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The effects of boron supplementation of layer diets varying in calcium and phosphorus concentrations on performance, egg quality, bone strength and mineral constituents of serum, bone and faeces a

b

K. Küçükyilmaz , R. Erkek & M. Bozkurt

c

a

Department of Animal Science, Faculty of Agriculture, Eskişehir Osmangazi University, Eskişehir, Turkey b

Department of Animal Science, Faculty of Agriculture, Ege University, İzmir, Turkey

c

Erbeyli Poultry Research Institute, Aydın, Turkey Accepted author version posted online: 20 Oct 2014.Published online: 18 Dec 2014.

Click for updates To cite this article: K. Küçükyilmaz, R. Erkek & M. Bozkurt (2014) The effects of boron supplementation of layer diets varying in calcium and phosphorus concentrations on performance, egg quality, bone strength and mineral constituents of serum, bone and faeces, British Poultry Science, 55:6, 804-816, DOI: 10.1080/00071668.2014.975782 To link to this article: http://dx.doi.org/10.1080/00071668.2014.975782

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British Poultry Science, 2014 Vol. 55, No. 6, 804–816, http://dx.doi.org/10.1080/00071668.2014.975782

The effects of boron supplementation of layer diets varying in calcium and phosphorus concentrations on performance, egg quality, bone strength and mineral constituents of serum, bone and faeces K. KÜÇÜKYILMAZ, R. ERKEK1,

AND

M. BOZKURT2

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Department of Animal Science, Faculty of Agriculture, Eskişehir Osmangazi University, Eskişehir, Turkey, 1Department of Animal Science, Faculty of Agriculture, Ege University, İzmir, Turkey, and 2Erbeyli Poultry Research Institute, Aydın, Turkey

Abstract 1. A 2 × 3 factorial arrangement of treatments was used to investigate the effects of dietary calcium (Ca), phosphorus (P), and supplemental boron (B) (0, 75, and 150 mg/kg) on the performance, egg quality, bone strength, and mineral constituents in bone, serum and faeces. 2. A reduction by 18% in the dietary Ca-P concentration from the recommended levels for the hen strain reduced (P < 0.01) faecal excretion of ash, Ca and P concentrations, and shear force with stress of the tibia in association with decreased feed intake, whereas improved albumen height and Haugh unit values in the egg. 3. Supplemental B significantly decreased the feed consumption, egg weight and final body weight in hens, as well as the albumen height, but had no effect on either the biomechanical characteristics of bones or the mineral profile of the bones and serum. However, there was a significant increase in the egg production rate and a reduction in the damaged and shell-less egg ratio, and in the feed conversion rate in hens fed adequate Ca-P with 150 mg/kg B compared to those of the unsupplemented controls. 4. The amount of B that accumulated in the bones and serum was correlated with the amount of B consumed. B increased the faecal excretion of ash, Ca and B. In general, dietary variables had no effect on mineral composition of serum and tibia. 5. The magnitude of the response to dietary B was much more pronounced in hens fed a diet deficient in Ca-P with 75 mg/kg B; these hens exhibited a production performance and an egg quality comparable to those given adequate Ca-P with no added B. 6. The data presented in this study describing the measured bone properties did not corroborate the hypothesis that B is a trace element playing an important role in mineral metabolism and bone strength through an interaction with Ca, P and Mg.

INTRODUCTION The metabolic and structural function of calcium and phosphorus in bone and eggshell formation is essential in laying hens. Supplying the hen with an optimal Ca intake is crucial for the proper calcification of the eggshell. Boron (B) has been known to be an essential element for plants since the 1920s, and there is now considerable evidence that it may also be essential for animals and humans. Plenty of data support the hypothesis that B is an essential

element and that it is involved in regulating parathormone action. Therefore, it is likely that B influences the metabolism of calcium, phosphorus, magnesium and cholecalciferol (Nielsen and Dietz, 1990; Wilson and Ruszler, 1996; Devirian and Volpe, 2003). Studies have demonstrated that B interacts with other nutrients and plays a regulatory role in the metabolism of minerals such as calcium, and subsequently bone metabolism, but the mechanism has not yet been clearly established (Nielsen et al., 1987; Brown et al., 1989; Hunt, 1989; Hegsted et al., 1991;

Correspondence to: K. Küçükyılmaz, Department of Animal Science, Faculty of Agriculture, Eskişehir Osmangazi University, Eskişehir, Turkey. E-mail: [email protected] Accepted for publication 26 August 2014.

© 2014 British Poultry Science Ltd

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BORON AND DIETARY CALCIUM-PHOSPHORUS LEVEL

Chapin et al., 1998). The particular mechanism through which B influences bone development is described as enhancing the macro mineral content of normal bone and some indices of growth cartilage maturation (Hunt et al., 1994). The effect of the dietary B given to laying hens on egg production performance, egg quality and bone mineralisation has been investigated by several researchers (Wilson and Ruszler, 1997, 1998; Mızrak et al., 2010; Olgun et al., 2013). In general, these researches have shown that B may affect the egg production performance, egg quality and bone mineral content of hens supplemented with up to 200 mg/kg, but egg production, feed consumption, feed conversion ratio and body weight were suppressed at a dietary B concentration of 400 mg/kg (Wilson and Ruszler, 1996; Eren, 2004). However, the magnitude of the positive response was much more pronounced in hens fed on diets with optimal concentrations of Ca and P. Preliminary studies with broiler chicks showed that B may improve growth performance and bone ash by increasing the availability of minerals when they were subjected to nutritional deficiency with Ca, Mg, P and Vit D (Nielsen and Dietz, 1990; Elliot and Edwards, 1992; Kurtoğlu et al., 2005; Bozkurt et al., 2012). However, the mechanism by which B modifies the use of these minerals when diets are lacking in these minerals has not been investigated in a laying hen model. Hence, there is a need to more clearly define the biochemical mechanism through which B could compensate for the dietary Ca and P deficiency in laying hens. The low Ca bone resorption hypothesis is based on the belief that when a bird becomes deficient in dietary Ca, this, in turn, stimulates the absorption and use of ingested Ca (Ballard and Edwards, 1988). The extent to which B could compensate for the deprivation of Ca as well as P under such circumstances in a layer hen model has not been evaluated. B satiety increases the serum ionised calcium, lowers the serum calcitonin concentrations, and improves bone mineralisation and strength by acting as a helper and/or facilitator to maintain bone integrity through its action on Ca and Mg (Nielsen and Dietz, 1990; Naghii and Samman, 1997). One hypothesis to explain the increase in bone adsorption may be the reaction of the borate anion in the molybdate assay, which may result in an apparent increase in P (Armstrong and Spears, 2001). The objective of the present study was to evaluate the effect of the addition of B at 75 and 150 mg/kg to a layer diet modified for Ca and P (at the recommended concentration and lower than the recommended concentration) on egg production performance, egg quality, the compositional and functional properties of bones, and the mineral content of the serum, tibia and faeces.

