Effect of Organic Acids and Prebiotics on Bone Quality in Laying Hens Fed Diets with Two Levels of Calcium and Phosphorus

In an experiment conducted on caged Bovans Brown hens, the effect of diet supplementation using organic acids and prebiotic fructans on the biomechanical and geometrical indicators of the tibia and femur bones was evaluated. At 25 weeks of age, layers were randomly assigned to 14 experimental groups, each comprising 6 hens kept in individual cages. A 2 × 7 factorial arrangement, with two dietary levels of calcium and phosphorus (normal – 37.0 g Ca/kg, 6.5 g P/kg, and reduced – 32.5 g Ca/kg, 6.0 g P/kg), and with diets supplemented by selected additives (none; inulin, 7.5 g/kg; oligofructose, 7.5 g/kg; short chain fatty acids (SCFA), 5.0 g/kg; medium chain fatty acid (MCFA), 2.5 g/kg; SCFA, 3.0 g/kg + MCFA, 2.0 g/kg; inulin, 3.0 g/kg + SCFA, 5.0 g/kg) was used. The experiment was conducted for 45 weeks and concluded when the hens were 70 weeks old. At 70 weeks of age, reducing the dietary levels of Ca and P had decreased the bone breaking strength by 8.9% (P ≤ 0.001) and the yielding load by 5.6% (P ≤ 0.05). A similar tendency for bone breaking strength (P ≤ 0.05) and stiffness (P ≤ 0.05) was found in the femur bones. The diet with a lower level of Ca and P negatively affected the geometrical indicators of the bones such as cortex thickness (P ≤ 0.05) and cross section area (P ≤ 0.05), but had no effect on bone weight and length. Hens fed diets supplemented with oligofructose, MCFA, SCFA + MCFA or inulin + SCFA displayed a significantly higher bone breaking strength and yield load in the tibia bone than that of the control group. In the case of femurs, a positive influence of MCFA or simultaneous addition of inulin + SCFA on bone breaking strength was found. The additives had no significant effects on the geometrical indicators of either bone. It was concluded that selected feed additives which lower the pH of the diet and intestinal content can beneficially affect the biomechanical indicators of the bones of high-productive laying hens. Laying hens, bone quality, calcium, organic acids, prebiotic fructans Symptoms of osteoporosis are often observed in modern flocks of high-productive layers, especially in the second part of the laying cycle. Osteoporosis can be defined as a decrease in the fully mineralized structural bone in which Ca is mobilized from the bone in order to contribute to eggshell formation (Whitehead and Fleming 2000). The condition leads to increased bone fragility and susceptibility to fracture. The consequences of this syndrome, also known as ‘cage layer fatigue’, i.e., poor bone quality, weakness, deformities and breakage, spinal bone collapse and paralysis, can be an important welfare problem, causing acute and chronic pain and distress to the birds (Webster 2004). In the United Kingdom, it was found that, in the end phase of lay, 29% of caged hens had sustained one or more broken bones during their lifetime (Gregory and Wilkins 1989). A study conducted by McCoy et al. (1996) attributed 35% of mortality in commercial caged laying hens to osteoporosis. Bone breakage is also a serious problem during the catching and transport of hens prior to slaughter, and during processing, which reduces the marketability of spent caged layers (Gregory and Wilkins 1989). Results of the study carried out by Jendral et al. (2008) indicate that hens caged in conventional cages, where the opportunity for movement and load-bearing exercises is limited, are particularly vulnerable to osteoporosis, exhibiting lower tibia and femur mineral density, bone mass, cortical bone area and mass and bone ACTA VET. BRNO 2010, 79: 185-193; doi:10.2754/avb201079020185 Address for correspondence: Doc. dr hab. Sylwester Świątkiewicz National Research Institute of Animal Science Department of Animal Nutrition and Feed Science ul. Krakowska 1, 32-083 Balice, Poland Phone: +48 666 081 343 Fax: +48 122 856 733 E-mail: sylwester.swiatkiewicz@izoo.krakow.pl http://www.vfu.cz/acta-vet/actavet.htm breaking strength than those kept in furnished colony cages, or cages modified with a nest box and perch. Optimization of nutrition is one of the strategies for prevention of osteoporosis in hens. Because of the high demand for Ca in highly producing layers, the supply of an adequate amount of this macroelement in the diet is the most important nutritional factor influencing