following antigen challenge in pigs, these authors BORON AND CHOLECALCIFEROL concluded that boron may affect the inflammatory response as well as serum thyroid hormone concentrations and growth. In the present study, boron supplementation did not significantly affect the proportions of ANAE-positive lymphocytes in the peripheral blood. However, splenic plasma cell counts significantly increased in the 25 mg/kg boron-supplemented adequate and inadequate vitamin D3 groups. This result shows that boron supplementation has no depressive effect on the chicken humoral immunity and is in accordance with the finding that boron did not affect the mitogenic response of lymphocytes to mitogenic stimulation or the humoral immune response against a sheep red blood cell suspension in pigs (Armstrong and Spears, 2003). In conclusion, evidence from the present study suggests that boron supplementations increased bone calcium and reduced bone zinc contents, reflecting a healthy bone condition, especially in vitamin-D3 deficient groups. Also, supplementation of 25 mg/kg boron for inadequate vitamin D3 groups caused significant increases in serum iron and copper concentrations. However, the possibility of cardiovascular problems, perhaps indicated by increased Hb and HCT levels in fast growing broilers, must be considered. In general, the findings of the present study support the hypothesis that boron has an important biological role, influencing mineral metabolism by biochemical and haematological mechanisms. ACKNOWLEDGEMENT The authors wish to thank Dr M.E. Tekin for assistance with the statistics. REFERENCES AITKEN, J.M. (1976) Factors affecting the distribution of zinc in the human skeleton. Calcification and Tissue Research, 20: 23—30. 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