Effect of the addition of glycosaminoglycans on bone and cartilaginous development of broiler chickens Sarah Sgavioli,∗,1 Elaine T. Santos,† Liliana L. Borges,† Giuliana M. Andrade-Garcia,† Diana M. C. Castiblanco,† Vitor R. Almeida,† Rodrigo G. Garcia,‡ Antônio C. Shimano,§ Irenilza A. Nääs,‡ and Silvana M. Baraldi-Artoni† ∗Brazil University, Descalvado, SP, Brazil; †Department of Morphology and Animal Physiology, São Paulo State University, Jaboticabal, SP, Brazil; ‡College of Agricultural Sciences, Federal University of Grande Dourados, Dourados, MS, Brazil; and §Department of Bioengineering, São Paulo State University, Ribeirão Preto, SP, Brazil ABSTRACT Locomotion issues in broiler production may decrease performance (carcass yield and traits) and lead to high financial losses. This study evalu- ates the addition of glucosaminoglycans in broiler di- ets to minimize the lack of proper bone development and joint weakening. The experiment was conducted using 2,160 broilers randomly distributed in a facto- rial pattern (3 × 3) using 3 levels of glucosamine sul- fate (0, 0.12, and 0.24%) and 3 levels of chondroitin sulfate addition (0, 0.08, and 0.16%). Eight repeti- tions were used for each treatment, distributed in 72 pens with 30 broilers each. There was a quadratic effect on feed conversion for broilers from 1 to 42 d old (P = 0.0123) for the addition of chondroitin, and better feed conversion was obtained by adding 0.08% of chondroitin. The relative tibia weight, the width of the proximal epiphysis and diaphysis pre- sented a linear increased effect in broilers at 42 d old. An interaction was found between the amount of chondroitin × glucosamine and the number of chon- drocytes in the proximal cartilage of the tibia (P = 0.0072). There was a quadratic effect of glucosamine levels (P = 0.0107) in the birds that had received the 0.16% addition of chondroitin, and the presence of 0.18% glucosamine increased the number chondro- cytes in the cartilage of broilers. These results provide the first evidence that broilers may benefit from in- creased dietary chondroitin sulfate. These results in- dicate that the addition of glucosamine and chon- droitin sulfates in broiler feed rations might alleviate leg conditions and decrease financial losses in the broiler industry. Key words: bone development, chondroitin, glucosamine, leg condition, performance 2017 Poultry Science 96:4017–4025 http://dx.doi.org/10.3382/ps/pex228 INTRODUCTION Modern broiler chickens have been specially bred for rapid growth. However, this accelerated growth may lead to metabolic disorders resulting in high financial losses. Examples of these disorders include locomotion problems from weak legs (Su et al., 2003; Nääs et al., 2012), bone deformations, fractures, and related struc- tural lesions. These are severe health issues that re- duce broiler performance because the affected birds have difficulty in accessing water and feed (Almeida Paz et al., 2008). These conditions are not limited to Brazil, but account for broiler production losses world- wide (Almeida Paz et al., 2010). Pharmaceutical products can prevent the develop- ment of structural lesions or reduce the progression C© 2017 Poultry Science Association Inc. Received April 3, 2017. Accepted August 7, 2017. 