bone quality. Use of particulate limestone, as compared to fine particle CaCO3, as a source of Ca for hens was of benefit to bone strength and structure (Koreleski and Świątkiewicz 2004; Fleming 2008). In our earlier experiment, supplementation of the diet with 25-hydroxycholecalciferol, active metabolite of vitamin D3 (25-OH-D3), which is necessary for the hen’s proper Ca metabolism, was also shown to have a positive effect on selected mechanical properties of tibia bones (Świątkiewicz and Koreleski 2005). The results of several studies carried out on rats have indicated that by lowering intestinal pH, such additives as organic acids or prebiotic fructans (inulin, oligofructose) had a beneficial effect on Ca absorption (Lutz and Scharrer 1991; Delzenne et al. 1995; Ohta et al. 1995; Morohashi et al. 1998; Demigne et al. 2008), bone mineralization (Roberfroid et al. 2002; Kruger et al. 2003; Zafar et al. 2004; Nzeusseu et al. 2006; Demigne et al. 2008) and bone architecture measured as the femoral bone volume (Takahara et al. 2000). Sacakli et al. (2006) indicated that the addition of short chain organic acids (lactic + formic acid) to the diet for quail improved utilization of dietary phosphorus and increased the content of crude ash in tibia bones. Inulin or organic acids added to the diet for broilers increased the length of intestinal villus (Rehman et al. 2007; Senkoylu et al. 2007), which might stimulate the absorption of minerals. The aim of the experiment was to study the effect on the biomechanical and geometrical properties of tibia and femur bones when short (SCFA), or medium chain fatty acids (MCFA), or prebiotic fructans with different lengths of chain (inulin or oligofructose) are added to the layer’s diet at different levels of Ca and P. Materials and Methods The Local Krakow Ethics Committee for Animal Experiments gave its approval to all the experimental procedures relating to the use of live animals. Eighty-four, 18-week-old Bovans Brown hens, obtained from a commercial source, were placed in individual cages, on a wire-mesh floor under controlled climate conditions in the poultry house at the Experimental Station of the National Research 186 Table 1. Composition of experimental diets in g/kg Ingredients Control Reduced level of Ca and P

breaking strength than those kept in furnished colony cages, or cages modified with a nest box and perch.
Optimization of nutrition is one of the strategies for prevention of osteoporosis in hens.Because of the high demand for Ca in highly producing layers, the supply of an adequate amount of this macroelement in the diet is the most important nutritional factor influencing bone quality.Use of particulate limestone, as compared to fine particle CaCO 3 , as a source of Ca for hens was of benefit to bone strength and structure (Koreleski and Świątkiewicz 2004;Fleming 2008).In our earlier experiment, supplementation of the diet with 25-hydroxycholecalciferol, active metabolite of vitamin D 3 (25-OH-D 3 ), which is necessary for the hen's proper Ca metabolism, was also shown to have a positive effect on selected mechanical properties of tibia bones (Świątkiewicz and Koreleski 2005).
The results of several studies carried out on rats have indicated that by lowering intestinal pH, such additives as organic acids or prebiotic fructans (inulin, oligofructose) had a beneficial effect on Ca absorption (Lutz and Scharrer 1991;Delzenne et al. 1995;Ohta et al. 1995;Morohashi et al. 1998;Demigne et al. 2008), bone mineralization (Roberfroid et al. 2002;Kruger et al. 2003;Zafar et al. 2004;Nzeusseu et al. 2006;Demigne et al. 2008) and bone architecture measured as the femoral bone volume (Takahara et al. 2000).Sacakli et al. (2006) indicated that the addition of short chain organic acids (lactic + formic acid) to the diet for quail improved utilization of dietary phosphorus and increased the content of crude ash in tibia bones.Inulin or organic acids added to the diet for broilers increased the length of intestinal villus (Rehman et al. 2007;Senkoylu et al. 2007), which might stimulate the absorption of minerals.The aim of the experiment was to study the effect on the biomechanical and geometrical properties of tibia and femur bones when short (SCFA), or medium chain fatty acids (MCFA), or prebiotic fructans with different lengths of chain (inulin or oligofructose) are added to the layer's diet at different levels of Ca and P.

