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The effect of long-term acidifying feeding on digesta organic acids, mineral balance, and bone mineralization in growing pigs
Accepted Manuscript Title: The effect of long-term acidifying feeding on digesta organic acids, mineral balance, and bone mineralization in growing pigs Author: J.V. Nørgaard O. Højberg K.U. Sørensen J. Eriksen J.M.S. Medina H.D. Poulsen PII: DOI: Reference:
S0377-8401(14)00180-1 http://dx.doi.org/doi:10.1016/j.anifeedsci.2014.05.010 ANIFEE 13088
To appear in:
Animal
Received date: Revised date: Accepted date:
6-2-2014 9-5-2014 23-5-2014
Feed
Science
and
Technology
Please cite this article as: Norgaard, J.V., Hojberg, O., Sorensen, K.U., Eriksen, J., Medina, J.M.S., Poulsen, H.D.,The effect of long-term acidifying feeding on digesta organic acids, mineral balance, and bone mineralization in growing pigs, Animal Feed Science and Technology (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Animal Feed Science and Technology Ms. No. ANIFEE-14-5460 Highlights
Benzoic acid had no significant effects on digesta concentrations of organic acids
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Replacing CaCO3 with CaCl2 suppressed organic acids in the stomach and colon
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Urinary pH was reduced by feeding benzoic acid and replacing CaCO3 with CaCl2
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Benzoic acid or CaCl2 alone did not affect bone strength during long-term feeding Bone minerals were reduced when feeding both benzoic acid and CaCl2.
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The effect of long-term acidifying feeding on digesta organic acids,
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mineral balance, and bone mineralization in growing pigs
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J.V. Nørgaarda,*, O. Højberga, K.U. Sørensena, J. Eriksenb, J.M.S. Medinac, H.D.
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Poulsena
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a
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Denmark
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Department of Agroecology, Aarhus University, Foulum, P.O. Box 50, DK-8830 Tjele, Denmark
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Orthopaedic Research Laboratory, Aarhus University Hospital, DK-8000 Aarhus C, Denmark
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Department of Animal Science, Aarhus University, Foulum, P.O. Box 50, DK-8830 Tjele,
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Abbreviations: BA, benzoic acid; CaCl2, calcium chloride; CaCO3, calcium carbonate; DCAD,
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dietary cation-anion difference; dEB, dietary electrolyte balance; dUA, dietary undetermined anion;
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GIT, gastrointestinal tract.
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* Corresponding author at: Blichers Alle 20, DK-8830 Tjele, Denmark. Tel.: +45 87157816. E-mail
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address: [email protected] (J.V. Nørgaard).
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Abstract
Acidification of slurry through dietary manipulation of urinary pH is a means of mitigating
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nitrogen emission from pig production, but long-term effects of diet acidification on bone
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mineralization and mineral balance is less investigated. The objective was therefore to study the
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long-term effects of feeding benzoic acid (BA) and calcium chloride (CaCl2) on the mineral balance
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and microbial activity in the gastrointestinal tract of pigs. Four diets containing the combinations of
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0 or 10 g/kg BA and 0 or 20 g/kg CaCl2 were fed to 24 pigs in a factorial design. For the diets
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without CaCl2, calcium carbonate (CaCO3) was added to provide equimolar levels of Ca. The pigs
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were fed the diets from 36 kg until slaughter at 113 kg BW, and they were housed in balance cages
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for 12 d from 60 to 66 kg BW. Supplementation of BA and/or CaCl2 had only minor effect on
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accumulation of digesta organic acids (acetate, propionate, butyrate and lactate) throughout the
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gastrointestinal tract. A reduction (P<0.01) in digesta concentration was, however, observed for
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acetate and propionate in colon digesta for the CaCl2 supplemented diet. Moreover, CaCl2
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supplementation reduced (P<0.01) the gastric concentration of lactic acid. In the urine, pH was
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reduced by both BA (P=0.04) and CaCl2 (P<0.01). The diet with combined BA and CaCl2 resulted
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in the lowest retentions of both P (P=0.03) and Ca (P<0.01). Metacarpal III bones were lighter
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(P=0.04) and shorter (P=0.02) when replacing CaCO3 with CaCl2, but BA supplementation had no
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effect. Both replacing CaCO3 with CaCl2 and BA supplementation reduced the concentration in
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bone of P (P<0.05), whereas the Ca concentration was not affected. Total mineral density in bones
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measured by CT-scanning was reduced (P<0.01) in the medial and distal sections by BA and
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reduced (P<0.01) in the proximal, medial, and distal sections by CaCl2. In conclusion, the dietary
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supplementation of BA and the replacement of CaCO3 with CaCl2 affected the nutrient balances of
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P and Ca. Mineral concentration and total mineral density of metacarpal III bones was reduced both
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by BA supplementation and by replacing CaCO3 with CaCl2. The combined effect of BA and by
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replacing CaCO3 with CaCl2 may reduce bone strength during long-term feeding.
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Keywords: Pig, Benzoic acid, Calcium chloride, Acidification
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1. Introduction Emission from intensive pig production is of great concern in many parts of the world. Volatilization of ammonia can cause eutrophication of natural ecosystems (Fangmeier et al., 1994),
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and affects local environmental conditions. On-farm acidification of slurry can alleviate the
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environmental impact of pig production (Kai et al., 2008). Feeding 10 g benzoic acid (BA) per kg
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diet to pigs has been shown to reduce ammonium emission by up to 40% through lower pH in urine
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(Guiziou et al., 2006; Murphy et al., 2011), as well as it may elicit antimicrobial effects and
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increased gain (Kluge et al., 2006). Absorption of anions such as BA affects both physiology and
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nutrient metabolism (Patience and Chaplin, 1997), but the long-term effect of acidifying feeding
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with BA on the mineral balance in pigs is scarcely studied. Another strategy to control slurry acidity
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is to modify the dietary electrolyte balance (dEB) (Mongin, 1981). When dEB is reduced, blood pH
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and bicarbonate concentration go down indicating metabolic acidosis (Patience et al., 1987;
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Patience and Chaplin, 1997; Dersjant-Li et al., 2002). Feed intake and gain, and also DM
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digestibility are reduced when feeding a low dEB diet mediated though CaCl2 inclusion (Dersjant-
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Li et al., 2001). It is therefore hypothesized that feeding an acidogenic diet by replacing calcium
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carbonate (CaCO3) with calcium chloride (CaCl2) will result in metabolic acidosis, mineral
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excretion, and reduced urinary pH. Metabolic acidosis results in reduced osteoblast activity
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(Brandao-Burch et al., 2005), and it is hypothesized that long-term use of BA and/or CaCl2 in pig
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diets will result in lower mineralization of bones and, consequently, weaker bones and reduced
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animal welfare.
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The objectives of the present study were to examine the long-term effects on gastrointestinal
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microbial activity, mineral balance, as well as mineralization of bones in pigs when supplementing
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feed with BA and/or replacing CaCO3 with CaCl2 from 35 to 113 kg BW.
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2. Materials and Methods
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2.1.
Twenty-four female pigs (Landrace-Yorkshire x Duroc) were randomly assigned to four
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Diets and experimental design
experimental diets (Table 1) in a 2 by 2 factorial arrangement. The basal diet (major ingredients:
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barley, wheat, soy bean meal, and fat) was optimized according to the Danish 30-105 kg BW
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recommendations for amino acids, P, and Ca (Tybirk et al., 2012). To obtain the four experimental
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diets, the basal diet was divided into four batches and supplemented with BA (VevoVitall®, DSM
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Special Products, Rotterdam, Holland), CaCO3 (40% Ca; CaCO3) or CaCl2 (28% Ca; CaCl2·H2O,
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CaCl2·2H2O) as follows: diet -BA/CaCO3 (no BA, 14 g/kg CaCO3, considered as a control diet),
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diet +BA/CaCO3 (10 g/kg BA, 14 g/kg CaCO3), diet -BA/CaCl2 (no BA, 20 g/kg CaCl2), and diet
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+BA/CaCl2 (10 g/kg BA and 20 g/kg CaCl2). The level of 10 g BA/kg diet was chosen because it is
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the maximum level of supplementation allowed in the EU (Commission, 2007). The CaCO3 level
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supplied Ca to meet the recommendations (Tybirk et al., 2012), and the CaCl2 diets were designed
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to supply equimolar amounts of Ca. On the basis of the chemical composition (Table 1), dEB in
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meq was calculated as (Na+/23.0 + K+/39.1 – Cl-/35.5) x 1000, where Na+, K+ and Cl- were in g/kg
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DM to be 185, 182, -124, and -91 meq/kg DM for -BA/CaCO3, +BA/CaCO3, -BA/CaCl2, and
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+BA/CaCl2 diets, respectively.
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2.2.
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Animals and husbandry
The pigs were subjected to feeding from 36 ± 2.0 kg BW until slaughter, when the average
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weight of the fastest growing treatment group reached 113 kg. During this period, in the weight
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range from 60 to 66 kg BW, the pigs were used in a mineral balance study. All experimental
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procedures have been carried out in accordance with the Danish Ministry of Justice, Law no. 253 of
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8 March 2013 concerning experiments with animals and care of experimental animals issued by the
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Danish Animal Experiments Inspectorate.
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The pigs were housed individually in pens with concrete floor and straw as rooting material. Diets were fed ad libitum as dry coarse diets. There was free access to water through the entire
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experimental period. During the balance study from 60 to 66 kg BW, the pigs were housed in
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stainless steel balance cages for total collection of urine and feces. After 5 d of adaptation, all pigs
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were fitted with urine bladder catheters allowing collection of urine directly into a closed container.
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The pigs were fed twice daily at 0800 and 1430 h and had free access to demineralized water. The
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offered daily rations were 1800, 1818, 1810, and 1828 g feed/d for the treatments -BA/CaCO3,
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+BA/CaCO3, -BA/CaCl2, and +BA/CaCl2, respectively, which supplied all animals with the same
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amount of basal diet. The pigs were weighed at the beginning and at the end of the 12 d period.
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Feed refusals were collected and weighed daily. Urine was weighed and samples collected once
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daily. Feces were collected twice daily. Urine and feces were pooled over the 7 d period and were
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stored at 5°C until representative feces samples were prepared after homogenization at d 7. Samples
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were stored at -20°C until analysis.