1Corresponding author: sarahsgavioli@yahoo.com.br of pathological changes in an animal’s bone structure (Smith et al., 1999). One such product, glycosaminogly- can (GAGs) polysulfate, has shown promise in treating such lesions (Vaughan-Scott and Taylor, 1997). The active principle in GAGs polysulfate and simi- lar agents have an anti-inflammatory action, may re- duce the loss of proteoglycan and collagen by stim- ulating their synthesis, and can increase proliferation of chondrocytes and the biosynthesis of the matrix (Wollenweber et al., 2006). This supports the hypothe- sis that changes in joint cartilage can be alleviated, and the use of GAGs might complement treatment (Kantor et al., 2014). Among the GAGs most used to try to minimize joint deterioration and prevent leg conditions are glu- cosamine sulfate and chondroitin sulfate. Furthermore, the use of GAGs to minimize locomotion issues has been studied in several species, including bovine (de Mattei et al., 2002), equine (Hanson et al., 1997; Fenton et al., 2000a), and canine (Gonçalves et al., 2008; Eleotério et al., 2012). In this context, the use of GAGs as 4017 D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/11/4017/4157254 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 24 June 2019 mailto:sarahsgavioli@yahoo.com.br 4018 SGAVIOLI ET AL. poultry feed supplements might be of substantial im- portance in commercial broiler production. However, no research has been published regarding the use of GAGs in preventing broiler locomotion issues. This research aimed to study the use of supplemen- tal GAGs in broiler diets to see if they might amelio- rate the structure of the locomotor system (bones and joints), thus preventing locomotion problems in com- mercial broilers. MATERIALS AND METHODS This experiment was approved by the Animal Ethics Committee (CEUA) of the College of Agriculture and Veterinary Sciences of the São Paulo State University (UNESP—Jaboticabal), Jaboticabal, SP, Brazil, protocol number 0,04732/13. Birds, Housing, and Experimental Design A total of 2,160 1-day-old, male, Cobb R© broiler chicks were reared in 72 pens with 30 birds each until 42 d old. The birds were distributed in a completely ran- domized 3 × 3 factorial design (chondroitin sulfate A in the amounts of 0, 0.08, and 0.16%, and glucosamine in the amounts of 0, 0.12, and 0.24%), with 8 repeti- tions. The chondroitin sulfate [(C14H21NO14S)n, In- finiti Nutraceuticals, Inc., Lake Forest, CA] had a pu- rity of 91.35% and the potassium glucosamine sulfate [(C6H14NO5)2SO4 × 2KCl, Zhejiang Freeman Shinfuda Co. Ltd., China] had a sulfate content of 15.7%. The birds received feed and water ad libitum dur- ing the entire experimental period. Temperatures were maintained by using brooders in the first 3 wk of grow- out and natural ventilation during the remaining 4 wk of grow-out. The chicks were vaccinated in the hatch- ery against Marek’s disease, infectious bursal disease (IBD), and avian pox. The following vaccination pro- gram was completed during the experimental period: IBD (mild strain) on d 7 using eye drops; Newcas- tle disease and IBD (hot strain), through the drink- ing water, using powdered milk as a carrier (2 g L−1) on d 14. The broilers were raised on wood-shaving litter us- ing 1.2 kg of dry substrate per bird. The adopted light regimen was 24L:0D (light: dark). Temperature and relative humidity were recorded daily using 2 digital thermo-hygrometers (Instrutemp, ITHT-2250, temperature scale from –50◦C to 70◦C, pre- cision: ±1◦C, São Paulo, Brazil), placed at the birds’ height in 2 equidistant places. The average and ab- solute maximum temperatures were 30◦C and 34.1◦C, respectively, while the average and absolute minimum temperatures were 18.6◦C and 13.4◦C, respectively. The average and absolute maximum relative humidity were 73.36% and 36.18%, and the average and absolute minimum relative humidity was 27.82% and 15.00%, respectively. Table 1. Percent and calculated nutritional composition of the feed ration for the experimental initial (1–21 d old) and growth (21–42 d old) phases. Ingredient (%) Initial Growth Corn 49.54 55.11 Soybean meal - 45% 40.75 34.39 Soybean oil 5.00 6.20 Dicalcium phosphate 1.70 1.25 Limestone particle 1.10 1.20 Salt 0.40 0.40 L-Lysine HCL (78%) 0.22 0.23 DL- Methionine (99%) 0.32 0.27 L- Threonine 0.07 0.05 Nutritional supplementa 0.50 0.50 Variable portionb 