805

MATERIALS AND METHODS Birds and housing A total of 576 commercial white layer hens (Super Nick), 46 weeks old with a uniform body weight, were randomly assigned to one of the 6 groups of 144 hens each. The experiment used a 2 × 3 factorial design consisting of two types of feed, namely adequate and deficient in Ca and P, and 3 diets supplemented with 0, 75, or 150 mg/kg of B. Four replicates were randomly assigned to each of the 6 dietary treatments. The experimental house was a three-tier cage facility, and the hens were housed six per cage (60 × 50 × 56 cm). The hens in four adjacent cages were considered an experimental protocol. The cages were located in a well-ventilated, open-sided house situated between 36° and 38° northern latitude and 26° and 28° eastern longitude, in Aydın, Western Turkey. The experiment took place over a 20-week period between October 2009 and February 2010. A 16-h photoperiod (from 6 a.m. to 10 p.m.) and 8 h of darkness/24 h were provided. Routine farm practices were followed throughout the experimental period. All the birds were given the respective experimental feeds ad libitum from 46 to 65 weeks of age. The Adnan Menderes University Animal Care and Use Committee approved the techniques and procedures involved in the animal care and handling. Experimental diets Experimental feed mixtures were prepared with maize, wheat, soybean meal, and sunflower meal. To establish the experimental deficiency, dietary Ca and available P concentrations were reduced by 17 and 19%, respectively, from those values recommended by the breeder for this age period. This diet was described as the DEF diet (for having a Ca and P deficiency), while the diet including the recommended concentrations of Ca and P is denoted as the REC diet throughout the paper. The analysed Ca and available P concentrations in the REC diet were 3.94% and 0.42%, respectively, which corresponded to 3.28% Ca and 0.34% P in the DEF diet. B was supplemented in both the REC and DEF diets at the concentration of 0, 75, or 150 mg/kg. Boric acid (H3BO3) was used as the B source and was provided by a government institute (BOREN, Ankara, Turkey) engaged in B research. The boric acid contained 18.03% B; thus, 417 g and 834 g boric acid per ton of basal diet were included to supply B at the 75 mg/kg and 150 mg /kg concentrations, respectively. Boric acid was added to an equal amount of fine ground soybean meal and homogenised by mixer, and then the pre-mixture was added to main

806

Table 1.

K. KÜÇÜKYILMAZ ET AL.

Ingredients and chemical composition of the diets

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Ingredients (g/kg) Maize meal Wheat meal Soybean meal (48% CP) Sunflower meal (31% CP) Soybean oil Limestone Dicalcium phosphate Sodium chloride Vitamin-mineral premix3 DL-methionine L-lysine HCl Sodium bicarbonate Choline chloride Wheat bran4 Chemical composition, g/kg Dry matter Crude protein Ether extract Crude fibre Crude ash Calcium Total phosphorus Boron (mg/kg) Metabolisable energy MJ/kg Calculated composition, g/kg Available phosphorus Lysine Methionine+cystine

REC diet1

DEF diet2

405.91 200.00 168.06 80.00 35.84 86.44 15.04 2.17 2.50 1.35 0.86 0.50 0.50 0.83

441.29 200.00 162.09 80.00 25.14 71.69 11.04 2.17 2.50 1.30 0.95 0.50 0.50 0.83

890.4 160.5 60.7 46.3 127.3 39.4 6.6 2.71 11.55

887.8 161.2 51.2 46.8 109.7 32.8 4.6 3.15 11.52

4.2 8.0 6.8

3.4 8.0 6.8

1

Contains recommended concentrations of Ca and available P. Contains lower concentrations of Ca and available P than those recommended. 3 Provides per kg of diet: trans-retinol 12 000 IU, cholecalciferol 2400 IU, α-tocopherol acetate 30 mg, vitamin K3 2.5 mg, vitamin B1 3 mg, vitamin B2 7 mg, vitamin B6 5 mg, vitamin B12 0.02 mg, nicotine amide 40 mg, calcium D-pantothenate 10 mg, folic acid 1 mg, D-biotin 0.050 mg, choline chloride, 125 mg, vitamin C 50 mg, Mn 80 mg, Fe 40 mg, Zn 60 mg, Cu 5 mg, Co 0.1 mg, I 0.4 mg, Se, 0.15 mg, antioxidant 10 mg. 4 B was included in the diet by replacing the same amount of bran in the basal diet. 2