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At the end of the experiment, the pigs were slaughtered in random order and 2-3 h after
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being fed in the morning. The metacarpal bone no. III of the right foreleg was dissected and stored
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at -20°C. The gastrointestinal tract (GIT) was removed immediately after slaughter and divided into
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eight segments: the entire stomach, three segments of small intestine (uniform by length), caecum,
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and three segments of colon (uniform by length). Digesta was collected from the stomach, the third
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(distal) segment of the small intestine (ileum and part of jejunum), the caecum, and the second
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segment of colon (mid colon). After homogenization of digesta from the individual segments,
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samples (~10 g) for analysis of organic acid content (see further below) were transferred to
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stomacher bags and stored at -20ºC. Digesta pH was measured directly in the homogenized digesta
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samples using a pH meter (Radiometer, Copenhagen, Denmark), and samples (5 - 10 g) were
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maintained for determination of DM content.
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2.3.
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Dry matter content, total nitrogen and crude fat in diets, and DM in feces were analyzed as
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described by Nørgaard et al. (2011). Phosphorus and Ca was analyzed in diets, non-defatted bone,
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urine, and feces after ashing at 450°C and hydrochloric acid/nitric acid treatment. Phosphorus was
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analyzed as a vanadomolybdate-complex by spectrophotometry (Lambda 900, Perkin Elmer,
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Überlingen, Germany) (Stuffins, 1967) and Ca by atomic absorption spectrophotometry (model
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SP9, Pye Unicam Ltd., Cambridge, UK). For analysis of Na and K in diets, lithium chloride was
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added to the samples and measured by flame photometry (FLM3 Flame Photometer, Radiometer,
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Copenhagen, Denmark). Chloride was extracted with nitric acid and analyzed by potentiometric
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titration with silver nitrate using a titrator (T70, Mettler Toledo, OH). Phytate bound P and phytase
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activity was analyzed as described by Carlson et al. (2012). The pH in diets, urine, and feces was
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measured by a pH meter (Radiometer, Copenhagen, Denmark). The concentration of organic acids,
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i.e. short-chain fatty acids (acetate, propionate, and butyrate), sorbic acid, lactic acid, succinic acid,
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BA, and hippuric acid, were measured as described by Canibe et al. (2007), and expressed per kg of
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fresh digesta to reflect in situ concentrations of the organic acids.
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Bone mineralization was measured by quantitative computed tomography (CT-scanning)
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which is an in vivo method for measuring bone mass in the peripheral skeleton where bone density
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is slow to change; it directly relates bone densitometry to actual bone density and is therefore
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suggested by Castellanos et al. (2011) as a standard in bone densitometry. Bone density denotes the
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solid constituents per cubic centimeter of bones, which is interpreted as mineral content or
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mineralization. A Stratec XCT 2000 Research+ scanner (Stratec Medizintechnik GmbH, Pforzheim,
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Germany) was used, performing 1 mm slices at the proximal and distal epiphysis and in the middle
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of the diaphysis. Identical locations were identified for all bones at the proximal and distal
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trochanter protuberances, then a third slice was selected from the middle point between them. The
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results were scored by two measures: the T-score and the Z-score. These scores indicate the amount
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of mineral density; negative scores show the lower bone density, whereas positive scores indicate
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higher amounts of minerals. In this study, only positive scores were taken into consideration,
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because it was not intended to establish risk-fracture parameters (Winzenberg and Jones, 2011).
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Negative scores were not considered as they, by consensus of the World Health Organization
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(WHO), are used to asses fracture risk (Unnanuntana et al., 2010) and, to our knowledge, this has
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not been established nor validated for animals.
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2.4.
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The dietary cation-anion difference (DCAD) is given in meq/kg DM and is calculated as the
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difference between the sum of cations and anions. In many practical situations, DCAD is called
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dEB and is calculated as meq Na+ + K+ - Cl- which is a simplified equation for the dietary elements
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in maintaining acid-base homeostasis (Mongin, 1981). The equation for calculating dEB in meq per
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kg DM was: (Na+/23.0 + K+/39.1 – Cl-/35.5) x 1000, where Na+, K+ and Cl- were in g/kg DM. The
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results on animal performance, mineral balance, and mineral density of bones were statistically
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analyzed by GLM in SAS (SAS Institute Inc. Cary, NC) according to a model including main
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effects (+/- BA and Ca-source) and the interaction between main effects. Multivariate analysis of
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variance was done on responses in bones using the MANOVA statement in the GLM procedure of
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SAS. The statistical model used to estimate the effect of diet on various response variables in
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digesta was a mixed linear model which included diet, segment of the GIT, and diet × segment as
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fixed effects. To capture the correlation between measurements in different segments of the GIT of
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each pig, the random errors were allowed to be correlated by a compound symmetry covariance
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structure using the repeated statement in SAS. Statistical significance was accepted at P<0.05 and
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tendencies at P<0.10. For pairwise comparisons, Fisher’s protected LSD test was used to separate
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treatment means. Presented data are least squares means and standard error of means.
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3. Results
None of the animals showed symptoms of impaired health and wellbeing. The concentration of
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BA in the stomach content of pigs receiving the BA supplemented diets was approx. 10 mmol/kg
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fresh digesta; BA was neither detectable in other GIT segments of these animals nor in any
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segments of animals receiving the non-supplemented diets (data not shown). In general, digesta
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contents of the 3 major short-chain fatty acids (acetate, propionate, and butyrate) were not
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significantly affected by the BA and CaCl2 supplementations (Table 2). A reduction in digesta
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concentration was observed for acetate (P<0.01) and propionate (P=0.03) in the colon for the CaCl2
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supplemented diet, whereas no significant effects were observed for digesta concentrations of
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butyrate (Table 2). Lactate was only detectable in the proximal gastrointestinal segments (stomach
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and small intestine) and was lower (P<0.01) in stomach contents for the CaCl2 diets. No further
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significant effects of the dietary treatments were observed neither for the rest of the analyzed
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organic acids (P>0.05, data not shown) nor for the digesta pH (Table 2). The dry matter content of
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the digesta was affected by diet and was higher (P<0.05) in all GIT segments for pigs receiving the
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CaCl2 diets.
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The pH in feces was increased (P<0.01) when feeding pigs CaCl2 compared to CaCO3. In
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urine, both BA (P=0.04) and CaCl2 (P<0.01) decreased pH, and the effect of both BA and CaCl2
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was a decrease in two pH units compared to the -BA/CaCO3 treatment (Table 3).
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The mineral balance was affected by BA and especially by CaCl2 supplementation. On the P balance, interactions (P<0.03) were observed between the two factors. The apparent digestibility of
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P was the lowest and the urinary P excretion was the highest in pigs fed the +BA/CaCl2 diet
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resulting in the lowest P retentions. The apparent digestibility of Ca was the lowest in pigs fed the
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+BA/CaCl2 diet. The urinary excretion of Ca was the greatest (P<0.01) in pigs fed CaCl2. Ca
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retention was the lowest (P<0.05) in pigs fed the +BA/CaCl2 diet.
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The weight and length of metacarpal bone no. III was lower (P=0.04) and shorter (P=0.02),
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respectively, when pigs were fed the CaCl2 diets, whereas there was no effect of BA
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supplementation. Bone ash and P concentration was reduced by both replacing CaCO3 with CaCl2
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(P=0.02) and by supplementing diets with BA (P<0.03). The concentration of Ca in metacarpal
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bones was affected by neither of the treatments.
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The density of minerals measured by CT-scanning was affected by both BA and CaCl2 (Table 4). Supplementation of BA resulted in lower mineral density in the medial (P=0.01) and distal
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(P<0.01) sections of the metacarpal bones, and replacing CaCO3 with CaCl2 resulted in lower
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mineral density in the proximal (P=0.01), medial (P=0.01), and distal (P<0.01) sections. When
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dividing the bone into trabecular and cortical tissue, the results were somewhat less clear.
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Trabecular tissue in the distal bones of pigs fed diets supplemented with CaCl2 had a greater
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(P=0.02) mineral density compared to pigs fed the diets supplemented with CaCO3, whereas it was
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lower in cortical tissue in both the medial (P<0.01) and distal (P<0.01) sections. Multivariate
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analysis of results on mineral densities showed an effect of replacing CaCO3 with CaCl2 (P=0.03),
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but not by BA supplementation (P=0.12). Considering both mineral concentrations and densities in
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the multivariate analysis, there was a tendency (P=0.06) of an effect by replacing CaCO3 with
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CaCl2, but not by BA supplementation (P=0.16).
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4. Discussion By mixing feces and urine collected in the balance study (Eriksen et al., unpublished), it was observed that ammonia emission was reduced by 24-37% in slurry from pigs fed the diets
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supplemented with BA or where CaCO3 was replaced with CaCl2. This confirms our previous
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findings (Eriksen et al., 2010) of feeding as an important and cost efficient mitigation option,
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provided that is does not imply negative effects on animal performance and welfare.
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The dEB and dietary undetermined anion (dUA) has pronounced effects on nutrient
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metabolism and the physiological status of the pigs (Patience and Chaplin, 1997). When feeding
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pigs with diets with negative dEB, the blood pH, HCO3-, and base excess are decreased (Patience et
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al., 1987; Budde and Crenshaw, 2003). The changes observed in these experiments were, however,
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not drastic enough to affect animal health.
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When replacing CaCO3 with CaCl2, the urinary pH decreased 1.3 units. Similar reductions in urinary pH after feeding pigs low-dEB diets have been observed before (Canh et al., 1998). Benzoic
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acid is efficiently absorbed from the small intestine and efficiently metabolized in the liver to
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hippuric acid and is excreted into urine (Kristensen et al., 2009). The reduction of urinary pH by
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dietary BA was in accordance with other studies (Canh et al., 1998; Sauer et al., 2009; Nørgaard et
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al., 2010b). The combined effect of CaCl2 and BA was a urinary pH reduction of 2.0 units.