0.40 0.40 TOTAL 100.00 100.00 Calculated nutritional compositionc Crude protein (%) 22.70 20.24 Metabolizable energy (kcal/kg) 3.032 3.180 Ca (%) 0.92 0.84 Na (%) 0.19 0.18 Available phosphorus (%) 0.44 0.35 Digestible methionine + cysteine (%) 0.90 0.80 Digestible methionine (%) 0.61 0.54 Digestible lysine (%) 1.25 1.10 Digestible threonine (%) 0.82 0.72 Digestible tryptophan (%) 0.25 0.21 Digestible arginine (%) 1.41 1.24 aNutrients per kilogram of diet: From 1 to 21 d of age—Vit. A 7,000 U.I., Vit. D3 3000 U.I., Vit. E 25 U.I., Vit. K 0.98 mg, Vit. B1 1.78 mg, Vit. B2 9.6 mg, Vit. B6 3.5 mg, Vit. B12 10 μg, Folic Acid 0.57 mg, Biotin 0.16 mg, Niacin 34.5 mg, Calcium Pantothenate 9.8 mg, Copper 0.12 g, Cobalt 0.02 mg, Iodine 1.3 mg, Iron 0.05 g, Manganese 0.07 g, Zinc 0.09 mg, Organic Zinc 6.75 mg, Selenium 0.27 mg, Choline 0.4 g, Growth promoter (Zinc bacitracin) 30 mg, (narasin+nicarbazin) 0.1 g, Methionine 1.68 g. From 21 to 42 d of age—Vit. A 7000 U.I., Vit. D3 3000 U.I., Vit. E 25 U.I., Vit. K 0.98 mg, Vit. B1 1.78 mg, Vit. B2 9.6 mg, Vit. B6 3.5 mg, Vit. B12 10 μg, Folic Acid 0.57 mg, Biotin 0.16 mg, Niacin 34.5 mg, Calcium Pantothenate 9.8 mg, Copper 0.12 g, Cobalt 0.02 mg, Iodine 1.3 mg, Iron 0.05 g, Manganese 0.07 g, Zinc 0.09 mg, Organic Zinc 6.75 mg, Selenium 0.27 mg, Choline 0.6 g, Growth promoter (avilamycin) 7.5 mg, (monensin sodium) 0.1 g, Me- thionine 1.4 g. bvariable portion is composed of glucosamine sulfate: [(C6H14NO5) 2SO4 × 2KCl, Zhejiang Freeman Shinfuda Co. Ltd.] had a sulfate content of 15.7% and/or chondroitin: [(C14H21NO14S)n, Infiniti Nutraceuticals, Inc.] had a purity of 91.35% and/or inert (kaolin) to meet the percentages proposed in the treatments. cThe analyzed values of crude protein, Ca, available phosphorus, di- gestible methionine + cysteine, digestible methionine, digestible lysine, digestible threonine, digestible tryptophan and digestible arginine were 22.85 and 20.32%; 0.89 and 0.82%; 0.45 and 0.37%; 0.87 and 0.81%; 0.63 and 0.55%; 1.28 and 1.13%; 0.80 and 0.69%; 0.27 and 0.25%; 1.48 and 1.27%, respectively for the 1–21 d old and 21–42 d old. The diets were based on corn and soybean meal (Table 1) and formulated for 2 phases: starter (1 to 21 d old) and grower (22 to 42 d old), as recommended by Rostagno et al. (2011). The total nitrogen content of the experimental diets was analyzed in a nitrogen distiller (LECO, St. Joseph, MI), using the Kjeldahl method (Method No. 2001.11) according to AOAC (2005). A factor of 6.25 was used in the conversion of the nitro- gen value to crude protein (CP). The total amino acids, other than tryptophan, were determined after hydrol- ysis of the protein under acidic conditions. These val- ues were corrected for digestible amino acids using the tabulated coefficients of digestibility (Rostagno et al., D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/11/4017/4157254 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 24 June 2019 GLYCOSAMINOGLYCANS EFFECT ON BONE AND CARTILAGINOUS 4019 2011). Calcium and phosphorus (%) were analyzed as suggested by Silva and Queiroz (2002). Performance At the end of the experimental period (42 d), the av- erage weight was recorded, and the weight gain, feed intake, and feed conversion were calculated. The feed provided and the feed residue were weighed at the be- ginning and the end of both the starter (1 to 21 d) and grower (22 to 42 d) phases. Mortality was recorded daily for the correction of performance parameters and to calculate viability. Broiler Selection and Slaughter On d 42 of grow-out, 81 birds (9 birds per treatment) with average body weights close to the experimental unit’s average body weight, were selected and identi- fied with numbered leg bands. The selected birds were fasted for 8 h and then slaughtered by neck displace- ment, drained, plucked, and eviscerated. The