mixture. Boric acid was included in the diet by replacing the same amount of bran in the basal diet. The basal mash diet was prepared every 2 weeks and was stored in sacks in a cool place. The ingredient and chemical composition of the basal diets are presented in Table 1. The basal diets adequate and deficient in Ca-P included 2.71 and 3.15 mg/kg B, respectively. The analysed values of B in the REC diets supplemented with 75 and 150 mg/kg B were 72.28 and 151.02 mg/kg, respectively, which corresponded to 75.19 and 149.34 mg/kg, respectively, in the DEF diet. The experimental diets were in mash form and met the nutrient requirements for layer hens according to the recommendations of the breeder (Nick Chick, Commercial Management Guide, 2008). The diets were isoenergetic and isonitrogenous. The hens were given 2 weeks to acclimate to the experimental diets. The standard techniques of the proximate analysis were used to determine the nutrient concentrations in the diets (Naumann and Bassler, 1993). The experimental diets were analysed for

starch, sugar, total calcium and phosphorus according to chemical analysis methods for feedstuff established by the Association of German Agricultural Analysis and Research Institutes (VDLUFA) (Naumann and Bassler, 1993). The metabolisable energy content of the diet was calculated from the chemical composition (Anonymous, 1991). The basal diets and the B-added experimental diets were analysed for B. The ground samples were dry-ashed (AOAC, 1990), and the concentrations of B were measured at a specific wavelength for this element (B, 249.677 nm) with an ICP (Perkin Elmer Optima 2100 DV). The calibration for the mineral assay was conducted with a series of mixtures containing graded concentrations of standard solutions (Merck, 170307 B ICP standard). Performance and egg quality parameters All hens were weighed individually at 46 and 65 weeks of age. The hen/d egg production (%) and the cracked-broken and shell-less egg ratio were recorded daily from 46 to 65 weeks of age. The shell-less egg ratio (%) was calculated by dividing the total number of eggs without shells (an egg without a shell but with an intact membrane) by the total number of eggs in each treatment. During this period, a random sample of 32 eggs/treatment/d was collected on three consecutive d every week (8 eggs per replicate per day). Therefore, a total of 1920 eggs were weighed in each treatment to determine the average egg weight throughout the trial. The feed consumption and feed conversion ratios were determined at 7-d intervals. The feed conversion ratio was expressed as kg of feed consumed per kg of egg produced (kg feed/kg egg). The egg mass was calculated by multiplying the egg weight by the egg production rate. All production variables were determined on a replicate basis. The magnitude of the production variables, such as feed intake and egg production, were adjusted for hen mortalities, which were recorded daily as they occurred. An additional sample of 24 eggs was randomly collected from each experimental group (6 eggs per replicate) every 28 d to assess the eggshell quality parameters. Therefore, 720 eggs in total were analysed for egg quality. The egg shape index was determined with equipment that measures the width:length ratio as a percentage. The eggshell quality characteristics were the eggshell weight, strength and thickness. The eggshell weight was defined as a percentage of the egg weight. The eggshell thickness (without the inner and outer shell membranes) was measured at three different points (top, middle, and bottom) using an ultrasonic micrometer (SANOVO Technology A/S, Odense NV, Denmark) without

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BORON AND DIETARY CALCIUM-PHOSPHORUS LEVEL

cracking the eggshell. The eggshell thickness was defined as an average of three different thickness measurements of an egg. The eggshell strength was measured using electronic eggshell tester equipment (Egg Force Reader, SANOVO) and expressed as a unit of compression force exposed to a unit of eggshell surface area (kg/cm2). Then, the eggs were cracked, and the eggshell was carefully separated; finally, the albumen height was obtained using a micrometer with an ultrasonic wave system (SANOVO). The albumen height, Haugh unit, and yolk colour score were described as the internal egg quality parameters. The Haugh unit was calculated using Rousch’s formula (1981). The egg yolk with the albumin was placed on a tray, which was enclosed in a completely dark module of the egg quality measuring equipment (SANOVO), and then the intensity of the yolk colour was compared with matching colour numbers in the Roche yolk colour fan (Vuilleumier, 1969) within 4 seconds. Serum biochemical components

Blood samples were collected by cardiac puncture and placed into non-additive blood collection tubes in order to separate the serum. The sera were separated by centrifugation at 1800 × g after one hour of incubation at room temperature and stored at −20 ºC until the analysis. The serum Ca, inorganic P, Mg, Fe, and Cu concentrations, as well as the alkaline phosphatase (ALP) and alanine transaminase (ALT) activities, were measured with a spectrophotometer (Hitachi, 911) using commercially available kits (Bayer). The B concentration in the blood was measured using ICP-OES (Perkin Elmer Optima 2100 DV) (Laakso et al., 2001). The calibrations for the B assays were conducted with a series of mixtures containing graded concentrations of standard solutions (Inorganic Ventures, CGB1-1B ICP Standard).

Measurement of bone mechanical properties Hens were killed by cervical dislocation, and then the left and right tibias with some attached flesh were collected. Bones were excised from the fresh carcasses, and all flesh and proximal cartilages were removed. While the right tibias were used for the determination of bone ash and mineral content, the left ones were used for measuring the bone mechanical properties. The tibias were individually sealed in plastic bags to minimise moisture loss. The sample bags were placed in a plastic container and stored at −20ºC until analysis. The bones were thawed at room temperature for 6 h in an air-conditioned room before the measurements began. The bone mechanical properties were determined from the load-deformation curve