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Organic acids are oxidized within the body and therefore do not affect the acid-base
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physiology. An exception is BA which is not oxidized but is efficiently converted into hippuric acid
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in the liver and subsequently excreted to urine by the kidneys (Kristensen et al., 2009). Benzoic acid
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and hippuric acid may therefore present a temporary acid load to the extracellular fluid
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compartment, thus increasing the demand for buffering capacity which among other mechanisms
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partly includes the intermediary metabolism of minerals. In this way, BA has effects similar to low
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dEB and dUA, and therefore it ought to be taken into account when calculating the dEB (Kristensen
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et al., 2009). A decrease in blood pH suppresses osteoblast activity and stimulates osteoclast
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activity, thereby stimulating bone mobilization of P and Ca along with ongoing proton buffering by
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the bone (Krieger et al., 2004). Prolonged mineral mobilization from bone will eventually lead to
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bone weakness. Even though the response of osteoclasts to changes in pH is steep (Arnett, 2003),
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previous studies have shown a reduction in blood pH caused by BA supplementation, but without
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effects on blood phosphate levels (Kristensen et al., 2009; Nørgaard et al., 2010b). The mechanism
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behind the small impact on acid-base status in pigs is likely to be a protracted absorption of BA
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(Kristensen et al., 2009). Sauer et al. (2009) observed increased blood pH and increased plasma P
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concentrations in pigs after feeding 20 g/kg BA for 21 d and argued for an increased renal distal
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tubule expression of carbonic anhydrase stimulated by long term and high levels of BA. This
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hypothesis seems only relevant for BA doses higher than 10 g/kg, which is the maximum inclusion
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level approved in the European Union, since other long term studies using 5 g/kg (Buhler et al.,
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2010) and 10 g/kg (present study) showed no changes or reductions in blood P and Ca
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concentrations (Buhler et al., 2010) and retentions (Buhler et al., 2010; present study).
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Calcium carbonate is typically supplemented to pig diets at approx. 15 g/kg to provide dietary
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Ca which mainly supports growth and maintenance of bone. The urinary pH can be reduced simply
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by lowering the amount of supplemented CaCO3 (Canh et al., 1998). By replacing CaCO3 with
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CaCl2 as the main source of Ca, several effects were obtained. Besides the lower dEB obtained
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through the increased inclusion of Cl, the change in carbonate content made the diets acidogenic
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and with a lower acid binding capacity. The inclusion of CaCO3 increases the acid binding capacity
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which is a measure reflecting pH buffering in the stomach, and it is important for especially protein
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digestion and animal health in general (Lawlor et al., 2006). The efficiency of Ca absorption is
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greatly dependent both the Ca and P concentrations in the diet, and the digestibility of CaCO3 is
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therefore not simple to estimate, but can be expected to be in the range from 0.45 to 0.70
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(González-Vega et al., 2013). Thus, large amounts of undigested CaCO3 are passing through the
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GIT ending up in the slurry, where it can be speculated to act as a buffering agent and counteract
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any biological or mechanical acidifying efforts. These concerns would argue for reducing the
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inclusion of CaCO3 and for including other and more digestible Ca sources. However, in the present
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study, the pH was for unknown reasons higher in feces from pigs fed CaCl2 compared to CaCO3 as
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the main Ca sources.
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The balances of P and Ca were clearly affected by the dietary treatments, also when the lower
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P and Ca intake of the groups of pigs fed both BA and CaCl2 is taken into account. The lower feed
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intake of this group corresponds well to early findings that low dUA reduces appetite (Patience and
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Wolynetz, 1990). In particular, the diets replacing CaCO3 with CaCl2 resulted in lower P and Ca
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retentions due to increased urinary excretions, and in relation to P, it was also due to decreased
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digestibility. Increased urinary Ca excretion was also observed when increasing dietary Cl-
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concentration, but no significant effects on urinary P excretion (Golz and Crenshaw, 1991; Patience
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and Chaplin, 1997). A positive effect by BA on P and Ca apparent faecal digestibility has been
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reported before (Sauer et al., 2009; Nørgaard et al., 2010a), although in the present study there were
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no significant differences. There was no effect on P and Ca excretion by supplementing BA to the
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CaCO3 based diet, and this is in accordance with several other studies on BA supplementation
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(Nørgaard et al., 2010a; Nørgaard et al., 2010b; Gräber et al., 2012) and in contrast to Sauer et al.
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(2009), who found reduced P and Ca excretions. The combined effect of main treatments, i.e. the
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pigs fed the +BA/CaCl2 diet, resulted in lower apparent faecal digestibility and retention of both P
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and Ca. Especially the 14% reduction in P apparent faecal digestibility could have strong practical
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relevance in pig production. Mineral solubility is generally thought to be increased through an
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acidified gastro-intestinal environment lowering the rate of gastric emptying (Jongbloed, 1987) and
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increasing the solubility of mineral chelates (Cao et al., 2000; Guo et al., 2001) and other
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complexes. An increased absorption of P and Ca by acidifying the gastro-intestinal environment has
297
recently been confirmed in sows fed citric acid (Liu et al., 2014). However, since the combination
298
of supplementing BA and replacing CaCO3 with CaCl2 reduced the apparent digestibility and
299
retention of P and Ca, it does not appear that an increased absorption is solely due to a lower
300
intestinal pH.
ip t
296
The weight, length, mineral content and mineral density of metacarpal bones was affected by
302
both BA and by replacing CaCO3 with CaCl2. Previous studies using pigs of similar weight ranges
303
showed tendencies of lower bone mineral density and content when fed 5 g/kg BA until 66 kg BW,
304
but no effect at greater live weight (108 kg) (Buhler et al., 2010). In pigs weighing 64 kg BW, no
305
effect was found of BA on bone mineral content and breaking strength (Gutzwiller et al., 2011).
306
The bone strength is affected by mineral density and bone structure, and there is a direct relation
307
between bone mineralization and bone strength in both human (Zumstein et al., 2012) and pigs
308
(Nielsen et al., 2007). The risk of fragility fractures in humans is indicated by the T score which
309
compares one individual’s bone mineral density in standard deviations with the mean peak density
310
in healthy young individuals. Osteoporosis is defined by the World Health Organization as 2.5 x
311
standard deviations or more below mean peak density (Brunader and Shelton, 2002). When
312
considering the bone mineral density of pigs in the control group (-BA/CaCO3) as representative for
313
healthy pigs, the T score was calculated for total bone mineral densities in the three sections. In the
314
distal sections, four of the six pigs of group +BA/CaCl2 were in risk of osteopenia, a precursor for
315
osteoporosis; in the medial section, 1, 0, and 2 of the six pigs in the groups +BA/CaCO3, -BACaCl2,
316
and +BA/CaCl2, respectively, were in risk of osteopenia; in the proximal section, 1, 1, and 2 of the
317
six pigs in the groups +BA/CaCO3, -BACaCl2, and +BA/CaCl2, respectively, were in risk of
318
osteopenia. Taking into account that the six pigs in the -BA/CaCO3 group can barely be considered
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14 Page 14 of 29
319
a population, the risk of bone fractures may be increased when both supplementing diets with BA
320
and replacing CaCO3 with CaCl2.
321
Benzoic acid is known to possess stronger antimicrobial properties than a range of other organic acids (Knarreborg et al., 2002). Of the dietary BA, 13% cannot be found in the portal blood,
323
and may therefore pass through the intestine (Kristensen et al., 2009), explaining previous results
324
that caecal microbial diversity was affected by feeding 5 g/kg BA (Halas et al., 2010). In the present
325
study, however, 10 g/kg BA did not significantly affect the digesta concentrations of lactate,
326
acetate, propionate, and butyrate throughout the GIT, indicating that the microbial activity was not
327
dramatically affected by the dietary treatment. The observed reduction in gastric lactate
328
accumulation of pigs receiving the CaCl2 diets resembles the observations of a recent study where a
329
high Ca (calcium monohydrogen phosphate) level was observed to reduce gastric lactate
330
accumulation (Metzler-Zebeli et al., 2011). The working mechanism behind the observed Ca effects
331
is unclear. Increased levels of dietary Ca have been shown to induce increased secretion of gastric
332
acids in rats (Floor et al., 1991) which may again lead to decreased stomach pH and, subsequently,
333
impaired microbial activity. However, neither the present study nor the study of Metzler-Zebeli et
334
al. (2011) demonstrated any reduction in stomach pH as a result of the Ca source and level,
335
respectively. On the other hand, Ca source and level may be argued to influence the buffer capacity
336
of the feed and thereby counteract any pH-decreasing effect of gastric acid secretion (Lawlor et al.,
337
2006). Therefore, even though the gastric pH levels did not differ by the time of slaughter and
338
digesta sampling, stomach acidification (inhibiting bacterial activity) after feed intake may have
339
occured more rapidly for the animals receiving the CaCl2 diets (low buffer capacity), explaining the
340
lower accumulation of organic acids observed for these animals. It was suggested and supported by
341
the observations of Metzler-Zebeli et al. (2011) that a high Ca level may moreover stimulate
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15 Page 15 of 29
342
fermentation of lactate to butyrate by e.g. Megasphaera elsdenii a mechanism like this is not
343
indicated by the digesta butyrate concentrations in the present study.
344 5. Conclusion
ip t
345
BA was efficiently absorbed in the proximal GIT, and had no significant effects on digesta
347
concentrations of organic acids. Replacing CaCO3 with CaCl2 was observed to suppress organic
348
acids in the stomach and colon. Reductions in urinary pH were found both after feeding BA and
349
after replacing CaCO3 with CaCl2. The dietary supplementation of BA and the replacement of
350
CaCO3 with CaCl2 affected the nutrient balances of P and Ca. Mineral concentration and total
351
mineral density of metacarpal III bones were reduced both by BA supplementation and by replacing
352
CaCO3 with CaCl2. Taking the limited number of animals into account, the changes in mineral
353
balance and density of bones caused by feeding either BA or by replacing CaCO3 with CaCl2 did
354
not appear to severely affect bone strength, but their combined effect may affect bone strength
355
during long-term feeding.
357 358
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Acknowledgements
This project was partly funded by the Danish Food Industry Agency under the Danish Ministry
359
of Food, Agriculture and Fisheries and partly by the Department of Animal Science, Aarhus
360
University.
