​Boron Supplementation to Calcium Inadequate Diet Influences Mineral Content of Serum, Tissue and Antioxidant Status in White Leghorn Layers

Reports suggest a possible role of Boron (B) in various physiological functions, especially in improving the utilization of minerals like calcium (Ca), phosphorus (P) and magnesium (Mg) (Vijaybhasker et al., 2017). Boron’s distinctive chemical properties allow it to form complexes with organic molecules to influence cellular activity (Park et al., 2005). Reports indicate that dietary B is beneficial in improving Ca level of serum and tibia in broiler chicks fed diets deficient in Vitamin D and Ca (Bozkurt et al., 2012). Multiple roles of B in animal biology still needs further investigation, especially during nutrition stress. Laying hens during peak of egg production are more responsive to dietary changes. Hence, this study was undertaken in layers to understand the influence of B supplementation to diet with inadequate Ca level.

Experimental design, management and diets
 
The study was conducted at ICAR-National Institute of Animal Nutrition and Physiology (NIANP), Bengaluru, India during the year 2017 (April-July). The animal experimental protocol was approved by Animal Ethics Committee of Institute. Eighty White Leghorn breed of layers of 23 weeks of age were procured. During the experiment, the hens were maintained in a well-ventilated layer house, open sided with windows covered with wire mesh. Hens were reared in individual laying cages (Size: 35cm x 35cm x 40cm) fitted with individual feeder and waterer. They were provided with minimum of 12 h artificial lighting. Birds were fed with ad libitum standard layer diet till 24 wk of age and then shifted to experimental diet from 25^th wk of age. Birds were randomly assigned to four treatment groups and each treatment group had 20 hens. The experiment was conducted with corn-soybean meal based layer diet (CP: 161g/kg, ME: 11.2 MJ/kg) with a 2x2 factorial design in a completely randomized design consisting of two diets with adequate in calcium (Normal calcium, NC) and inadequate in Ca (Low Ca, LC), and another 2 diets supplemented with 40 mg/kg of  B to diet adequate in Ca (NC+B) and inadequate in Ca (LC+B), over and above basal level of B. Calcium level (35g/kg) in the standard layer diet was maintained (NC) as per ICAR recommendations (2013) and Ca level was reduced by 10% for Ca-inadequate diet group(LC). Sodium borate (Na₂B₄O₇.10H₂O, Feed grade) was used as B source.
 
Collection of blood and tissue sample
 
At the end of the 16 wk of feeding trial, eight birds from each of the four dietary groups were randomly selected and blood samples were collected by cardiac puncture and placed in to blood collection tubes with no anticoagulant. After one hour, the serum was separated by centrifugation at 1800 x g and stored in deep freezer for further analysis. After blood collection, the same birds were sacrificed by cervical dislocation. Tibia bone and soft tissues (breast muscle, liver) collected were excised and all flesh and proximal cartilage was removed and washed with phosphate buffer saline, collected in polythene covers and preserved in deep freezer.
 
Estimation of serum minerals, biochemicals and total antioxidant activity
 
Serum samples were digested as per procedure described by Kolmer et al., (1951). 1 ml of serum sample with equal volume of concentrated nitric acid was mixed in the digestion tube. The samples were kept overnight at room temperature followed by digestion on low heat (70-80°C) using heating bench, until the volume of samples was reduced to about 1ml. To this 3ml of double acid mixture (3 parts concentrated HNO3 and 1 part 70% H2SO4) was added and digestion continued until the digested samples became watery clear. Final volume of filtrate was made up to 10 ml with Millipore water. Minerals (Ca, P, Mg, B) in digested serum samples were analyzed using inductively coupled plasma optical emission spectrometer (ICP-OES).