807

generated from a three-point bending test (ASAE Standard S459, 2001) using an Instron Universal Testing Instrument (Model 1122; Instron, Canton, MA) and the Test Works 4 software package (version 4.02; MTS System Corporation, Eden Prairie, MN). The crosshead speed was constant at 5 mm per min. The fullscale load of the load cell was 5000 Newtons (N). Shear tests were performed on the tibias using a double shear block apparatus. The shear force was exerted over a 6.35 mm (0.25 inch) section located at the centre of the diaphysis. These tests resulted in the ultimate shear force and shear stress being evaluated for each bone. The bone diameter was measured with a caliper (0.01 mm). The average wall thickness (cortex thickness) of the tibia was measured at two points on the central axis of the broken tibia used in determining the mechanical properties using digital calipers with a precision of 0.001 mm. These mechanical properties of bone are described by Wilson and Ruszler (1996) and Armstrong et al. (2002). Preparation of tibia and faeces for mineral analysis At the beginning of 65 weeks of age, twelve birds per treatment (two hens per replicate whose body weight was close to group mean) were randomly selected and transferred to an offsite cage facility. The birds were placed in individual cages that allowed for the collection of faeces. The hens were maintained on their respective experimental treatments and had ad libitum access to feed and water. The hens were allowed to adjust to individual cage management for 4 d, followed by a 3 d total collection of faeces. After the faeces collection procedure was terminated, these hens were used for further analysis including serum biochemical constituents, mineral composition of faeces and bone, and bone mechanical properties. Trays covered with plastic were placed under the experimental cages for collecting excretions twice per day. The faecal samples, which were collected for 3 consecutive d (minimum 500 g/d/treatment), were homogenously mixed and stored at −20 ºC until analysis. Before performing the analyses, the samples were thawed, homogenised, and dried at 100ºC for 24 h in a forced air oven. Three g of dried sample was ashed in a furnace at 550°C for 24 h, and the ash content was measured by dividing the ash weight by the initial weight to describe the percentage weight. Each tibia (i.e., the right ones) was broken into small pieces, weighed, oven-dried at 105 ºC for 24 h, cooled in a desiccator, weighed, dryashed at 600 ºC for 12 h, cooled in a desiccator, and weighed (AOAC, 1990). The ash content was expressed as a percentage of the dry bone weight.

0.0001 0.1146 0.0001 0.8091 0.0001 0.0308 0.8913 0.0001 0.3389 0.5317 0.6078 0.0001 0.0005 0.5374 0.0001 0.0399 0.0001 0.1031 0.7400 0.0418 0.4987 0.0133 0.1343 0.0806 0.0997 0.6028 0.0168 0.0735 0.3999 0.4601 0.22 0.11 0.07 0.92 0.11 0.48 0.60 0.009 14.7 17.6 91.99 1.45a 0.65a 98.95 63.47c 58.30b 107.40b 1.840a 1505.41 1612.81b 92.92 1.70a 0.66a 97.91 64.08b 59.46ab 107.54b 1.806cd 1509.27 1609.27b 93.26 1.56a 0.22b 97.91 64.85a 60.59a 110.14a 1.818bc 1498.64 1632.29ab

a

150

c

SEM

Ca-P

P

75

92.86 1.66a 0.76a 100.00 63.54c 58.89b 106.14b 1.802cd 1496.97 1604.68b

Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Contains recommended concentrations of Ca and available P. 2 Contains lower concentrations of Ca and available P than the recommended concentrations.

93.36 0.97b 0.27b 100.00 63.85b 59.54ab 106.12b 1.781d 1517.08 1616.25b

0

a

150 75

ab

92.31 1.41a 0.77a 98.95 64.57a 59.47ab 109.28a 1.834ab 1529.61 1665.41a

DEF diet2 Supplementary B (mg/kg)

a

1

The egg production performance, body weight and survival of the hens are shown in Table 2. Significant B by Ca-P level interactions characterised the egg production rate, shell-less egg

a–d

Laying hen performance

Egg production rate (%) Cracked-broken egg (%) Shell-less egg ratio (%) Liveability (%) Egg weight (g) Egg mass (g/d) Feed intake (g/d) FCR (g feed/g egg) Body weight at 46 weeks of age Body weight at 65 weeks of age

RESULTS

bc

The experiment used a completely randomised design. The data were analysed with a two-factorial ANOVA using the GLM procedure found in the JMP software (SAS Institute, 2002). The main effects of the Ca-P concentration, B, and B by Ca-P concentration interaction were tested. An arc-sin transformation was applied to the percentage values before testing for differences. Duncan’s multiple range test was carried out to detect differences among the treatments. All differences were considered significant at P < 0.05.

REC diet1

Statistical analyses

Performance parameters of laying hens fed on diets adequate or deficient in Ca-P with or without supplementary boron (B)

The mineral contents of the faeces and tibias of 12 samples per treatment were analysed. The calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), zinc (Zn), copper (Cu), and B (B) concentrations were determined using the following method. Ultrapure HNO3 (5 ml, Merck) was added to each ash sample until it was completely dissolved; afterwards, 20 ml of de-ionised water was added to each sample. The samples were filtered using WH 42 filter paper. The obtained solutions were diluted with de-ionised water to a final volume of 100 ml. The concentrations of minerals were measured at specific wavelengths for each element with an ICP-OES (Perkin Elmer Optima 2100 DV). The calibrations for the mineral assays were conducted with a series of mixtures containing graded concentrations of standard solutions of each element (Merck, 170 308 Ca ICP Standard; Merck, 170 340 P ICP Standard; Merck, 170331 Mg ICP Standard; Merck, 170 326 Fe ICP Standard; Merck, 170 369 Zn ICP Standard; Merck, 170 314 Cu ICP Standard; Merck, 170 307 B ICP standard). The detection and quantification limits of the analytical methodology were determined on the basis of 10 measurements of element concentrations in a reagent blank solution taken during the sample preparation procedure. The spike recoveries typically varied between 92% and 106%. The limits of detection were as follows: Ca, 0.55 mg/kg; P, 0.01 mg/kg; Mg, 0.01 mg/kg; Fe, 0.20 mg/kg; Zn, 0.05 mg/kg; Cu, 0.02 mg/kg; and B, 0.01 mg/kg. The quantification limits for Ca, P, Mg, Fe, Zn, Cu and B were 0.68, 0.01, 0.01, 0.21, 0.05, 0.02 and 0.01, respectively.