361 362
References
363
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490
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d te
492
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Animal Feed Science and Technology Ms. No. ANIFEE-14-5460 Conflict of interests statement
The authors have no conflicts of interests regarding this work
ip t
523 524 525 526 527 528
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29 Page 23 of 29
Table 1 Feedstuff and analyzed chemical composition of diets supplemented with 0 or 10 g/kg benzoic acid (BA, +BA) and two Ca-sources (CaCO3 or CaCl2). Diet +BA/
-BA/
CaCO3
CaCO3
CaCl2
Barley
562
562
Wheat
230
Soybean meal, toasted dehulled
177
Fat
20.0
cr 562
us
Feedstuff composition (g/kg)
+BA/
ip t
-BA/
CaCl2
562
230
230
177
177
177
20.0
20.0
20.0
1.7
1.7
1.7
0.4
0.4
0.4
0.4
0.5
0.5
0.5
0.5
2.7
2.7
2.7
2.7
3.5
3.5
3.5
3.5
Vitamin and mineral mixa
2.0
2.0
2.0
2.0
Phytaseb
0.3
0.3
0.3
0.3
14.0
14.0
0
0
0
0
20.0
20.0
0
10.0
0
10.0
Dry matter (DM)
868
863
863
860
Crude protein
199
197
197
198
Ash
48.5
47.0
48.6
46.4
Crude fat
47.9
49.2
47.8
45.3
1.7
M
L-lysine HCl (78%) DL-methionine (99%)
CaCl2c
Ac
CaCO3c
ce pt
NaCl
ed
L-threonine (99%) Monocalciumphosphate
Benzoic acidc
an
230
Chemical composition (g/kg DM)
22 Page 24 of 29
8.1
8.1
8.2
8.2
Calcium
7.7
7.8
8.2
7.2
Phosphorus
4.3
4.2
4.3
4.2
Chloride
3.4
2.9
14.3
13.0
Sodium
1.7
1.3
1.6
1.5
Phytate bound P
2.6
2.5
Phytase, FTU/g
2941
2673
Benzoic acid
0.0
8.2
pH
6.1
dEB, meq/kg DMd
185
ip t
Potassium
2.3
2302
2446
cr
2.4
us
0.4
8.9
5.2
4.7
182
-124
-91
an
5.5
482
a
483
vitamin E, 0.6 mg vitamin K3, 0.6 mg vitamin B1, 1.2 mg vitamin B2, 3 mg D-panthotenic acid, 6.4 mg
484
niacin, 0.060 mg biotin, 21.3 mg α-tocopherol, 0.006 vitamin B12, 0.6 mg vitamin B6, 50 mg Fe (Fe(II)
485
sulphate), 41.3 mg Cu (Cu(II) sulphate), 50 mg Zn (Zn(II) oxide), 13.9 mg Mn (Mn(II) oxide), 0.076 mg KI,
486
0.075 mg Se (Se-selenite).
487
b
Natuphos 5000 5641 FTU/g, BASF, 2300 Copenhagen, DK.
488
c
Supplemented to batches of the basal diet. Benzoic acid was VevoVitall, DSM Special Products,
489
Rotterdam, NL.
490
d
M
ed
ce pt
dEB, dietary electrolyte balance.
Ac
491
Trouw Nutrition Denmark A/S. Content per g premix: 2500 IU vitamin A, 500 IU vitamin D3, 23.4 IU
23 Page 25 of 29
492
Table 2
493
Concentrations (mmol/kg fresh digesta) of organic acids and pH of digesta samples taken
494
immediately after slaughtering animals fed diets supplemented with 0 or 10 g/kg benzoic acid (-BA,
495
+BA) and two Ca-sources (CaCO3 or CaCl2) from 35 to 113 kg BW. Dieta -BA/
+BA/
-BA/
ip t
Segmentb
Ca
BA×Ca
cr
Item
P-value
+BA/
BA
0.17
0.78
SEM
Butyrate
CaCl2
CaCl2
St
26.1
22.6
21.5
19.6
Si
28.1
24.5
26.1
22.3
4.7
0.45
0.67
0.98
Ce
96.3
94.0
86.2
89.4
4.0
0.91
0.09
0.50
Co
96.8
92.9
79.0
82.7
3.7
0.97
<0.01
0.31
St
8.6
7.3
8.4
7.8
1.1
0.41
0.92
0.77
Si
bdc
bd
bd
bd
-
-
-
-
Ce
54.9
Co
47.1
us
an
M
47.5
49.9
3.7
0.10
0.30
0.79
38.5
35.2
35.5
3.1
0.20
0.03
0.17
8.1
7.9
5.8
6.3
1.0
0.86
0.08
0.73
2.8
3.1
2.9
2.1
0.8
0.69
0.56
0.49
16.6
15.7
18.4
21.0
1.9
0.65
0.08
0.38
Co
19.3
18.9
20.5
21.4
1.7
0.88
0.29
0.70
St
91.6
76.9
45.3
43.3
13.4
0.54
<0.01
0.64
Si
65.2
56.1
67.9
64.1
13.9
0.65
0.70
0.85
Ce
bd
bd
bd
bd
-
-
-
-
Co
bd
bd
bd
bd
-
-
-
-
St
3.8
3.8
3.7
3.9
0.1
0.33
0.83
0.16
St
Ac
Ce
pH
0.33
44.5
Si
Lactate
2.7
ed
Propionate
CaCO3
ce pt
Acetate
CaCO3
24 Page 26 of 29
Si
6.3
6.2
5.9
6.0
0.2
0.90
0.07
0.84
Ce
5.7
5.6
5.5
5.6
0.1
0.98
0.20
0.18
Co
6.2
6.0
6.3
6.1
0.1
0.11
0.36
0.70
a
Values are least square means and standard errors of the means (SEM). n = 6.
497
b
Samples were taken from stomach (St), small intestine (Si), cecum (Ce), and mid colon (Co).
498
c
bd, below detection limit.
ip t
496
cr
499
Ac
ce pt
ed
M
an
us
500
25 Page 27 of 29
Table 3
502
Feed intake, pH in feces and urine, daily intake, apparent digestibility, urinary excretion, retention, and
503
retention in percent of intake of P and Ca of pigs in a 12 days balance study fed diets supplemented with 0 or
504
10 g/kg benzoic acid (-BA, +BA) and two Ca-sources (Ca) (CaCO3 or CaCl2).a Dieta
P-value -BA/
+BA/
CaCO3
CaCO3
CaCl2
CaCl2
1497
1500
1417
1361
Feces pH
6.8
6.6
7.3
7.6
Urine pH
8.2
7.9
6.9
Intake, g/d
5.57
5.41
5.27
App. diges.
0.64a
0.65a
0.64a
Urine, g/d
0.09b
0.03b
Retention, g/d
3.47a
Retention, %
Feed intake, g/d
SEM
BA
BA×Ca
68
0.66
0.08
0.62
0.2
0.87
<0.01
0.22
0.3
0.04
<0.01
0.40
4.96
0.25
0.28
0.10
0.73
0.55b
0.03
0.08
0.03
0.03
0.09b
0.28a
0.05
0.11
<0.01
<0.01
3.47a
3.13a
2.42b
0.17
0.03
<0.001
0.03
62.2a
64.2a
60.9a
49.3b
2.8
0.07
<0.01
0.01
10.1a
10.1a
10.0a
8.4b
0.46
0.08
0.05
0.05
0.74ab
0.79a
0.79a
0.68b
0.04
0.50
0.46
0.04
Urine, g/d
0.15
0.27
0.90
0.95
0.15
0.52
<0.01
0.81
Retention, g/d
7.20a
7.70a
6.95a
4.79b
0.42
0.04
<0.001
0.002
Retention, %
72.0a
76.3a
70.0a
57.3b
5.0
0.33
0.02
0.05
Intake, g/d
Ac
App. diges.
505
a
506
a,b
ed
ce pt
Ca balance
M
P balance
6.2
Ca
cr
+BA/
us
-BA/
an
Item
ip t
501
Values are least squares means and standard errors of the means (SEM). n = 6. Within a row for items with a significant interaction, means without a common superscript differ (P<0.05).
507
26 Page 28 of 29
Table 4
509
Bone weight and size, ash, Ca, and P concentration, and mineral density measured by CT-scanning in
510
metacarpal III bones of pigs fed diets supplemented with 0 or 10 g/kg benzoic acid (-BA, +BA) and two Ca-
511
sources (Ca) (CaCO3 or CaCl2) from 35 to 113 kg BW.a Itemb
P-value
-BA/
+BA/
-BA/
+BA/
BA×Ca
CaCO3
CaCO3
CaCl2
CaCl2
Weight, g
28.7
30.7
28.0
27.0
1.0
0.64
0.04
0.15
Length, mm
78.7
78.5
76.4
75.1
1.1
0.53
0.02
0.60
Ash, g/100 g
28.2
26.4
26.2
24.7
0.7
0.02
0.02
0.81
Ca, g/100 g
10.7
10.1
10.1
9.8
0.4
0.31
0.28
0.61
P, g/100 g
5.1
4.7
4.8
4.5
0.1
0.02
0.03
0.70
Proximal, total, mg/cm3
347.4
330.8
306.7
298.1
13.6
0.37
0.01
0.77
Proximal, trabec., mg/cm3
168.2
176.8
181.1
183.8
12.4
0.66
0.43
0.81
Proximal, cortical, mg/cm3
605.3
575.9
565.8
564.6
16.9
0.38
0.15
0.41
Medial, total, mg/cm3
364.8
336.6
337.5
296.9
12.5
0.01
0.01
0.62
Medial, trabec., mg/cm3
43.5
43.2
42.8
37.4
2.4
0.25
0.19
0.30
Medial, cortical, mg/cm3
857.7
864.5
839.0
841.1
6.0
0.46
<0.01
0.70
Distal, total, mg/cm3
447.8
427.0
409.5
368.2
11.4
<0.01
<0.01
0.35
Distal, trabec., mg/cm3
244.0
240.5
254.0
250.7
4.2
0.40
0.02
0.97
Distal, cortical, mg/cm3
583.2
571.1
557.1
542.3
6.8
0.06
<0.01
0.84
BA
us
an
ed
ce pt
Ac
SEM
cr
Ca
M
Diet
ip t
508
512
a
Values are least squares means and standard errors of the means (SEM). n = 6.
513
b
Mineral density of trabecular and cortical tissues or in total was measured in the proximal, medial and distal
514
metacarpal III bone of the right foreleg. Trabec = trabecular.