left and right tibia of each bird were removed and marked. Bone Densitometry Bone mineral density (g/cm2), bone area (cm2), and mineral composition (g) were determined on the right tibia (Sgavioli et al., 2016). Bone mineral den- sity was measured in the proximal, distal, and diaphy- seal region using dual-energy radiograph absorptiom- etry calibrated by the manufacturer (Discovery DXA System—Hologic, Bedford, MA), and a small animal software (Discovery DXA System—Hologic, Bedford, MA). Clean bones were placed in an acrylic container with deionized water and scanned using the densitome- ter. The small animal software was used to select the region for subsequent densitometry analysis. Bone Strength The right tibia was used for the mechanical bone strength tests (3-point bending and axial compression) (Sgavioli et al., 2016). The tests were conducted using an EMIC R© (DL 10,000, cell Trd 21, software, Tesc 3.13, São José dos Pinhais, Brazil) universal testing machine (UTM). The pre-load force applied was 10 N, and the adaptation time was 5 s. The power speed was 5 mm min−1 using the cell force of 50 kg to determine the maximum force (N), the maximum deformation at full force (mm), and the rigidity (N m−1). The equipment was calibrated to allow the diaphysis length of 6 cm for all bone samples, as this value was the maximum duration of support found amongst the smallest select sample. The bones were fixed on a 2-point support with the span adjusted according to the size of the smallest bone. Force was then applied at the bone’s geometric mean point between the 2 supports (the middle third of the bone), and the equipment recorded the results. These variables express the bone strength at the ends of the bone and at the middle third. Mineral Profile The left tibia was used to determine bone calcium, phosphorus, and ash content. The soft tissue was re- moved and the bones boiled in deionized water for 5 min. After drying at room temperature, the samples were immersed in petroleum ether for 48 h, dried in a forced-ventilation oven at 60◦C for 48 h, and then ground in a ball mill. Bone mineral content was ana- lyzed at the Feed Analysis Laboratory of the College of Agriculture and Veterinary Sciences of the Sao Paulo State University (UNESP—Jaboticabal), Jaboticabal, SP Brazil, using wet analyses. Ash content was deter- mined by burning the samples at 600◦C. The methods were applied according to Silva & Queiroz (2002), and expressed as a percentage of defatted dry matter. Macroscopic Analysis of the Bone Before the mineral profile analysis, the tibiae were weighed to obtain the absolute weight and the relative weight (in relation to the carcass). The length of the tibia, the width of the proximal epiphysis, the diaphysis and the distal epiphysis were measured using a digital caliper (Toledo –Adventure ARD 110, São Bernardo do Campo, Brazil) (Sgavioli et al., 2016). The Seedor index was determined by calculating the relationship between the bone weight (mg) and the bone length (mm). Histopathological Evaluation of the Joint Cartilage For the joint cartilage histological evaluation, 1 cm from the proximal epiphysis cartilage was collected from the right tibia and the chondrocytes cartilage number measured. After the macroscopic evaluation, the joint carti- lage was fixed in a solution of formaldehyde (10%) for 24 h and decalcified in a solution of formic acid (5%) for 14 d at room temperature, then washed in dis- tilled water and processed according to the usual meth- ods for light microscopy. The samples were dehydrated in a series of increasing ethanol concentrations (70%, 80%, 90%, 95%, and 100%). Afterward, they were di- aphanized in an ethanol-xylene solution (1:1), followed by xylene (100%, 3×), and embedded in a mixture of xylene-paraffin (1:1), followed by paraffin, spend- ing