Table 2.

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Mineral analysis

B

Ca-P x B

K. KÜÇÜKYILMAZ ET AL.

0

808

0.1297 0.5755 0.0390 0.0002 0.0049 0.8808 0.9800 0.8072 0.0065 0.3751 0.0039 0.0351 0.0016 0.0479 0.2232 0.4624 0.2991 0.1749 0.0004 0.9099 0.0017 0.0105 0.0352 0.0260 0.39 0.19 0.063 0.11 2.23 0.076 0.54 0.086 63.20c 76.59 10.47ab 4.386a 399.8a 6.94bc 81.90ab 5.64a 64.47ab 76.64 10.33b 4.525a 404.8a 7.07ab 82.27ab 5.60ab 1

a–c

63.84bc 76.84 10.51a 4.306ab 403.5a 6.90bc 81.45ab 5.39b 64.25abc 76.68 10.45ab 4.399a 403.1a 6.97abc 81.84ab 5.51ab

Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Contains recommended concentrations of Ca and available P. 2 Contains lower concentrations of Ca and available P than the recommended concentrations.

65.18a 76.26 10.10c 3.875c 389.9b 7.17a 82.84a 5.66a

75 0 150

Supplementary B (mg/kg)

63.76bc 76.60 10.49ab 4.050bc 405.1a 6.82c 80.88b 5.54ab

Ca-P x B B 150

SEM

Ca-P

P DEF diet2 REC diet1

75

Egg weight (g) Egg shape index Egg shell weight (%) Shell breaking strength (kg/cm2) Shell thickness (μ) Albumen height (mm) Haugh Unit (score) Yolk colour (score)

The effects of dietary B supplementation and Ca-P concentration on the external and internal egg quality characteristics are shown in Table 3. The egg-shape index was not affected by these dietary variables (P>0.05). Significant B by Ca-P concentration interactions (P < 0.05) were present for the shell weight, shell breaking strength, and shell thickness. B at both inclusion levels increased (P < 0.05) the eggshell weight, shell breaking strength, and shell thickness in conjunction with the DEF diet, but this positive effect of B disappeared when the hens were given the REC diet. B supplementation reduced (P < 0.05) the albumen

809

0

Egg quality

The effect of dietary modifications with Ca-P and boron (B) on egg quality characteristics

ratio, egg weight, FCR (P < 0.01) and egg mass (P < 0.05). Supplementation with 150 mg/kg B decreased egg production compared with the unsupplemented group in hens given the DEF diet, whereas significant increases were observed when birds were given 150 mg/kg B on a REC diet. B at 150 mg/kg decreased the cracked-broken egg ratio (P < 0.01); however, the effect was more pronounced when the hens were given the REC diet. More cracked-broken eggs were obtained from hens given a diet deficient in Ca-P than those given diets adequate in Ca-P (P < 0.05). The fortification diet with B at both concentrations induced three-fold increases (P < 0.01) in the shell-less egg ratio in hens treated with the DEF diet, while a discrepancy was observed when the hens were administered the REC diet with B at 150 mg/kg. B at 75 and 150 mg/kg reduced (P < 0.01) the egg weight to different degrees in combination with the DEF or REC diet. The egg weights of the hens that consumed the DEF diet with 75 and 150 mg/kg B were 0.87 and 1.38 g lighter, respectively, than those of hens that were not given supplementary B. However, these differences were 1.03 and 0.72 g in hens fed on the REC diet. The relative reduction in egg mass related to boron supplementation was much higher in the DEF diet than in the REC diet. B supplementation induced a decrease (P < 0.01) in the feed intake that was similar for both diets; the observed reduction was approximately 2%. A reduction in the dietary Ca-P concentration increased (P < 0.05) the feed intake of the hens by 1.1%. B at 150 mg/kg worsened (P < 0.01) the FCR when administered the DEF diet, whereas the opposite was true when the hens were fed on the REC diet. The hens responded to both supplemental concentrations of B with a reduction (P < 0.05) in final body weight (65 weeks of age). The survival of the hens through the 20-week period was very good and was not affected (P > 0.05) by the dietary variables tested (i.e., the B and Ca-P concentrations).

Table 3.

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BORON AND DIETARY CALCIUM-PHOSPHORUS LEVEL

0.1608 0.8370 0.0934 0.5798 0.6075 0.6574 0.1995 0.3339 0.3197 0.9116 0.6249 0.2156 0.8387 0.3973 0.9809 0.0058 0.0101 0.3289 0.07 0.02 0.49 25.84 1.50 58.18 6.36 0.62 25.22 521.41b 20.74b 628.46 Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Contains recommended concentrations of Ca and available P. 2 Contains lower concentrations of Ca and available P than the recommended concentrations. 1

6.24 0.59 24.22 521.63b 21.70ab 580.95 6.20 0.62 24.07 583.96ab 24.30a 733.94 6.19 0.66 24.18 573.62ab 24.27a 622.33

Ca-P SEM 150 75 0 150

6.16 0.63 23.86 542.80ab 23.02ab 594.58 a,b

Table 7 presents the ash and mineral contents of the tibia and faeces. While the tibia of the hens did not differ in terms of the Fe content when fed on the REC diet, a significant decrease was observed when the hens were given 150 mg/kg B in the DEF diet (P < 0.05). The Ca, P and Mg concentrations of the faeces were strongly (P < 0.0001) affected by the change in dietary Ca-P, but B only influenced the faecal Ca concentration. Hens consuming the DEF diet excreted less Ca and P than hens fed on the REC diet, but the opposite was observed

6.33 0.62 25.07 605.29a 24.32a 586.58

Ash and mineral contents of tibia and faeces

Bone diameter (mm) Cortex thickness (mm) Profile area (mm2) Shear force (N) Shear stress (N/mm2) Fracture energy (N-mm)

The serum mineral concentration, as well as the ALP and ALT activity were not influenced by the treatments (P>0.05) (Table 6).