515
27 Page 29 of 29
S0377-8401(14)00180-1 http://dx.doi.org/doi:10.1016/j.anifeedsci.2014.05.010 ANIFEE 13088
To appear in:
Animal
Received date: Revised date: Accepted date:
6-2-2014 9-5-2014 23-5-2014
Feed
Science
and
Technology
Please cite this article as: Norgaard, J.V., Hojberg, O., Sorensen, K.U., Eriksen, J., Medina, J.M.S., Poulsen, H.D.,The effect of long-term acidifying feeding on digesta organic acids, mineral balance, and bone mineralization in growing pigs, Animal Feed Science and Technology (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Animal Feed Science and Technology Ms. No. ANIFEE-14-5460 Highlights
Benzoic acid had no significant effects on digesta concentrations of organic acids
ip t
1 2 3 4 5 6
Replacing CaCO3 with CaCl2 suppressed organic acids in the stomach and colon
8
Urinary pH was reduced by feeding benzoic acid and replacing CaCO3 with CaCl2
9
Benzoic acid or CaCl2 alone did not affect bone strength during long-term feeding Bone minerals were reduced when feeding both benzoic acid and CaCl2.
us
10
cr
7
Ac ce p
te
d
M
an
11 12
1 Page 1 of 29
12
The effect of long-term acidifying feeding on digesta organic acids,
13
mineral balance, and bone mineralization in growing pigs
ip t
14
J.V. Nørgaarda,*, O. Højberga, K.U. Sørensena, J. Eriksenb, J.M.S. Medinac, H.D.
16
Poulsena
cr
15
us
17 18
a
19
Denmark
20
b
Department of Agroecology, Aarhus University, Foulum, P.O. Box 50, DK-8830 Tjele, Denmark
21
c
Orthopaedic Research Laboratory, Aarhus University Hospital, DK-8000 Aarhus C, Denmark
M
an
Department of Animal Science, Aarhus University, Foulum, P.O. Box 50, DK-8830 Tjele,
22
Abbreviations: BA, benzoic acid; CaCl2, calcium chloride; CaCO3, calcium carbonate; DCAD,
24
dietary cation-anion difference; dEB, dietary electrolyte balance; dUA, dietary undetermined anion;
25
GIT, gastrointestinal tract.
te
Ac ce p
26
d
23
27
* Corresponding author at: Blichers Alle 20, DK-8830 Tjele, Denmark. Tel.: +45 87157816. E-mail
28
address: [email protected] (J.V. Nørgaard).
29 30 31
Abstract
Acidification of slurry through dietary manipulation of urinary pH is a means of mitigating
32
nitrogen emission from pig production, but long-term effects of diet acidification on bone
33
mineralization and mineral balance is less investigated. The objective was therefore to study the
34
long-term effects of feeding benzoic acid (BA) and calcium chloride (CaCl2) on the mineral balance
2 Page 2 of 29
and microbial activity in the gastrointestinal tract of pigs. Four diets containing the combinations of
36
0 or 10 g/kg BA and 0 or 20 g/kg CaCl2 were fed to 24 pigs in a factorial design. For the diets
37
without CaCl2, calcium carbonate (CaCO3) was added to provide equimolar levels of Ca. The pigs
38
were fed the diets from 36 kg until slaughter at 113 kg BW, and they were housed in balance cages
39
for 12 d from 60 to 66 kg BW. Supplementation of BA and/or CaCl2 had only minor effect on
40
accumulation of digesta organic acids (acetate, propionate, butyrate and lactate) throughout the
41
gastrointestinal tract. A reduction (P<0.01) in digesta concentration was, however, observed for
42
acetate and propionate in colon digesta for the CaCl2 supplemented diet. Moreover, CaCl2
43
supplementation reduced (P<0.01) the gastric concentration of lactic acid. In the urine, pH was
44
reduced by both BA (P=0.04) and CaCl2 (P<0.01). The diet with combined BA and CaCl2 resulted
45
in the lowest retentions of both P (P=0.03) and Ca (P<0.01). Metacarpal III bones were lighter
46
(P=0.04) and shorter (P=0.02) when replacing CaCO3 with CaCl2, but BA supplementation had no
47
effect. Both replacing CaCO3 with CaCl2 and BA supplementation reduced the concentration in
48
bone of P (P<0.05), whereas the Ca concentration was not affected. Total mineral density in bones
49
measured by CT-scanning was reduced (P<0.01) in the medial and distal sections by BA and
50
reduced (P<0.01) in the proximal, medial, and distal sections by CaCl2. In conclusion, the dietary
51
supplementation of BA and the replacement of CaCO3 with CaCl2 affected the nutrient balances of
52
P and Ca. Mineral concentration and total mineral density of metacarpal III bones was reduced both
53
by BA supplementation and by replacing CaCO3 with CaCl2. The combined effect of BA and by
54
replacing CaCO3 with CaCl2 may reduce bone strength during long-term feeding.
Ac ce p
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an
us
cr
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35
55 56
Keywords: Pig, Benzoic acid, Calcium chloride, Acidification
57
3 Page 3 of 29
58 59
1. Introduction Emission from intensive pig production is of great concern in many parts of the world. Volatilization of ammonia can cause eutrophication of natural ecosystems (Fangmeier et al., 1994),
61
and affects local environmental conditions. On-farm acidification of slurry can alleviate the
62
environmental impact of pig production (Kai et al., 2008). Feeding 10 g benzoic acid (BA) per kg
63
diet to pigs has been shown to reduce ammonium emission by up to 40% through lower pH in urine
64
(Guiziou et al., 2006; Murphy et al., 2011), as well as it may elicit antimicrobial effects and
65
increased gain (Kluge et al., 2006). Absorption of anions such as BA affects both physiology and
66
nutrient metabolism (Patience and Chaplin, 1997), but the long-term effect of acidifying feeding
67
with BA on the mineral balance in pigs is scarcely studied. Another strategy to control slurry acidity
68
is to modify the dietary electrolyte balance (dEB) (Mongin, 1981). When dEB is reduced, blood pH
69
and bicarbonate concentration go down indicating metabolic acidosis (Patience et al., 1987;
70
Patience and Chaplin, 1997; Dersjant-Li et al., 2002). Feed intake and gain, and also DM
71
digestibility are reduced when feeding a low dEB diet mediated though CaCl2 inclusion (Dersjant-
72
Li et al., 2001). It is therefore hypothesized that feeding an acidogenic diet by replacing calcium
73
carbonate (CaCO3) with calcium chloride (CaCl2) will result in metabolic acidosis, mineral
74
excretion, and reduced urinary pH. Metabolic acidosis results in reduced osteoblast activity
75
(Brandao-Burch et al., 2005), and it is hypothesized that long-term use of BA and/or CaCl2 in pig
76
diets will result in lower mineralization of bones and, consequently, weaker bones and reduced
77
animal welfare.
78
Ac ce p
te
d
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an
us
cr
ip t
60
The objectives of the present study were to examine the long-term effects on gastrointestinal
79
microbial activity, mineral balance, as well as mineralization of bones in pigs when supplementing
80
feed with BA and/or replacing CaCO3 with CaCl2 from 35 to 113 kg BW.
81
4 Page 4 of 29
82
2. Materials and Methods
83
2.1.
Twenty-four female pigs (Landrace-Yorkshire x Duroc) were randomly assigned to four
ip t
84
Diets and experimental design
experimental diets (Table 1) in a 2 by 2 factorial arrangement. The basal diet (major ingredients:
86
barley, wheat, soy bean meal, and fat) was optimized according to the Danish 30-105 kg BW
87
recommendations for amino acids, P, and Ca (Tybirk et al., 2012). To obtain the four experimental
88
diets, the basal diet was divided into four batches and supplemented with BA (VevoVitall®, DSM
89
Special Products, Rotterdam, Holland), CaCO3 (40% Ca; CaCO3) or CaCl2 (28% Ca; CaCl2·H2O,
90
CaCl2·2H2O) as follows: diet -BA/CaCO3 (no BA, 14 g/kg CaCO3, considered as a control diet),
91
diet +BA/CaCO3 (10 g/kg BA, 14 g/kg CaCO3), diet -BA/CaCl2 (no BA, 20 g/kg CaCl2), and diet
92
+BA/CaCl2 (10 g/kg BA and 20 g/kg CaCl2). The level of 10 g BA/kg diet was chosen because it is
93
the maximum level of supplementation allowed in the EU (Commission, 2007). The CaCO3 level
94
supplied Ca to meet the recommendations (Tybirk et al., 2012), and the CaCl2 diets were designed
95
to supply equimolar amounts of Ca. On the basis of the chemical composition (Table 1), dEB in
96
meq was calculated as (Na+/23.0 + K+/39.1 – Cl-/35.5) x 1000, where Na+, K+ and Cl- were in g/kg
97
DM to be 185, 182, -124, and -91 meq/kg DM for -BA/CaCO3, +BA/CaCO3, -BA/CaCl2, and
98
+BA/CaCl2 diets, respectively.
99 100 101
2.2.
Ac ce p
te
d
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an
us
cr
85
Animals and husbandry
The pigs were subjected to feeding from 36 ± 2.0 kg BW until slaughter, when the average
102
weight of the fastest growing treatment group reached 113 kg. During this period, in the weight
103
range from 60 to 66 kg BW, the pigs were used in a mineral balance study. All experimental
104
procedures have been carried out in accordance with the Danish Ministry of Justice, Law no. 253 of
5 Page 5 of 29
105
8 March 2013 concerning experiments with animals and care of experimental animals issued by the
106
Danish Animal Experiments Inspectorate.
107
The pigs were housed individually in pens with concrete floor and straw as rooting material. Diets were fed ad libitum as dry coarse diets. There was free access to water through the entire
109
experimental period. During the balance study from 60 to 66 kg BW, the pigs were housed in
110
stainless steel balance cages for total collection of urine and feces. After 5 d of adaptation, all pigs
111
were fitted with urine bladder catheters allowing collection of urine directly into a closed container.
112
The pigs were fed twice daily at 0800 and 1430 h and had free access to demineralized water. The
113
offered daily rations were 1800, 1818, 1810, and 1828 g feed/d for the treatments -BA/CaCO3,
114
+BA/CaCO3, -BA/CaCl2, and +BA/CaCl2, respectively, which supplied all animals with the same
115
amount of basal diet. The pigs were weighed at the beginning and at the end of the 12 d period.
116
Feed refusals were collected and weighed daily. Urine was weighed and samples collected once
117
daily. Feces were collected twice daily. Urine and feces were pooled over the 7 d period and were
118
stored at 5°C until representative feces samples were prepared after homogenization at d 7. Samples
119
were stored at -20°C until analysis.
cr
us
an
M
d
te
Ac ce p
120
ip t
108
At the end of the experiment, the pigs were slaughtered in random order and 2-3 h after
121
being fed in the morning. The metacarpal bone no. III of the right foreleg was dissected and stored
122
at -20°C. The gastrointestinal tract (GIT) was removed immediately after slaughter and divided into
123
eight segments: the entire stomach, three segments of small intestine (uniform by length), caecum,
124
and three segments of colon (uniform by length). Digesta was collected from the stomach, the third
125
(distal) segment of the small intestine (ileum and part of jejunum), the caecum, and the second
126
segment of colon (mid colon). After homogenization of digesta from the individual segments,
127
samples (~10 g) for analysis of organic acid content (see further below) were transferred to
128
stomacher bags and stored at -20ºC. Digesta pH was measured directly in the homogenized digesta
6 Page 6 of 29
129
samples using a pH meter (Radiometer, Copenhagen, Denmark), and samples (5 - 10 g) were
130
maintained for determination of DM content.