Materials and Methods
The Local Krakow Ethics Committee for Animal Experiments gave its approval to all the experimental procedures relating to the use of live animals.Eighty-four, 18-week-old Bovans Brown hens, obtained from a commercial source, were placed in individual cages, on a wire-mesh floor under controlled climate conditions in the poultry house at the Experimental Station of the National Research 186 39.0 32.5 P 6.6 6.0 1 Premix provided per 1 kg of diet: vitamin A, 10,000 IU; vitamin D 3 , 3,000 IU; vitamin E, 50 IU; vitamin K 3 , 2 mg; vitamin B 1 , 1 mg; vitamin B 2 , 4 mg; vitamin B 6 , 1.5 mg; vitamin B 12 , 0.01 mg; Ca-pantotenate, 8 mg; niacin, 25 mg; biotin, 0.1 mg; folic acid, 0.5 mg; choline chloride, 250 mg; manganese, 100 mg; zinc, 50 mg; iron, 50 mg; copper, 8 mg; iodine, 0.8 mg; selenium, 0.2 mg, cobalt, 0.2 mg. 2 Calculated from tables of feed composition on the basis of component nutrient content. 3According to the Janssen (1989) as a sum of ME content of feed components calculated on the basis of nutrient content.
Institute of Animal Production in Balice, Poland.The cage dimensions were 40 × 40 cm, equalling to 1600 cm 2 of total floor space.During the pre-experimental period, up to the 25 th week of the hens' age, a commercial laying hen diet was offered for ad libitum consumption (170 g crude protein/kg, 11.3 MJl/kg AME N , 37.0 g Ca/kg and 3.8 g available P/kg).
At 25 weeks of age, the hens were randomly assigned to one of 14 experimental treatments comprising 6 individually caged layers for each treatment.During the experiment, the hens were provided with water and feed ad libitum, and were exposed to a 14 L:10 D lighting schedule with the light intensity of 10 lux.
A 2 × 7 factorial arrangement, with two dietary levels of Ca and P, and with the diets being supplemented with experimental additives, was used.The basal experimental diets (Table 1) contained normal (37.0 and 6.5 g/kg) or lowered (32.5 and 6.0 g/kg) levels of Ca and P, respectively.These diets were either unsupplemented or supplemented with additives as follows (per kg of diet): 7.5 g inulin (Beneo TM IPS, Orafti, Belgium), 7.5 g oligofructose (Beneo TM OPS, Orafti, Belgium), 5.0 g SCFA (2.0, 1.5 and 1.5 g of formic, propionic and acetic acid, respectively), 2.5 g MCFA (1.25 g of caproic and 1.25 g of capric acid), 3.0 g SCFA + 2.0 g MCFA or 7.5 g inulin + 5.0 g SCFA.
The experimental diets were fed from 25 to 70 weeks of age.The nutrient content of the diets was calculated on the basis of the chemical composition of raw feedstuffs, and metabolizable energy value in line with equations from the European Tables (Janssen 1989).Samples of feed components were analyzed using standard methods (AOAC 1990) for moisture (method 930.15), crude protein (984.13),crude fat (920.39) and ash (942.05).Amino acids were analyzed in acid hydrolysates, after initial performic acid oxidation of sulphur amino acids and after alkaline hydrolysis of tryptophan (AOAC 1990;method 982.30).The Ca content of ingredients and the diets was analyzed by flame atomic absorption spectrophotometry (AOAC 1990;method 968.08) and the total P content by colorimetry using the molybdo-vanadate method (AOAC 1990;method 965.17).
At the end of the experiment all the hens were sacrificed by cervical dislocation.The tibia and femur from the right leg were collected, cleaned of soft tissues, weighed and frozen (-20 ºC) until analysis.Biomechanical properties of the bones were measured by means of three point bending test using an Instron 5542 testing machine (constant speed of the crosshead -10 mm/min and distance between supports -50 mm).Bone breaking strength and yield load were measured as a graphical record from post deformation curves.Stiffness in elastic conditions was calculated as the yield load/elastic deformation ratio.