75

Serum biochemical constituents

Supplementary B (mg/kg)

The B contents of the serum, faeces and tibia are presented in Table 5. The B concentration in the serum, faeces and tibia substantially increased (P < 0.01) in association with the increased supplemental B. A significant B by Ca-P interaction was found for faeces B concentration; the magnitude of the response was much more pronounced when the hens were fed on the DEF diet with 150 mg/kg B than when the hens were fed on the REC diet. The B concentrations in the faeces and tibia were positively correlated with increased dietary Ca-P concentrations (P < 0.05), but its content in serum showed no response to dietary Ca-P manipulation (P > 0.05).

DEF diet2

B concentration of serum, faeces and tibia

REC diet1

The biomechanical properties of the tibia bone in the laying hens are shown in Table 4. There were no significant differences in the bone diameter, cortex thickness, profile area and fracture energy among the treatment groups (P > 0.05). However, changes in the dietary Ca-P concentration affected the shear force (P < 0.01) and shear stress (P < 0.05) of the bone. The shear stress and shear force of bones from hens fed on the REC diet were significantly higher than for those fed on the DEF diet. These parameters were not influenced by B supplementation (P > 0.05).

Table 4. Measurement and mechanical properties of tibia in laying hens fed on Ca-P deficient diets with or without supplementary boron (B)

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Bone biomechanical properties

P

height, particularly at the 150 mg/kg dose level, while a slight decrease was observed after supplementation with 75 mg/kg B. An 18% reduction in the dietary Ca-P concentration from the recommended levels for the breed improved (P < 0.05) the internal egg quality parameters including the albumen height, Haugh Unit and yolk colour.

B

Ca-P x B

K. KÜÇÜKYILMAZ ET AL.

0

810

2.305b 30.86c 1.23b

0.489c 7.08d 0.47c

4.175a 61.07a 2.07a

150 0.295c 3.60e 0.62c

0

Supplementary B (mg/kg)

28.05 5.15 2.99 0.75 0.21 0.059 506.25 12.00

0

2

2.516b 32.62c 1.50b

75

DEF diet2

4.316a 57.42b 2.22a

150 0.215 0.94 0.11

SEM 0.7664 0.0228 0.0450

Ca-P

26.88 5.11 3.29 0.70 0.18 0.062 499.91 13.41

75

REC diet1

27.31 5.16 3.13 0.76 0.20 0.061 482.50 8.00

150 27.27 5.39 3.21 0.77 0.20 0.061 731.16 10.25

0

Supplementary B (mg/kg)

27.08 5.19 2.77 0.78 0.20 0.058 590.75 7.33

75

DEF diet2

26.47 4.69 2.91 0.73 0.21 0.061 493.25 9.50

150

0.79 0.25 0.16 0.027 0.013 0.0036 83.78 2.46

SEM

0.4688 0.8031 0.9583 0.3955 0.3660 0.7617 0.1164 0.2982

Ca-P

Table 6. Serum biochemical constituents of laying hens fed on diets differing in Ca and P with or without added boron (B)

Contains recommended concentrations of Ca and available P. Contains lower concentrations of Ca and available P than the recommended concentrations.

1

Ca (mg/dl) P (mg/dl) Mg (mg/dl) Fe (mg/dl) Zn (mg/dl) Cu (mg/dl) ALP (IU/l) ALT (U/l)

1

Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Contains recommended concentrations of Ca and available P. 2 Contains lower concentrations of Ca and available P than the recommended concentrations.

a–e

Serum (mg/l) Faeces (mg/kg) Tibia (mg/kg)

75

REC diet1

Boron (B) contents of serum, faeces and tibia of laying hens fed on diets adequate or deficient in Ca-P with or without supplementary B

0

Table 5.

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B

B 0.5708 0.3993 0.2916 0.7433 0.3757 0.9412 0.3003 0.6179

P

0.0001 0.0001 0.0001

P

0.7627 0.3375 0.4229 0.1057 0.3786 0.6462 0.4388 0.3105

Ca-P x B

0.6073 0.0071 0.8458

Ca-P x B

BORON AND DIETARY CALCIUM-PHOSPHORUS LEVEL 811

Ash (%) Ca (%) P (%) Mg (%) Fe (mg/kg) Zn (mg/kg) Cu (mg/kg) Ash (%) Ca (%) P (%) Mg (%) Fe (mg/kg) Zn (mg/kg) Cu (mg/kg)

46.08 15.80 7.59 0.22 65c 142 3.1 20.21b 6.09b 2.44ab 0.61b 575b 339c 43ab

0 45.08 17.21 8.21 0.23 74bc 142 3.2 22.37a 7.43a 2.59a 0.60b 541b 345bc 39b

75

REC diet1

45.64 16.78 7.94 0.22 69c 147 2.3 23.44a 8.07a 2.37b 0.60b 617a 337c 43ab

150 46.02 16.60 7.77 0.24 90a 148 3.1 18.06c 4.79c 2.19c 0.62b 559b 362b 49a

0

Supplementary B (mg/kg)

1

46.59 16.65 8.13 0.23 81ab 141 3.2 17.97c 5.22c 2.13c 0.66a 543b 393a 49a

75

DEF diet2

45.86 16.41 7.99 0.23 76bc 146 2.9 18.72bc 5.53bc 2.07c 0.62b 551b 339c 40b

150 0.71 0.74 0.31 0.008 3.91 6.07 0.62 0.54 0.30 0.056 0.012 13.37 6.65 2.61

SEM

0.3189 0.9409 0.8318 0.0689 0.0002 0.7786 0.7161 0.0001 0.0001 0.0001 0.0020 0.0185 0.0001 0.0603

Ca-P

B 0.8927 0.6145 0.3094 0.9008 0.3695 0.6384 0.5109 0.0028 0.0001 0.0549 0.3031 0.0089 0.0001 0.2343

P

The effect of supplemental boron (B) and dietary regimens with adequate or deficient Ca-P on mineral composition of tibia and faeces in laying hens

Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Contains recommended concentrations of Ca and available P. 2 Contains lower concentrations of Ca and available P than the recommended concentrations.

a–c

Feces

Tibia

Table 7.