132
2.3.
ip t
131 Chemical analysis
Dry matter content, total nitrogen and crude fat in diets, and DM in feces were analyzed as
134
described by Nørgaard et al. (2011). Phosphorus and Ca was analyzed in diets, non-defatted bone,
135
urine, and feces after ashing at 450°C and hydrochloric acid/nitric acid treatment. Phosphorus was
136
analyzed as a vanadomolybdate-complex by spectrophotometry (Lambda 900, Perkin Elmer,
137
Überlingen, Germany) (Stuffins, 1967) and Ca by atomic absorption spectrophotometry (model
138
SP9, Pye Unicam Ltd., Cambridge, UK). For analysis of Na and K in diets, lithium chloride was
139
added to the samples and measured by flame photometry (FLM3 Flame Photometer, Radiometer,
140
Copenhagen, Denmark). Chloride was extracted with nitric acid and analyzed by potentiometric
141
titration with silver nitrate using a titrator (T70, Mettler Toledo, OH). Phytate bound P and phytase
142
activity was analyzed as described by Carlson et al. (2012). The pH in diets, urine, and feces was
143
measured by a pH meter (Radiometer, Copenhagen, Denmark). The concentration of organic acids,
144
i.e. short-chain fatty acids (acetate, propionate, and butyrate), sorbic acid, lactic acid, succinic acid,
145
BA, and hippuric acid, were measured as described by Canibe et al. (2007), and expressed per kg of
146
fresh digesta to reflect in situ concentrations of the organic acids.
us
an
M
d
te
Ac ce p
147
cr
133
Bone mineralization was measured by quantitative computed tomography (CT-scanning)
148
which is an in vivo method for measuring bone mass in the peripheral skeleton where bone density
149
is slow to change; it directly relates bone densitometry to actual bone density and is therefore
150
suggested by Castellanos et al. (2011) as a standard in bone densitometry. Bone density denotes the
151
solid constituents per cubic centimeter of bones, which is interpreted as mineral content or
152
mineralization. A Stratec XCT 2000 Research+ scanner (Stratec Medizintechnik GmbH, Pforzheim,
7 Page 7 of 29
Germany) was used, performing 1 mm slices at the proximal and distal epiphysis and in the middle
154
of the diaphysis. Identical locations were identified for all bones at the proximal and distal
155
trochanter protuberances, then a third slice was selected from the middle point between them. The
156
results were scored by two measures: the T-score and the Z-score. These scores indicate the amount
157
of mineral density; negative scores show the lower bone density, whereas positive scores indicate
158
higher amounts of minerals. In this study, only positive scores were taken into consideration,
159
because it was not intended to establish risk-fracture parameters (Winzenberg and Jones, 2011).
160
Negative scores were not considered as they, by consensus of the World Health Organization
161
(WHO), are used to asses fracture risk (Unnanuntana et al., 2010) and, to our knowledge, this has
162
not been established nor validated for animals.
M
163
an
us
cr
ip t
153
164
2.4.
165
The dietary cation-anion difference (DCAD) is given in meq/kg DM and is calculated as the
166
difference between the sum of cations and anions. In many practical situations, DCAD is called
167
dEB and is calculated as meq Na+ + K+ - Cl- which is a simplified equation for the dietary elements
168
in maintaining acid-base homeostasis (Mongin, 1981). The equation for calculating dEB in meq per
169
kg DM was: (Na+/23.0 + K+/39.1 – Cl-/35.5) x 1000, where Na+, K+ and Cl- were in g/kg DM. The
170
results on animal performance, mineral balance, and mineral density of bones were statistically
171
analyzed by GLM in SAS (SAS Institute Inc. Cary, NC) according to a model including main
172
effects (+/- BA and Ca-source) and the interaction between main effects. Multivariate analysis of
173
variance was done on responses in bones using the MANOVA statement in the GLM procedure of
174
SAS. The statistical model used to estimate the effect of diet on various response variables in
175
digesta was a mixed linear model which included diet, segment of the GIT, and diet × segment as
176
fixed effects. To capture the correlation between measurements in different segments of the GIT of
Ac ce p
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Calculations and statistical analysis
8 Page 8 of 29
each pig, the random errors were allowed to be correlated by a compound symmetry covariance
178
structure using the repeated statement in SAS. Statistical significance was accepted at P<0.05 and
179
tendencies at P<0.10. For pairwise comparisons, Fisher’s protected LSD test was used to separate
180
treatment means. Presented data are least squares means and standard error of means.
ip t
177
181
cr
183
3. Results
None of the animals showed symptoms of impaired health and wellbeing. The concentration of
us
182
BA in the stomach content of pigs receiving the BA supplemented diets was approx. 10 mmol/kg
185
fresh digesta; BA was neither detectable in other GIT segments of these animals nor in any
186
segments of animals receiving the non-supplemented diets (data not shown). In general, digesta
187
contents of the 3 major short-chain fatty acids (acetate, propionate, and butyrate) were not
188
significantly affected by the BA and CaCl2 supplementations (Table 2). A reduction in digesta
189
concentration was observed for acetate (P<0.01) and propionate (P=0.03) in the colon for the CaCl2
190
supplemented diet, whereas no significant effects were observed for digesta concentrations of
191
butyrate (Table 2). Lactate was only detectable in the proximal gastrointestinal segments (stomach
192
and small intestine) and was lower (P<0.01) in stomach contents for the CaCl2 diets. No further
193
significant effects of the dietary treatments were observed neither for the rest of the analyzed
194
organic acids (P>0.05, data not shown) nor for the digesta pH (Table 2). The dry matter content of
195
the digesta was affected by diet and was higher (P<0.05) in all GIT segments for pigs receiving the
196
CaCl2 diets.
197
Ac ce p
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184
The pH in feces was increased (P<0.01) when feeding pigs CaCl2 compared to CaCO3. In
198
urine, both BA (P=0.04) and CaCl2 (P<0.01) decreased pH, and the effect of both BA and CaCl2
199
was a decrease in two pH units compared to the -BA/CaCO3 treatment (Table 3).
9 Page 9 of 29
200
The mineral balance was affected by BA and especially by CaCl2 supplementation. On the P balance, interactions (P<0.03) were observed between the two factors. The apparent digestibility of
202
P was the lowest and the urinary P excretion was the highest in pigs fed the +BA/CaCl2 diet
203
resulting in the lowest P retentions. The apparent digestibility of Ca was the lowest in pigs fed the
204
+BA/CaCl2 diet. The urinary excretion of Ca was the greatest (P<0.01) in pigs fed CaCl2. Ca
205
retention was the lowest (P<0.05) in pigs fed the +BA/CaCl2 diet.
cr
The weight and length of metacarpal bone no. III was lower (P=0.04) and shorter (P=0.02),
us
206
ip t
201
respectively, when pigs were fed the CaCl2 diets, whereas there was no effect of BA
208
supplementation. Bone ash and P concentration was reduced by both replacing CaCO3 with CaCl2
209
(P=0.02) and by supplementing diets with BA (P<0.03). The concentration of Ca in metacarpal
210
bones was affected by neither of the treatments.
M
211
an
207
The density of minerals measured by CT-scanning was affected by both BA and CaCl2 (Table 4). Supplementation of BA resulted in lower mineral density in the medial (P=0.01) and distal
213
(P<0.01) sections of the metacarpal bones, and replacing CaCO3 with CaCl2 resulted in lower
214
mineral density in the proximal (P=0.01), medial (P=0.01), and distal (P<0.01) sections. When
215
dividing the bone into trabecular and cortical tissue, the results were somewhat less clear.
216
Trabecular tissue in the distal bones of pigs fed diets supplemented with CaCl2 had a greater
217
(P=0.02) mineral density compared to pigs fed the diets supplemented with CaCO3, whereas it was
218
lower in cortical tissue in both the medial (P<0.01) and distal (P<0.01) sections. Multivariate
219
analysis of results on mineral densities showed an effect of replacing CaCO3 with CaCl2 (P=0.03),
220
but not by BA supplementation (P=0.12). Considering both mineral concentrations and densities in
221
the multivariate analysis, there was a tendency (P=0.06) of an effect by replacing CaCO3 with
222
CaCl2, but not by BA supplementation (P=0.16).
Ac ce p
te
d
212
223
10 Page 10 of 29
224 225
4. Discussion By mixing feces and urine collected in the balance study (Eriksen et al., unpublished), it was observed that ammonia emission was reduced by 24-37% in slurry from pigs fed the diets
227
supplemented with BA or where CaCO3 was replaced with CaCl2. This confirms our previous
228
findings (Eriksen et al., 2010) of feeding as an important and cost efficient mitigation option,
229
provided that is does not imply negative effects on animal performance and welfare.
cr
The dEB and dietary undetermined anion (dUA) has pronounced effects on nutrient
us
230
ip t
226
metabolism and the physiological status of the pigs (Patience and Chaplin, 1997). When feeding
232
pigs with diets with negative dEB, the blood pH, HCO3-, and base excess are decreased (Patience et
233
al., 1987; Budde and Crenshaw, 2003). The changes observed in these experiments were, however,
234
not drastic enough to affect animal health.
M
235
an
231
When replacing CaCO3 with CaCl2, the urinary pH decreased 1.3 units. Similar reductions in urinary pH after feeding pigs low-dEB diets have been observed before (Canh et al., 1998). Benzoic
237
acid is efficiently absorbed from the small intestine and efficiently metabolized in the liver to
238
hippuric acid and is excreted into urine (Kristensen et al., 2009). The reduction of urinary pH by
239
dietary BA was in accordance with other studies (Canh et al., 1998; Sauer et al., 2009; Nørgaard et
240
al., 2010b). The combined effect of CaCl2 and BA was a urinary pH reduction of 2.0 units.