Tibia length, cortex thickness, external and internal diameters (for cross-section area calculations) were measured in the breaking place using an electronic slide caliper.The cross-section area was calculated from the equation: 3.14 (HB -hb)/4, where H = external, vertical diameter; B = external, horizontal diameter; h = internal, vertical diameter; and b = internal, horizontal diameter.
The data were statistically analyzed using a completely randomized design in accordance with the GLM procedure of Statistica 5.0 (Statsoft, Inc., Tulsa, OK).All the data were analyzed using two-way ANOVA.When significant differences in treatment means were detected by ANOVA, Duncan's multiple range test was applied to separate means.Significance was considered at P ≤ 0.05.

Results and Discussion
The bone breaking strength of tibias and femurs of Bovans Brown hens obtained in this study (160 and 166 N averaged across all dietary treatments) were lower than the values obtained in our earlier experiments with Hy-Line Brown and Lohman Brown hens of a similar age (Świątkiewicz and Koreleski 2005;2007).These differences may indicate the influence of genetic selection on the biomechanical indicators of bones.Similarly, Newman and Leeson (1999) reported differences in bone quality among four strains of layers.They found the highest value of tibia breaking strength and cross sectional area for Hy-Line and the lowest for DeKalb hens.Riczu et al. (2004) noticed that the bone breaking strengths of the modern brown-egg strain (Shaver 579) were greater by 22% (femur) and 18% (humerus) than in the white-egg strain hens (Shaver 2000).The effects of the hen's genetic line on bone quality were also found by Rennie et al. (1997) who reported that highly productive modern strain layers (Hisex Brown) were more susceptible to osteoporosis than the older J-line Brown Leghorn strain.Clark et al. (2008) found genetic line differences in fracture incidence in layers at 65 weeks of age and concluded that these differences were not related to egg laying performance, but rather to a different Ca metabolism, bone structure and body weight.
Dietary levels of Ca and P and the use of feed additives affected the biomechanical indicators of the tibia (Table 2).Reducing the levels of dietary Ca and P decreased the tibia breaking strength by 8.9% (P ≤ 0.001), the yield load by 5.6% (P ≤ 0.05) and tibia stiffness by 7.6% (P ≤ 0.01).Similar tendency was observed for the bone breaking strength (P ≤ 0.05) and stiffness (P ≤ 0.05) of femur bones (Table 2).Diet with a lower level of Ca and P had a negative effect on geometrical indicators of tibias and femurs such as cortex thickness (P ≤ 0.05) and cross-section area (P ≤ 0.05), but had no effect on tibia and femur weight and length (Tables 3 and 4).The effect of dietary Ca level on hen bone quality was also observed by Schreiweis et al. (2003) who reported that tibia and humerus density, mineral content, bone breaking force and modulus of elasticity decreased as dietary Ca was lowered in the diet.Cheng and Coon (1990ab) found a linear increase in bone ash concentration and bone breaking strength in hens fed Ca, in amounts increasing on a daily basis from 2.0 to 4.5 g.A similar tendency for tibia breaking strength was observed when the dietary Ca concentration increased from 28 to 42 g/kg (Narvaez-Solarte et al. 2006).
Some of the additives used had a positive effect on the biomechanical characteristics of bones.Hens fed diets with the addition of oligofructose, MCFA, SCFA + MCFA or inulin + SCFA had a significantly higher bone breaking strength and yield load of tibia than in the control group (Table 2).In the femurs, the positive influence of MCFA or the simultaneous addition of inulin + SCFA was found for breaking strength (Table 2).There were no significant effects of additives on the geometrical indicators of both bones (Tables 3 and 4).Lack of interaction between experimental factors (Tables 2, 3 and 4 Experimental data on the effect of prebiotic fructans or organic acids on the quality of bones in poultry are limited.Results corresponding to our findings were obtained by Chen and Chen (2004), who noted that supplementing the diet with 10 g/kg of oligofructose or inulin increased the total ash, Ca and P in the tibia of layers.In molted hens, supplementation of alfalfa diet with fructooligosaccharides (7.5 g/kg of diet) prevented a decrease in tibia and femur breaking strength and in tibia mineral content during molting (Kim et al. 2006).The authors of this study have concluded that, probably due to their beneficial effect on Ca absorption, fructooligosaccharides have the potential to maintain bone strength, which is often reduced by structural bone loss during molting.In a study on rats, dehydrated chicory, a rich source of inulin, increased the distal bone mineral density and breaking load and was more effective in this effect than purified inulin (Demigne et al. 2008).