approximately 45 min in each solution. Histologic semi-serial cartilage cuts 6 μm thick were stained with hematoxylin-eosin. Eighty-one slides were made, with 9 replica- tions/treatment. Four semi-serial cuts were placed on each slide. Four images per cut were obtained for a total of 144 images per treatment. The pictures were D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/11/4017/4157254 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 24 June 2019 4020 SGAVIOLI ET AL. taken using a system of registering and analyzing the image (Leica QWin; Leica Imaging Systems, Wetzlar, Germany) with the 10× objective lens. For the cell count in the images, an area of 86.8 μm2 area was stip- ulated (Xiao et al., 2010). Statistical Analysis The effects of the addition of chondroitin sulfate A (CO) (0, 0.08, and 0.16%) and glucosamine (GLU) (0, 0.12, and 0.24%), as well as their interaction (CO × GLU), on the variables were analyzed according to the experimental model: Yijk = μ + (CO)i + (GLU)J + (CO × GLU) ij + eijk. Data for all variables were checked for the presence of outliers, assumptions of normality of observational mistakes, and homogeneity of variances. After corrections, the data were submitted to analy- sis of variance using the General Linear Model proce- dure (GLM) of SAS R© (SAS Institute, 2002, Cary, NC). When means differed significantly by the F test, the or- thogonal analysis was performed to test the linear and quadratic effects of chondroitin and glucosamine con- centrations on the studied parameters. RESULTS Performance parameters for d 1 to 21, 22 to 42, and 1 to 42 are summarized in Table 2. Tibia calcium, phos- phorus, and ash percentage and the number of chondro- cytes in proximal tibia cartilage for broilers at 42 d old are summarized in Table 3 and tibia length, absolute and relative weight, the width of the proximal epiph- ysis, diaphysis and distal epiphysis for broilers at 42 d old are summarized in Table 4. Performance Feed conversion (FC) of broilers from 1 to 21 d old showed an interaction between addition of chondroitin × glucosamine (P = 0.0189) (Table 2). A quadratic ef- fect was identified (P = 0.0022) with the addition of 0.24% glucosamine, with a better FC obtained by the addition of 0.08% chondroitin, as shown in Equation 3 (Table 2). For the inclusion of 0.08% chondroitin, a linear decreasing effect of glucosamine levels (P = 0.0077) was found, as shown in Equation 4 (Table 2), therefore increasing the addition of glucosamine will improve the bird’s FC1–21d. A quadratic effect was ob- served on the feed conversion of broilers from 22 to 42 d old (P = 0.0082), and from 1 to 42 d old (P = 0.0123) (Table 2), with better FC observed at chondroitin ad- ditions of 0.10% and 0.08%, respectively, as shown in Equations 5 and 6 (Table 2). Adding chondroitin and glucosamine did not change (P > 0.05) broiler performance (body weight, weight gain, feed intake) (Table 2). No effect (P > 0.05) for the addition of chondroitin or glucosamine was identified in the via- bility (Table 2). Densitometry, Bone Strength, and Mineral profile No effect on density and bone strength was found with the addition of chondroitin and glucosamine (P > 0.05) when the following traits were analyzed at 42 d old; bone mineral density, area and mineral composition, maximum force, deformation at maxi- mum force and rigidity. A quadratic effect was found in treatments with chondroitin (CO) for calcium (P < 0.0001) and ash (P = 0.0057) concentrations in the tibia of broilers at 42 d old (Equations 7 and 10, Ta- ble 3). The maximum calcium concentration was ob- served with the inclusion of 0.11% of chondroitin, and 0.09% of chondroitin led to a lower amount of ash in the tibia. An interaction was identified between the treatments (P = 0.0122) and the amount of phosphorus in the tibia (Table 3). A