241
Organic acids are oxidized within the body and therefore do not affect the acid-base
Ac ce p
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236
242
physiology. An exception is BA which is not oxidized but is efficiently converted into hippuric acid
243
in the liver and subsequently excreted to urine by the kidneys (Kristensen et al., 2009). Benzoic acid
244
and hippuric acid may therefore present a temporary acid load to the extracellular fluid
245
compartment, thus increasing the demand for buffering capacity which among other mechanisms
246
partly includes the intermediary metabolism of minerals. In this way, BA has effects similar to low
247
dEB and dUA, and therefore it ought to be taken into account when calculating the dEB (Kristensen
11 Page 11 of 29
et al., 2009). A decrease in blood pH suppresses osteoblast activity and stimulates osteoclast
249
activity, thereby stimulating bone mobilization of P and Ca along with ongoing proton buffering by
250
the bone (Krieger et al., 2004). Prolonged mineral mobilization from bone will eventually lead to
251
bone weakness. Even though the response of osteoclasts to changes in pH is steep (Arnett, 2003),
252
previous studies have shown a reduction in blood pH caused by BA supplementation, but without
253
effects on blood phosphate levels (Kristensen et al., 2009; Nørgaard et al., 2010b). The mechanism
254
behind the small impact on acid-base status in pigs is likely to be a protracted absorption of BA
255
(Kristensen et al., 2009). Sauer et al. (2009) observed increased blood pH and increased plasma P
256
concentrations in pigs after feeding 20 g/kg BA for 21 d and argued for an increased renal distal
257
tubule expression of carbonic anhydrase stimulated by long term and high levels of BA. This
258
hypothesis seems only relevant for BA doses higher than 10 g/kg, which is the maximum inclusion
259
level approved in the European Union, since other long term studies using 5 g/kg (Buhler et al.,
260
2010) and 10 g/kg (present study) showed no changes or reductions in blood P and Ca
261
concentrations (Buhler et al., 2010) and retentions (Buhler et al., 2010; present study).
te
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Calcium carbonate is typically supplemented to pig diets at approx. 15 g/kg to provide dietary
263
Ca which mainly supports growth and maintenance of bone. The urinary pH can be reduced simply
264
by lowering the amount of supplemented CaCO3 (Canh et al., 1998). By replacing CaCO3 with
265
CaCl2 as the main source of Ca, several effects were obtained. Besides the lower dEB obtained
266
through the increased inclusion of Cl, the change in carbonate content made the diets acidogenic
267
and with a lower acid binding capacity. The inclusion of CaCO3 increases the acid binding capacity
268
which is a measure reflecting pH buffering in the stomach, and it is important for especially protein
269
digestion and animal health in general (Lawlor et al., 2006). The efficiency of Ca absorption is
270
greatly dependent both the Ca and P concentrations in the diet, and the digestibility of CaCO3 is
271
therefore not simple to estimate, but can be expected to be in the range from 0.45 to 0.70
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12 Page 12 of 29
(González-Vega et al., 2013). Thus, large amounts of undigested CaCO3 are passing through the
273
GIT ending up in the slurry, where it can be speculated to act as a buffering agent and counteract
274
any biological or mechanical acidifying efforts. These concerns would argue for reducing the
275
inclusion of CaCO3 and for including other and more digestible Ca sources. However, in the present
276
study, the pH was for unknown reasons higher in feces from pigs fed CaCl2 compared to CaCO3 as
277
the main Ca sources.
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The balances of P and Ca were clearly affected by the dietary treatments, also when the lower
279
P and Ca intake of the groups of pigs fed both BA and CaCl2 is taken into account. The lower feed
280
intake of this group corresponds well to early findings that low dUA reduces appetite (Patience and
281
Wolynetz, 1990). In particular, the diets replacing CaCO3 with CaCl2 resulted in lower P and Ca
282
retentions due to increased urinary excretions, and in relation to P, it was also due to decreased
283
digestibility. Increased urinary Ca excretion was also observed when increasing dietary Cl-
284
concentration, but no significant effects on urinary P excretion (Golz and Crenshaw, 1991; Patience
285
and Chaplin, 1997). A positive effect by BA on P and Ca apparent faecal digestibility has been
286
reported before (Sauer et al., 2009; Nørgaard et al., 2010a), although in the present study there were
287
no significant differences. There was no effect on P and Ca excretion by supplementing BA to the
288
CaCO3 based diet, and this is in accordance with several other studies on BA supplementation
289
(Nørgaard et al., 2010a; Nørgaard et al., 2010b; Gräber et al., 2012) and in contrast to Sauer et al.
290
(2009), who found reduced P and Ca excretions. The combined effect of main treatments, i.e. the
291
pigs fed the +BA/CaCl2 diet, resulted in lower apparent faecal digestibility and retention of both P
292
and Ca. Especially the 14% reduction in P apparent faecal digestibility could have strong practical
293
relevance in pig production. Mineral solubility is generally thought to be increased through an
294
acidified gastro-intestinal environment lowering the rate of gastric emptying (Jongbloed, 1987) and
295
increasing the solubility of mineral chelates (Cao et al., 2000; Guo et al., 2001) and other
Ac ce p
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13 Page 13 of 29
complexes. An increased absorption of P and Ca by acidifying the gastro-intestinal environment has
297
recently been confirmed in sows fed citric acid (Liu et al., 2014). However, since the combination
298
of supplementing BA and replacing CaCO3 with CaCl2 reduced the apparent digestibility and
299
retention of P and Ca, it does not appear that an increased absorption is solely due to a lower
300
intestinal pH.
ip t
296
The weight, length, mineral content and mineral density of metacarpal bones was affected by
302
both BA and by replacing CaCO3 with CaCl2. Previous studies using pigs of similar weight ranges
303
showed tendencies of lower bone mineral density and content when fed 5 g/kg BA until 66 kg BW,
304
but no effect at greater live weight (108 kg) (Buhler et al., 2010). In pigs weighing 64 kg BW, no
305
effect was found of BA on bone mineral content and breaking strength (Gutzwiller et al., 2011).
306
The bone strength is affected by mineral density and bone structure, and there is a direct relation
307
between bone mineralization and bone strength in both human (Zumstein et al., 2012) and pigs
308
(Nielsen et al., 2007). The risk of fragility fractures in humans is indicated by the T score which
309
compares one individual’s bone mineral density in standard deviations with the mean peak density
310
in healthy young individuals. Osteoporosis is defined by the World Health Organization as 2.5 x
311
standard deviations or more below mean peak density (Brunader and Shelton, 2002). When
312
considering the bone mineral density of pigs in the control group (-BA/CaCO3) as representative for
313
healthy pigs, the T score was calculated for total bone mineral densities in the three sections. In the
314
distal sections, four of the six pigs of group +BA/CaCl2 were in risk of osteopenia, a precursor for
315
osteoporosis; in the medial section, 1, 0, and 2 of the six pigs in the groups +BA/CaCO3, -BACaCl2,
316
and +BA/CaCl2, respectively, were in risk of osteopenia; in the proximal section, 1, 1, and 2 of the
317
six pigs in the groups +BA/CaCO3, -BACaCl2, and +BA/CaCl2, respectively, were in risk of
318
osteopenia. Taking into account that the six pigs in the -BA/CaCO3 group can barely be considered
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14 Page 14 of 29
319
a population, the risk of bone fractures may be increased when both supplementing diets with BA
320
and replacing CaCO3 with CaCl2.
321
Benzoic acid is known to possess stronger antimicrobial properties than a range of other organic acids (Knarreborg et al., 2002). Of the dietary BA, 13% cannot be found in the portal blood,
323
and may therefore pass through the intestine (Kristensen et al., 2009), explaining previous results
324
that caecal microbial diversity was affected by feeding 5 g/kg BA (Halas et al., 2010). In the present
325
study, however, 10 g/kg BA did not significantly affect the digesta concentrations of lactate,
326
acetate, propionate, and butyrate throughout the GIT, indicating that the microbial activity was not
327
dramatically affected by the dietary treatment. The observed reduction in gastric lactate
328
accumulation of pigs receiving the CaCl2 diets resembles the observations of a recent study where a
329
high Ca (calcium monohydrogen phosphate) level was observed to reduce gastric lactate
330
accumulation (Metzler-Zebeli et al., 2011). The working mechanism behind the observed Ca effects
331
is unclear. Increased levels of dietary Ca have been shown to induce increased secretion of gastric
332
acids in rats (Floor et al., 1991) which may again lead to decreased stomach pH and, subsequently,
333
impaired microbial activity. However, neither the present study nor the study of Metzler-Zebeli et
334
al. (2011) demonstrated any reduction in stomach pH as a result of the Ca source and level,
335
respectively. On the other hand, Ca source and level may be argued to influence the buffer capacity
336
of the feed and thereby counteract any pH-decreasing effect of gastric acid secretion (Lawlor et al.,
337
2006). Therefore, even though the gastric pH levels did not differ by the time of slaughter and
338
digesta sampling, stomach acidification (inhibiting bacterial activity) after feed intake may have
339
occured more rapidly for the animals receiving the CaCl2 diets (low buffer capacity), explaining the
340
lower accumulation of organic acids observed for these animals. It was suggested and supported by
341
the observations of Metzler-Zebeli et al. (2011) that a high Ca level may moreover stimulate
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15 Page 15 of 29
342
fermentation of lactate to butyrate by e.g. Megasphaera elsdenii a mechanism like this is not
343
indicated by the digesta butyrate concentrations in the present study.
344 5. Conclusion
ip t
345
BA was efficiently absorbed in the proximal GIT, and had no significant effects on digesta
347
concentrations of organic acids. Replacing CaCO3 with CaCl2 was observed to suppress organic
348
acids in the stomach and colon. Reductions in urinary pH were found both after feeding BA and
349
after replacing CaCO3 with CaCl2. The dietary supplementation of BA and the replacement of
350
CaCO3 with CaCl2 affected the nutrient balances of P and Ca. Mineral concentration and total
351
mineral density of metacarpal III bones were reduced both by BA supplementation and by replacing
352
CaCO3 with CaCl2. Taking the limited number of animals into account, the changes in mineral
353
balance and density of bones caused by feeding either BA or by replacing CaCO3 with CaCl2 did
354
not appear to severely affect bone strength, but their combined effect may affect bone strength
355
during long-term feeding.
357 358
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346
Acknowledgements
This project was partly funded by the Danish Food Industry Agency under the Danish Ministry
359
of Food, Agriculture and Fisheries and partly by the Department of Animal Science, Aarhus
360
University.