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0.5166 0.6127 0.9194 0.5379 0.0389 0.8134 0.8559 0.0409 0.1168 0.1684 0.0807 0.0350 0.0030 0.0357

Ca-P x B

812 K. KÜÇÜKYILMAZ ET AL.

BORON AND DIETARY CALCIUM-PHOSPHORUS LEVEL

for faecal Mg excretion. Significant B by Ca-P interactions was observed for the faecal contents of ash, Fe, Zn, and Cu (P < 0.05). The excretion of Fe through faeces was increased in response to 150 mg/kg B when the hens were fed on the REC diet; however, no such effect was observed with the DEF diet. The faecal excretion of Zn was highest in birds fed on the DEF diet with 75 mg/kg B compared with all other dietary treatments. Supplementation of the DEF diet with 150 mg/kg B induced significant reductions in the faecal Cu concentration, whereas no change was observed for the hens on the REC diet.

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DISCUSSION The significant B by Ca-P interactions characterising most of the performance indices studied indicates that B follows different metabolic pathways in response to modifications of dietary Ca and P levels. In the present study, the most pronounced beneficial effect of both levels of dietary B supplementation was the marked improvement (P < 0.01) in FCR by 2 to 4% when the hens were fed on a diet with adequate Ca and P. The regulatory role of B in the metabolism of Ca, P and Mg (Nielsen and Dietz, 1990; Chapin et al., 1998) may be partly responsible for this improvement, but this effect is most likely due to the role of B in regulating enzymatic activity in pathways involved in substrate metabolism, insulin release, antioxidant action and the immune system. Thus, B may be capable of modifying energy and mineral metabolism in man and animals (Nielsen et al., 1987, 1988). Unfortunately, the role of B in energy metabolism has not been verified by experimental studies in birds. Previous research has not demonstrated improvements in the efficiency of feed conversion in laying hens as a result of B supplementation at doses ranging between 50 and 400 mg/kg (Kurtoğlu et al., 2002; Yeşilbağ and Eren, 2008; Olgun et al., 2009). These findings differ from the present findings, which demonstrated that B leads to a decrease in FCR even at a supplemental level of 75 mg/kg. The reduction in the feed intake (P < 0.01) and final body weight of the hens (P < 0.05) by approximately 3% in response to B addition is noticeable. However, such a reduction in these traits in laying hens was not observed with the supplemental concentrations of B at 400 mg/kg (Wilson and Ruszler, 1996, 1998; Kurtoğlu et al., 2002; Eren, 2004). Reflecting the reduction in feed intake, hens fed 75 and 150 mg/kg B produced eggs that were lighter than those of hens that were not fed supplementary B. Hence, the reduction in all these performance features was associated with a decrease in the feed intake of

813

the birds. However, the process by which B causes reduced feed consumption remains unclear. Overall, survival was in excess of 98% and no treatment-related differences in survival were observed. Thus, B did not have a negative effect on bird mortality in the present study. This lack of effect was also observed in an earlier study in which supplementation with B was tested up to a limit of 400 mg/kg (Wilson and Ruszler, 1998). In this study, another beneficial attribute of B at 150 mg/kg was the reduction in the crackedbroken egg ratio and the shell-less egg ratio in hens fed on the REC diet. This result agrees with the reports of Kurtoğlu et al. (2002) and Yeşilbağ and Eren (2008), who fed hens B at concentrations ranging from 25 to 250 mg/kg. Noticeably, this reduction in the proportion of damaged eggs in this study was not correlated with the measured eggshell quality parameters. Unlike the eggs from hens fed a REC diet, B increased eggshell thickness, shell weight and shell breaking strength when added to a DEF diet. Studies in post-menopausal women verified the increased bioavailability and absorbability of dietary B in conjunction with increased bone mineralisation and the prevention of osteoporosis (Nielsen et al., 1987; Nielsen and Dietz, 1990; Hunt et al., 1997). Based on this evidence, we assumed that the beneficial effect of B on bones would be reflected in shell mineralisation as a result of interactions between B and Ca, P and Mg. This beneficial effect was observed for hens fed on the DEF diet with B, but no effect was observed in hens that consumed the REC diet. One possible explanation for this increase in bone adsorption may be the reaction of the borate anion in the molybdate assay, which may have resulted in an apparent increase in P concentrations. This speculative theory is based on the similarity between the configuration of the borate anion and phosphate anion (Armstrong and Spears, 2001). The deterioration in albumen height and Haugh units, the main determinants of egg quality, with respect to dietary B supplementation is noteworthy. This effect was most likely due to the decline in feed intake induced by B supplementation rather than a direct effect of the B. One promising piece of evidence from this study is that hens fed a diet deficient in Ca and P with 75 mg/kg B had comparable performance indices compared with hens receiving the recommended concentrations of Ca-P with no B without compromising egg quality and bone strength. Therefore, as postulated by Nielsen and Dietz (1990), it may be possible that B has a pronounced influence on mineral metabolism only when animals are subjected to nutritional or other stressors. These observations could open new avenues for research and might have economic and environmental implications because