Based their work on a model study with ovariectomized rats, Zafar et al. (2004) indicated that protective effects of fructans on bone were established through increased Ca absorption and Ca balance, increased bone mineralization and decreased bone turnover rate.In an in vitro study, fructooligosaccharides increased the net transport of Ca in the small and large intestines of rats (Mineo et al. 2001).Kruger et al. (2003) reported that the effects of fructooligosaccharides with various degree of polymerization (DP) on Ca bioavailability and bone mineralization in rats were   2007), the mechanism of the positive effect of fructans on mineral utilization complex can be related to such factors as high solubility of minerals because of increased production of short chain fatty acids by probiotic bacteria through an increased supply with substrate (fructans), an alteration of intestinal mucosa and an increase of the absorption surface by beneficial effect of bacterial fermentation products on the proliferation of enterocytes, increased expression of Cabinding proteins, release of bone modulating factors, degradation of phytates by probiotic bacteria enzymes, and overall improvement of gut health.
In our study, the addition of MCFA, and the simultaneous addition SCFA + MCFA or SCFA + inulin had a beneficial effect on the selected biomechanical indicators of the tibia bones.This influence can be probably attributed to improved availability of Ca and P by virtue of a decrease in pH in the upper part of the intestine and the stimulating effect of organic acids on villus height, which was observed in broilers by Senkoylu et al. (2007).It has also been proposed that organic acids (citric acid) improved Ca availability by chelating Ca and reducing the formation of insoluble Ca-phytate-complexes (Boling et al. 2000).
Findings corresponding to our results were obtained by Nezhad et al. (2008) who indicated that tibia mineralization (ash content) in 64-week-old laying hens was  increased after the addition of citric acid.The positive effect of citric acid on the bone crude ash was also found in broilers; however, there was no influence of malic acid or fumaric acid (Liem et al. 2008).Orban et al. (1993) found that the addition of ascorbic acid to the broilers' diet increased the femur breaking strength.In a study on pigs, Radcliffe et al. (1998) observed no effect of citric acid supplementation on bone breaking strength, but reported a tendency (P < 0.08) for a linear increase in bone ash content with an increasing content of acid in the diet.Abdel-Fattah et al. (2008) reported that chicks fed a diet supplemented with organic acids had significantly higher Ca and P blood concentration, which the authors attributed to the lowering of gut pH and the increase in the absorption of these macroelements.Dietary acetic acid increased the Ca absorption and Ca content of the femur of ovariectomized rats suggesting that this acid may reduce the bone turnover caused by ovariectomy and may be helpful in preventing osteoporosis (Kishi et al. 1999).
In conclusion, the results of this study indicate that selected feed additives with the mode of action to lower the pH of the diet and intestinal content could have a positive effect on the mechanical properties of tibia and femur bones in aged, highly producing laying hens.Significant improvement in bone quality was obtained by the use of oligofructose, medium chain fatty acids, short + medium chain fatty acids or inulin + short chain fatty acids.
Effects of experimental factors on biomechanical indicators of tibia and femur bones a, b (x, y) -values in the columns (rows) with different letters differ significantly (P ≤ 0.05); NS -P > 0.05, * -P ≤ 0.05, ** -P ≤ 0.01, *** -P ≤ 0Normal Reduced Mean Normal Reduced Mean Normal Reduced Mean Normal Reduced Mean Normal Reduced used feed additives in improving bone quality is not connected with the dietary level of Ca and P.

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values in the rows with different letters differ significantly (P ≤ 0.05); NS -P > 0.05, * -P ≤ 0. Long chain inulin (DP > 23) increased the bone mineral density and bone mineral content in the femur and the spine to a considerably greater extent than oligofructose.According toScholz-Ahrens et al. (

Table 1 .
Composition of experimental diets in g/kg

Table 3 .
Effects of experimental factors on geometrical indicators of tibia bones x, y

Table 4 .
Effects of experimental factors on geometrical indicators of tibia bones