361 362
References
363
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Animal Feed Science and Technology Ms. No. ANIFEE-14-5460 Conflict of interests statement
The authors have no conflicts of interests regarding this work
ip t
523 524 525 526 527 528
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29 Page 23 of 29
Table 1 Feedstuff and analyzed chemical composition of diets supplemented with 0 or 10 g/kg benzoic acid (BA, +BA) and two Ca-sources (CaCO3 or CaCl2). Diet +BA/
-BA/
CaCO3
CaCO3
CaCl2
Barley
562
562
Wheat
230
Soybean meal, toasted dehulled
177
Fat
20.0
cr 562
us
Feedstuff composition (g/kg)
+BA/
ip t
-BA/
CaCl2
562
230
230
177
177
177
20.0
20.0
20.0
1.7
1.7
1.7
0.4
0.4
0.4
0.4
0.5
0.5
0.5
0.5
2.7
2.7
2.7
2.7
3.5
3.5
3.5
3.5
Vitamin and mineral mixa
2.0
2.0
2.0
2.0
Phytaseb
0.3
0.3
0.3
0.3
14.0
14.0
0
0
0
0
20.0
20.0
0
10.0
0
10.0
Dry matter (DM)
868
863
863
860
Crude protein
199
197
197
198
Ash
48.5
47.0
48.6
46.4
Crude fat
47.9
49.2
47.8
45.3
1.7
M
L-lysine HCl (78%) DL-methionine (99%)
CaCl2c
Ac
CaCO3c
ce pt
NaCl
ed
L-threonine (99%) Monocalciumphosphate
Benzoic acidc
an
230
Chemical composition (g/kg DM)
22 Page 24 of 29
8.1
8.1
8.2
8.2
Calcium
7.7
7.8
8.2
7.2
Phosphorus
4.3
4.2
4.3
4.2
Chloride
3.4
2.9
14.3
13.0
Sodium
1.7
1.3
1.6
1.5
Phytate bound P
2.6
2.5
Phytase, FTU/g
2941
2673
Benzoic acid
0.0
8.2
pH
6.1
dEB, meq/kg DMd
185
ip t
Potassium
2.3
2302
2446
cr
2.4
us
0.4
8.9
5.2
4.7
182
-124
-91
an
5.5
482
a
483
vitamin E, 0.6 mg vitamin K3, 0.6 mg vitamin B1, 1.2 mg vitamin B2, 3 mg D-panthotenic acid, 6.4 mg
484
niacin, 0.060 mg biotin, 21.3 mg α-tocopherol, 0.006 vitamin B12, 0.6 mg vitamin B6, 50 mg Fe (Fe(II)
485
sulphate), 41.3 mg Cu (Cu(II) sulphate), 50 mg Zn (Zn(II) oxide), 13.9 mg Mn (Mn(II) oxide), 0.076 mg KI,
486
0.075 mg Se (Se-selenite).
487
b
Natuphos 5000 5641 FTU/g, BASF, 2300 Copenhagen, DK.
488
c
Supplemented to batches of the basal diet. Benzoic acid was VevoVitall, DSM Special Products,
489
Rotterdam, NL.
490
d
M
ed
ce pt
dEB, dietary electrolyte balance.
Ac
491
Trouw Nutrition Denmark A/S. Content per g premix: 2500 IU vitamin A, 500 IU vitamin D3, 23.4 IU
23 Page 25 of 29
492
Table 2
493
Concentrations (mmol/kg fresh digesta) of organic acids and pH of digesta samples taken
494
immediately after slaughtering animals fed diets supplemented with 0 or 10 g/kg benzoic acid (-BA,
495
+BA) and two Ca-sources (CaCO3 or CaCl2) from 35 to 113 kg BW. Dieta -BA/
+BA/
-BA/
ip t
Segmentb
Ca
BA×Ca
cr
Item
P-value
+BA/
BA
0.17
0.78
SEM
Butyrate
CaCl2
CaCl2
St
26.1
22.6
21.5
19.6
Si
28.1
24.5
26.1
22.3
4.7
0.45
0.67
0.98
Ce
96.3
94.0
86.2
89.4
4.0
0.91
0.09
0.50
Co
96.8
92.9
79.0
82.7
3.7
0.97
<0.01
0.31
St
8.6
7.3
8.4
7.8
1.1
0.41
0.92
0.77
Si
bdc
bd
bd
bd
-
-
-
-
Ce
54.9
Co
47.1
us
an
M
47.5
49.9
3.7
0.10
0.30
0.79
38.5
35.2
35.5
3.1
0.20
0.03
0.17
8.1
7.9
5.8
6.3
1.0
0.86
0.08
0.73
2.8
3.1
2.9
2.1
0.8
0.69
0.56
0.49
16.6
15.7
18.4
21.0
1.9
0.65
0.08
0.38
Co
19.3
18.9
20.5
21.4
1.7
0.88
0.29
0.70
St
91.6
76.9
45.3
43.3
13.4
0.54
<0.01
0.64
Si
65.2
56.1
67.9
64.1
13.9
0.65
0.70
0.85
Ce
bd
bd
bd
bd
-
-
-
-
Co
bd
bd
bd
bd
-
-
-
-
St
3.8
3.8
3.7
3.9
0.1
0.33
0.83
0.16
St
Ac
Ce
pH
0.33
44.5
Si
Lactate
2.7
ed
Propionate
CaCO3
ce pt
Acetate
CaCO3
24 Page 26 of 29
Si
6.3
6.2
5.9
6.0
0.2
0.90
0.07
0.84
Ce
5.7
5.6
5.5
5.6
0.1
0.98
0.20
0.18
Co
6.2
6.0
6.3
6.1
0.1
0.11
0.36
0.70
a
Values are least square means and standard errors of the means (SEM). n = 6.
497
b
Samples were taken from stomach (St), small intestine (Si), cecum (Ce), and mid colon (Co).
498
c
bd, below detection limit.
ip t
496
cr
499
Ac
ce pt
ed
M
an
us
500
25 Page 27 of 29
Table 3
502
Feed intake, pH in feces and urine, daily intake, apparent digestibility, urinary excretion, retention, and
503
retention in percent of intake of P and Ca of pigs in a 12 days balance study fed diets supplemented with 0 or
504
10 g/kg benzoic acid (-BA, +BA) and two Ca-sources (Ca) (CaCO3 or CaCl2).a Dieta
P-value -BA/
+BA/
CaCO3
CaCO3
CaCl2
CaCl2
1497
1500
1417
1361
Feces pH
6.8
6.6
7.3
7.6
Urine pH
8.2
7.9
6.9
Intake, g/d
5.57
5.41
5.27
App. diges.
0.64a
0.65a
0.64a
Urine, g/d
0.09b
0.03b
Retention, g/d
3.47a
Retention, %
Feed intake, g/d
SEM
BA
BA×Ca
68
0.66
0.08
0.62
0.2
0.87
<0.01
0.22
0.3
0.04
<0.01
0.40
4.96
0.25
0.28
0.10
0.73
0.55b
0.03
0.08
0.03
0.03
0.09b
0.28a
0.05
0.11
<0.01
<0.01
3.47a
3.13a
2.42b
0.17
0.03
<0.001
0.03
62.2a
64.2a
60.9a
49.3b
2.8
0.07
<0.01
0.01
10.1a
10.1a
10.0a
8.4b
0.46
0.08
0.05
0.05
0.74ab
0.79a
0.79a
0.68b
0.04
0.50
0.46
0.04
Urine, g/d
0.15
0.27
0.90
0.95
0.15
0.52
<0.01
0.81
Retention, g/d
7.20a
7.70a
6.95a
4.79b
0.42
0.04
<0.001
0.002
Retention, %
72.0a
76.3a
70.0a
57.3b
5.0
0.33
0.02
0.05
Intake, g/d
Ac
App. diges.
505
a
506
a,b
ed
ce pt
Ca balance
M
P balance
6.2
Ca
cr
+BA/
us
-BA/
an
Item
ip t
501
Values are least squares means and standard errors of the means (SEM). n = 6. Within a row for items with a significant interaction, means without a common superscript differ (P<0.05).
507
26 Page 28 of 29
Table 4
509
Bone weight and size, ash, Ca, and P concentration, and mineral density measured by CT-scanning in
510
metacarpal III bones of pigs fed diets supplemented with 0 or 10 g/kg benzoic acid (-BA, +BA) and two Ca-
511
sources (Ca) (CaCO3 or CaCl2) from 35 to 113 kg BW.a Itemb
P-value
-BA/
+BA/
-BA/
+BA/
BA×Ca
CaCO3
CaCO3
CaCl2
CaCl2
Weight, g
28.7
30.7
28.0
27.0
1.0
0.64
0.04
0.15
Length, mm
78.7
78.5
76.4
75.1
1.1
0.53
0.02
0.60
Ash, g/100 g
28.2
26.4
26.2
24.7
0.7
0.02
0.02
0.81
Ca, g/100 g
10.7
10.1
10.1
9.8
0.4
0.31
0.28
0.61
P, g/100 g
5.1
4.7
4.8
4.5
0.1
0.02
0.03
0.70
Proximal, total, mg/cm3
347.4
330.8
306.7
298.1
13.6
0.37
0.01
0.77
Proximal, trabec., mg/cm3
168.2
176.8
181.1
183.8
12.4
0.66
0.43
0.81
Proximal, cortical, mg/cm3
605.3
575.9
565.8
564.6
16.9
0.38
0.15
0.41
Medial, total, mg/cm3
364.8
336.6
337.5
296.9
12.5
0.01
0.01
0.62
Medial, trabec., mg/cm3
43.5
43.2
42.8
37.4
2.4
0.25
0.19
0.30
Medial, cortical, mg/cm3
857.7
864.5
839.0
841.1
6.0
0.46
<0.01
0.70
Distal, total, mg/cm3
447.8
427.0
409.5
368.2
11.4
<0.01
<0.01
0.35
Distal, trabec., mg/cm3
244.0
240.5
254.0
250.7
4.2
0.40
0.02
0.97
Distal, cortical, mg/cm3
583.2
571.1
557.1
542.3
6.8
0.06
<0.01
0.84
BA
us
an
ed
ce pt
Ac
SEM
cr
Ca
M
Diet
ip t
508
512
a
Values are least squares means and standard errors of the means (SEM). n = 6.
513
b
Mineral density of trabecular and cortical tissues or in total was measured in the proximal, medial and distal
514
metacarpal III bone of the right foreleg. Trabec = trabecular.
515
27 Page 29 of 29