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814

K. KÜÇÜKYILMAZ ET AL.

the use of B would lead to marked savings when compared to the use of inorganic dietary supplements that are used to supply Ca and P. Studies confirm that bone-breaking strength, bone ash and bone mineral retention, criteria for assessing the bone quality, are increased in layer hens with regard to dietary B supplementation ranging from 50 to 200 mg/kg (Wilson and Ruszler, 1997, 1998). The above evidence suggests that B can also play a vital role in bone development. However, in our study, no improvements in the overall tibia mechanical properties were observed in hens treated with 75 mg/kg B, even those subjected to dietary Ca and P deprivation that was approximately 18%. B is known to interact with Ca, Mg and vitamin D, which are all important in bone metabolism (Nielsen et al., 1988). It is postulated that this interaction is mediated by the influence of B on cell membranes (Naghii and Samman, 1993). However, none of these observations were corroborated by this study. As observed in the case of magnesium deficiency (Nielsen and Dietz, 1990), we believed that supplemental B would alleviate the physiologically adverse effects of dietary Ca deficiency as a result of the ability of B to interact with Ca. Nonetheless, the significant (P < 0.01) reduction in the shear force and shear fracture energy, a measure of bone resistance, could not be ameliorated by dietary B supplementation with 75 or 150 mg/kg when hens were subjected to Ca and P deficiency. This study demonstrated that a moderate reduction in Ca and P relative to the recommended concentrations reduces the bone strength of a modern layer hen strain without compromising bone measurements including the bone diameter, bone wall thickness, and cross– sectional area of tibia. Therefore, the magnitude of the effect of dietary Ca and P concentrations on bone strength was significant, while that of B was not. One possible explanation for the lack of an effect of B on bone mineralisation and strength is that B was naturally acquired from the feed mixture, which was composed of maize, wheat and soybean, at a concentration of 5–6 mg/kg diet. This naturally occurring B may have provided for normal bone development and bone strength. Supplementation of the hen diet with 75 and 150 mg B as orthoboric acid did not influence the mineral concentration of serum and bone. Likewise, the bone ash content, which is a measure of bone density, was not affected by supplemental levels of Ca-P and B in this investigation. The observation that the mineral concentrations of the serum and tibia were unchanged does not conflict with the observations regarding the bone ash and bone mechanical properties, as none of these measures were affected by B. This observation is also supported by the assumption of

Armstrong and Spears (2001) that some compositional changes other than mineralisation may affect the mechanics of bone. While some earlier reports corroborate our findings, other studies produced results that conflict with the findings of this study. In their consecutive works (Kurtoğlu et al., 2005, 2007) no differences in the tibia weight and tibia ash levels were noted after supplementation with B at 50 to 250 mg/kg. Rossi et al. (1993) reported similar findings when using diets containing 0, 60, 120, 180, 240, and 300 mg/kg B. In another study, no significant effect of 0, 50, 100, 200, and 400 mg/kg B addition on bone ash was observed (Wilson and Ruszler, 1998). However, Wilson and Ruszler (1997) and Mızrak et al. (2010) presented evidence that B increased bone ash in laying hens. In the present study, the serum mineral profile was not affected by dietary modification with B or Ca-P. In contrast, B affected the serum mineral constituents of laying hens in previous studies (Qin and Klandorf, 1991; Kurtoğlu et al., 2002; Eren, 2004). Such discrepancies among studies may be due to the use of different experimental protocols, which could have differed in terms of breed, age and reproductive performance of the hens; the composition and nutritive value of the experimental diets; and, most importantly, the concentration of B that was naturally acquired via feed ingredients, hardly evaluated in earlier works. B supplementation had no effect on serum ALP and ALT activities; this finding was consistent with the observations of Eren (2004) and Eren and Uyanik (2007). Nielsen and Dietz (1990) also suggested that B plays a role in Ca metabolism because higher dietary concentrations of B have been shown to decrease faecal Ca excretion and increase plasma ionised Ca concentrations. However, in the present study conducted with layer hens, B increased the faecal excretion of Ca in comparison with the unsupplemented treatment; the difference was more marked in hens fed on the REC diet than in hens fed on the DEF diet. This indicates that B may act well in the presence of nutritional deficiency (Hunt and Nielsen, 1981; Nielsen and Dietz, 1990; Elliot and Edwards, 1992; Kurtoĝlu et al., 2001). Consequently, no deterioration in egg production rate, efficiency of feed conversion and egg quality was observed in the hens fed B. The data describing the serum and bone mineral profiles do not indicate increased circulatory retention or movement into the bone. However, the present data suggest that dietary B may favour eggshell quality indices in hens fed on diets that were moderately (18%) deprived of Ca and P. These findings suggest that dietary B might benefit calcium metabolism through different mechanisms in laying hens and may enable hens to utilise

BORON AND DIETARY CALCIUM-PHOSPHORUS LEVEL

Ca more efficiently in the case of dietary Ca deficiency. The marked reduction in feed intake in response to B intake without affecting feed conversion efficiency is conclusive. The feed-saving effect mediated by B suggests that this element has a role in regulating enzymatic activity in pathways involved in energy substrate metabolism, which merits further investigation.

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WILSON, J.H. & RUSZLER, P.L. (1996) Effects of dietary boron supplementation on laying hens. British Poultry Science, 37: 723–729. doi:10.1080/00071669608417902 WILSON, J.H. & RUSZLER, P.L. (1997) Effects of boron on growing pullets. Biological Trace Element Research, 56: 287–294. doi:10.1007/BF02785300 WILSON, J.H. & RUSZLER, P.L. (1998) Long term effects of boron on layer bone strength and production parameters. British Poultry Science, 39: 11–15. doi:10.1080/00071669 889312 Y EŞILBAĞ, D. & EREN, M. (2008) Effects of dietary boric acid supplementation on performance, eggshell quality and some serum parameters in aged laying hens. Turkish Journal of Veterinary and Animal Sciences, 32: 113–117.