quadratic effect on the chondroitin levels (P = 0.0040) (Equation 8) was observed with the ad- dition of 0.24% of glucosamine (Table 3) and the addi- tion of 0.14% chondroitin maximized the concentration of phosphorus in the tibia. A quadratic effect of glu- cosamine levels (P = 0.0035) was found for birds fed with no chondroitin (Table 3), and adding 0.09% glu- cosamine results in a higher concentration of phospho- rus in the tibiae (Equation 9). Number of Chondrocytes An interaction was found between chondroitin × glu- cosamine for the number of chondrocytes in proximal tibia cartilage (P = 0.0072) (Table 3). The inclusion of 0.12% and 0.24% of glucosamine had a quadratic effect of the chondroitin addition (P = 0.0005 and P = 0.0043, respectively, Table 4; Equations 11 and 12). Adding 0.04% and 0.02% chondroitin will respec- tively increase the number of chondrocytes in cartilage. There was a quadratic effect of glucosamine addition in the diet (P = 0.0107) for birds that had received the addition of 0.16% of chondroitin (Table 4; Equation 13). The inclusion of 0.18% of glucosamine increased the number chondrocytes in proximal tibia cartilage of broilers at 42 d old. Macroscopic Study of Bones There was a quadratic effect for glucosamine addition in the absolute tibia weight (P = 0.0340) in broilers 42 d old (Table 4). Higher absolute weight occurs with the glucosamine inclusion of 0.19%, as seen in Equa- tion 14. The inclusion of glucosamine had a linear in- creased effect (Table 4) for the relative weight, width of the proximal epiphysis, and width of the diaphysis tibia for 42-day-old broilers (Equation 15, 16 and 17 in Table 4, P = 0.0108, P = 0.0032 and P = 0.0091, respectively). D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/11/4017/4157254 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 24 June 2019 GLYCOSAMINOGLYCANS EFFECT ON BONE AND CARTILAGINOUS 4021 T ab le 2. P er fo rm an ce pa ra m et er s fo r br oi le rs 1 to 21 d, 22 to 42 d, an d 1 to 42 d ol d. C ho nd ro it in 1 (C O , % ) G lu co sa m in e2 (G L U ,% ) R eg re ss io n M ea n SE M P ro ba bi lit y 0 0. 12 0. 24 R eg re ss io n C O R eg re ss io n G L U C O × G L U 1– 21 d ol d Fe ed co nv er si on (g /g ) 0 1. 15 1. 15 1. 19 ns 1. 17 0. 01 ns ns 0. 01 89 0. 08 1. 17 1. 16 1. 15 L 4 (0 .0 07 7) 1. 16 0. 16 1. 16 1. 18 1. 20 ns 1. 19 R eg re ss io n ns ns Q 3 (0 .0 02 2) M ea n 1. 16 1. 17 1. 17 22 –4 2 d ol d Fe ed co nv er si on (g /g ) 0 1. 15 1. 16 1. 87 ns 1. 62 0. 02 Q 5 (0 .0 08 2) ns ns 0. 08 1. 17 1. 16 1. 45 ns 1. 57 0. 16 1. 16 1. 82 1. 20 ns 1. 59 R eg re ss io n ns ns ns M ea n 1. 58 1. 60 1. 59 1– 42 d ol d W ei gh t ga in (g ) 0 32 92 .3 8 33 43 .6 2 32 57 .1 0 ns 32 97 .7 0 38 .2 4 ns ns ns 0. 08 32 49 .5 1 33 41 .4 5 32 56 .8 1 ns 32 82 .5 9 0. 16 32 37 .5 9 32 18 .3 7 32 63 .0 6 ns 32 40 .2 7 R eg re ss io n ns ns ns M ea n 32 66 .1 8 33 01 .1 4 32 58 .9 9 Fe ed in ta ke (g ) 0 48 19 .9 7 49 20 .7 0 48 42 .1 6 ns 48 60 .9 4 69 .0 3 ns ns ns 0. 08 47 36 .9 8 48 66 .7 5 46 58 .9 3 ns 47 62 .1 5 0. 16 46 92 .0 9 47 25 .2 4 48 66 .4 5 ns 47 81 .0 2 R eg re ss io n ns ns ns M ea n 47 66 .1 3 48 37 .5 6 47 89 .1 8 Fe ed co nv er si on (g ) 0 1. 47 1. 47 1. 49 ns 1. 48 0. 02 Q 6 (0 .0 12 3) ns ns 0. 08 1. 44 1. 45 1. 43 ns 1. 44 0. 16 1. 45 1. 47 1. 49 ns 1. 48 R eg re ss io n ns ns ns M ea n 1. 45 1. 47 1. 47 V ia bi lit y (% ) 0 96 .7 1 98 .3 3 95 .8 3 ns 96 .9 6 1. 36 ns ns ns 0. 08 99 .1 7 97 .5 0 93 .3 3 ns 96 .6 7 0. 16 95 .0 0 96 .6 7 97 .5 0 ns 96 .7 9 R eg re ss io n ns ns ns M ea n 97 .5 2 97 .5 0 95 .5 6 SE M : st an da rd er ro r of th e m ea n. 1 [ (C 14 H 21 N O 14 S) n, In fin it i N ut ra ce ut ic al s, In c. ] ha d a pu ri ty of 91 .3 5% . 2 [ (C 6H 14 N O 5) 2S O 4 × 2K C l, Z he jia ng Fr ee m an Sh in fu da C o. L td .] ha d a su lfa te co nt en t of 15 .7 % . N s: no t si gn ifi ca nt . 