The serum alkaline phosphatase (ALP) was estimated with p-nitrophenyl phosphate (p-NPP) substrate method (Linhardt and Walter, 1965), Superoxide dismutase (SOD) activity in serum was determined by the method on the ability of the enzyme to inhibit the auto-oxidation of pyrogallol (Marklund and Marklund, 1974).Total antioxidant activity (TAA) in serum was measured by ferric reducing antioxidant power (FRAP) assay of Benzie and Strain (1999). For estimating total antioxidant activity in liver, frozen samples were thawed at room temperature and around 1.0g of liver sample was minced in homogenizer and prepared 10% tissue homogenate in PBS buffer and then centrifuged at 1800 x g for 15 minutes. The supernatant was collected and used for the analysis of TAA.
 
Estimation of minerals in bone and soft tissue
 
The samples (tibia, breast muscle and liver) were thawed
at room temperature. The soft tissues (liver and breast muscle) were taken in pre- weighed silica crucible and oven dried at 80°C for 24h, later subjected to de-carbonization followed by ashing at 550-600°C for 4h. The residue left in the crucible was taken as total ash which was digested with 5N HCl on a hot plate for 15 minutes and then cooled. The digested sample was then filtered through Whatman filter paper No: 41 in to a 100 ml volumetric flask followed by washing with deionized water and volume was made up to the mark.

The tibia bones of experimental birds were boiled in de-ionized water at 80°C for 30 min and cooled. Later, muscle tissues attached to bone were manually removed and air dried for 24h. Bone samples were solvent extracted in petroleum ether at 60°C for 8h. Mineral extract from bone was prepared as mentioned above. The minerals (Ca, P, Mg and B) were estimated using ICP-OES.
 
Statistical analysis
 
The data of the present study was analyzed using Statistical Package for Social Sciences with two-factorial ANOVA using the GLM procedure found in SPSS (2009) software.

Chemical composition of diets
 
The basal diet contained 11.26 MJ/kg ME and 161 g/kg CP in all the groups and is as per the recommended level for layers. The estimated mineral content in all 4 diets were: Diet 1, Normal calcium diet (NC): Ca 34.6 g/kg, P 6.00 g/kg, Mg 4.60g/kg, and B 19.7 ppm; Diet 2, Low calcium diet (LC): Ca 31.3 g/kg, P 6.50 g/kg, Mg 5.10 g/kg, and B 18.8 ppm; Diet 3, Normal calcium diet with 40 mg/kg B supplementation (NC+B): Ca 34.8 g/kg, P 6.10 g/kg, Mg 4.80 g/kg, and B 57.8 ppm; Diet 4, Low calcium diet with 40 mg/kg B supplementation (LC+B): Ca 31.6 g/kg, P 6.30 g/kg, Mg 5.00 g/kg, and B 57.9 ppm, respectively.
 
Mineral content in serum, bone, muscle and liver
 
The data on mineral content in serum of layer birds is presented in Table 1. In serum, macro minerals such as Ca, P and Mg concentrations did not differ significantly among different dietary groups, while the B content was significantly (P<0.01) higher in groups supplemented with 40 ppm B irrespective of level of Ca. Statistically, similar level of serum Ca, in spite of varied dietary Ca intake in the dietary groups might be due to the homeostatic mechanism involving bone and blood. Kurtoglu et al., (2002) reported that B supplementation (50, 100, 150, 200 and 250 mg/kg) in laying hens increased serum levels of Ca and P. Similarly, Eren et al., (2004) reported that the serum Ca, inorganic P and Mg levels were elevated in the laying hens fed on diets supplemented with boric acid for 8 weeks. Olgun et al., (2012) observed that the addition of boron (60, 120 and 240 mg/kg) to layer ration significantly increased plasma B concentration. Amina et al., (2017) reported that supplementing B at 100, 200 and 300 ppm significantly increased plasma Ca, P and B concentrations in laying hens. The increase of minerals in plasma was attributed to the interaction of B with macro-elements (Ca, P,Mg), thereby modifying their concentration in the plasma of chickens (Kurtoglu et al., 2001, 2005; Bozkurt et al., 2012). Moreover, Kucukyilmaz et al., (2017) confirmed that the addition of B to the diet resulted in significant increase in B concentration of serum. In contrast, Kaya and Macit (2018) reported that B supplementation did not change serum P and Mg level, while decreased serum Ca content in laying hens. Kabu et al., (2013) reported that dietary sodium borate @ 30 g/day in Holstein cattle resulted in higher serum concentrations of Ca and Mg at the time of calving and prevented metabolic disorders. This indicates that B has sufficient influence on Ca,P and Mg utilization.



Boron supplementation to diets increased the P content in bone (P<0.03). The B content in bone was significantly (P<0.01) higher in both the groups supplemented with 40 ppm B with significant (P<0.02) interaction with Ca levels, indicating that bone is a major storage site of B similar to Ca (Table 2). During the active phase of egg laying, much of the Ca in blood is diverted for egg shell formation and this is the probable reason for no significant change in Ca content of bone. The data on mineral content in breast muscle of layer birds is presented in Table 4. Boron supplementation did not significantly influence the Ca and B content of muscle. However, it significantly increased P (P<0.001) and Mg (P<0.019) content of muscle, indicating better gut absorption of P and Mg due to B supplementation (Table 3).

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Boron supplementation to Ca inadequate diet increased the Ca content in liver (Table 4). Feeding diet with inadequate Ca reduced the Ca content in liver and was restored to control value upon B supplementation with significant Ca x B interaction (P<0.04). Feeding diet with inadequate Ca resulted in higher P (P<0.006) and Mg (P<0.001) content in liver, indicating better P and Mg utilization when Ca intake is less. The P (P<0.003) and Mg (P<0.021) content in liver was further increased with B supplementation, confirming that B promotes P and Mg deposition in liver. The interaction effect for Ca (P<0.004) and B (P<0.001) was significant, indicating that B and Ca has a close relationship.

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As per the reports, B is a regulator of mineral metabolism and it is beneficial for Ca metabolism. Hunt et al., (1994) reported that chicks fed on diets with 15.60 ppb (adequate) Vitamin D3 supplemented with B exhibited enhanced content of macro minerals in bone compared to those fed on diet having inadequate Vitamin D3. Kurtoglu et al., (2007) reported that B supplementation in laying hen diets at 50, 100, 150, 200 and 250 mg/kg decreased the tibial Ca level, while gradually increasing the B concentration up to seven-fold without impacting the bone ash. Bozkurt et al., (2012) concluded that B supplementation @ 30 ppm was effective in increasing the Ca, P and ash content of tibia bone in total rearing period of 1-42 days of broiler birds fed on diets deficient in Ca and P.
 
Serum biochemicals and total antioxidant activity
 
Boron supplementation to diets did not significantly alter the TAA in serum and liver, as well as ALP activity in serum (Table 5). However, the reduced SOD activity due to feeding inadequate Ca diet was ameliorated with B supplementation and was restored to control level. Elkomy et al., (2015) reported that supplementing B as boric acid to rabbit significantly increased total antioxidant capacity. El-Saadany et al., (2016) reported that supplementing B @ 100 mg/kg in male chicks improved total antioxidant capacity. Turkez et al., (2007) reported that SOD activity was significantly higher in erythrocyte of human peripheral blood culture exposed to 15 ppm borax. However, few studies indicated that there was no significant difference in SOD activity of erythrocytes (Ince et al., 2010) and liver tissues (Zafar and Ali, 2013) with borax supplementation. Vijay Bhasker et al., (2016) reported that boron supplementation improved SOD activity in rats by upregulating SOD genes. Elkomy et al., (2015) reported that supplementing boron as boric acid at 100 mg/kg feed in rabbit bucks increased SOD activity.

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From the results, it is concluded that 40 ppm B supplementation positively influenced the tissue deposition of Ca, P, Mg and serum SOD activity and the response was enhanced when B was supplemented to Ca inadequate diet.

The authors thankfully acknowledge the support of Director, ICAR-NIANP, Bengaluru in conducting this work.