3 F ee d co nv er si on 1– 21 d – 0. 24 % G L = 7. 03 12 C O 2 – 1. 06 25 C O + 1. 19 ; R 2 = 0. 97 . 4 F ee d co nv er si on 1– 21 d – 0. 08 % C O = -0 .0 83 3 G L U + 1. 17 ; R 2 = 0. 98 . 5 F ee d co nv er si on 22 - 42 d ol d = 5. 46 87 C O 2 – 1. 06 25 C O + 1. 62 ; R 2 = 0. 98 . 6 F ee d co nv er si on 1 - 42 d ol d = 5. 22 48 C O 2 – 0. 82 67 C O + 1. 47 ; R 2 = 0. 98 . D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/11/4017/4157254 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 24 June 2019 4022 SGAVIOLI ET AL. T ab le 3. T ib ia ca lc iu m , ph os ph or us ,a sh pe rc en ta ge , an d nu m be r of ch on dr oc yt es in pr ox im al ti bi a ca rt ila ge fo r br oi le rs at 42 d ol d. C ho nd ro it in 1 (C O , % ) G lu co sa m in e2 (G L U , % ) R eg re ss io n M ea n SE M P ro ba bi lit y 0 0. 12 0. 24 R eg re ss io n C O R eg re ss io n G L U C O × G L U C al ci um (% ) 0 33 .6 1 35 .5 4 35 .6 5 ns 34 .9 4 0. 76 Q 7 (< .0 00 1) ns ns 0. 08 36 .7 5 37 .6 3 37 .4 2 ns 37 .2 5 0. 16 36 .9 1 37 .0 2 37 .3 3 ns 37 .0 9 R eg re ss io n ns ns ns M ea n 35 .7 7 36 .6 7 36 .8 0 P ho sp ho ru s (% ) 0 17 .0 0 17 .1 4 17 .3 9 Q 9 (0 .0 03 5) 17 .1 7 1. 10 ns ns 0. 01 22 0. 08 16 .9 9 17 .1 1 17 .0 9 ns 17 .0 6 0. 16 17 .0 7 17 .0 8 17 .0 3 ns 17 .0 6 R eg re ss io n ns ns Q 8 (0 .0 04 0) M ea n 17 .0 2 17 .1 1 17 .1 7 A sh (% ) 0 43 .3 4 41 .8 5 42 .0 9 ns 42 .4 3 1. 10 Q 10 (0 .0 05 7) ns ns 0. 08 39 .2 8 39 .7 1 40 .2 8 ns 39 .7 6 0. 16 41 .2 4 42 .2 2 41 .8 6 ns 41 .7 7 R eg re ss io n ns ns ns M ea n 41 .2 9 41 .2 6 41 .4 1 N um be r of ch on dr oc yt es 0 12 .2 6 14 .7 6 13 .5 0 ns 13 .4 1 0. 87 ns ns 0. 00 72 0. 08 14 .2 8 13 .3 9 13 .6 1 ns 13 .7 1 0. 16 12 .6 5 10 .2 3 10 .2 7 Q 13 (0 .0 10 7) 11 .1 1 R eg re ss io n ns Q 11 (0 .0 00 5) Q 12 (0 .0 04 3) M ea n 12 .8 8 12 .7 9 12 .3 9 SE M : st an da rd er ro r of th e m ea n. 1 [ (C 14 H 21 N O 14 S) n, In fin it i N ut ra ce ut ic al s, In c. ] ha d a pu ri ty of 91 .3 5% . 2 [ (C 6H 14 N O 5) 2S O 4 × 2K C l, Z he jia ng Fr ee m an Sh in fu da C o. L td .] ha d a su lfa te co nt en t of 15 .7 % . N s: no t si gn ifi ca nt . 7 C al ci um = −1 92 .9 7C O 2 + 44 .3 13 C O + 34 .9 4; R 2 = 0. 97 . 8 P ho sp ho ru s 0. 24 % G L U = 18 .7 5 G L 2 – 5. 25 G L + 17 .3 9; R 2 = 0. 97 . 9 P ho sp ho ru s 0% C O = 3. 81 94 G L 2 + 0. 70 83 G L + 17 ; R 2 = 0. 98 . 10 A sh = 36 5. 62 C O 2 – 62 .6 25 C O + 42 .4 3; R 2 = 0. 97 . 11 N um be r of ch on dr oc yt es 0. 12 % G L U = −1 39 .8 4 C O 2 − 5. 93 75 C O + 14 .7 6; R 2 = 0. 96 . 12 N um be r of ch on dr oc yt es 0. 24 % G L U = −2 69 .5 3 C O 2 − 22 .9 38 C O + 13 .5 0; R 2 = 0. 97 . 13 N um be r of ch on dr oc yt es 0. 16 % C O = 85 .4 17 G L U 2 − 30 .4 17 G L U + 12 .6 5; R 2 = 0. 98 . D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/11/4017/4157254 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 24 June 2019 GLYCOSAMINOGLYCANS EFFECT ON BONE AND CARTILAGINOUS 4023 Table 4. Tibia length, absolute and relative weight, the width of the proximal epiphysis, diaphysis, and distal epiphysis for broilers at 42 d old. Length (mm) Absolute weight (g) Relative weight (%) Width (mm) Proximal epiphysis Diaphysis Distal epiphysis Chondroitin1 (CO, %) 0 102.72 16.48 0.691 17.86 8.81 8.97 0.08 103.06 17.31 0.737 17.92 8.92 8.95 0.16 101.34 16.36 0.698 17.38 8.93 8.71 Glucosamine2 (GLU, %) 0 101.74 16.02 0.677 17.23 8.49 8.85 0.12 102.46 17.10 0.715 17.71 9.00 8.86 0.24 103.18 17.18 0.745 18.36 9.23 8.94 Probability Regression CO ns ns ns ns ns ns Regression GLU ns Q14 (0.0340) L15 (0.0108) L16 (0.0032) L17 (0.0091) ns CO X GLU ns ns ns ns ns ns SEM 1.25 0.59 0.03 0.45 0.34 0.27 SEM: standard error of the mean. 1[(C14H21NO14S)n, Infiniti Nutraceuticals, Inc.] had a purity of 91.35%. 2[(C6H14NO5)2SO4 × 2KCl, Zhejiang Freeman Shinfuda Co. Ltd.] had a sulfate content of 15.7%. Ns: not significant. 14Absolute weight = −34.722 GLU2 + 13.167 GLU + 16.02; R2 = 0.98. 15Relative weight = 0.2833 GLU + 0.6783; R2 = 0.95. 16Width of the proximal epiphysis = 4.7083 GLU + 17.202; R2 = 0.94. 17Width of the diaphysis = 3.0833 GLU + 8.5367; R2 = 0.92. DISCUSSION The addition of chondroitin sulfate and glucosamine were evaluated for the ability to promote changes in bone and cartilage development, thereby improving the bird’s performance. Difficulty in walking or the inabil- ity to reach feeders and drinkers will quickly decrease or even stop broiler growth, and within a few d the flock may become uneven causing welfare conditions to deteriorate (Almeida Paz et al., 2010). This study showed for the first time in the literature the effect of chondroitin on broiler performance; there was an improvement in feed conversion with inclusion of 0.08% of chondroitin in broiler diets fed from one to 42 d old and our findings suggest that higher concentra- tions of chondroitin sulfate could be used during broiler growth to improve performance. Growing birds require high concentrations of chon- droitin sulfate. The cartilage of the growing broiler keel has high concentrations of chondroitin sulfate, thus a readily available source of chondroitin (Nakano et al., 2012). However, according to the study’s feed conver- sion results, the quantity found in the cartilage of broil- ers is not sufficient, requiring its addition in the diet. The best feed conversion found in the present study might be related to an improvement in locomotion from better bone development (Almeida Paz et al., 2008). This assumption was justified in Table 3, where the data indicate that the tibiae of broilers fed an addition of chondroitin had higher percentages of calcium and phosphorus, as compared to broilers that had not in- gested chondroitin. This improvement in the tibia de- velopment may have been promoted by the addition of the product, leading to better locomotion as found by Henrotin et al. (2005), studying the use of chon- droitin sulfate and glucosamine as chondroprotection to promote the improvement of symptoms such as lame- ness and pain in canines. The GAGs can influence the cartilage macroscopy and long bones formation, inasmuch as these substances stimulate the synthesis of the proteoglycans and colla- gen and are capable of increasing the proliferation of the chondrocytes and the biosynthesis of the matrix (Clark, 1991), they are essential in the endochondral ossification process, responsible for the longitudinal growth of long bones due to epiphyseal disk calcification (Anderson et al., 2005), and therefore have a direct in- fluence on bone mineral concentrations. A higher percentage of bone calcium and phosphorus also results in higher bone mineralization. This affects the mechanical quality of the broiler bones, due to the relationship between calcium and bone fragility. Thorp and Waddington (1997) observed that broiler bones with a high amount of calcium had fewer fractures dur- ing processing and Crespo et al. (2002) found a lower fracture incidence in adult tom turkey femurs with a high percentage of calcium. However, in this study, the mineral composition of the tibia did not affect the den- sity and bone strength parameters (P > 0.05). According to Nääs et al. (2012), the current broiler strains selected for fast growth have little bone strength to support body weight, as their skeletons have not adapted to support the rapid weight gain. This could be related to the incidence of crooked legs or locomutation issues in broilers. However, in this study we found that as the amount of glucosamine added to the broiler diet increased, so did the relative weight, absolute weight, and width of the proximal epiphysis and diaphysis in the tibia. In addition to these effects, the inclusion of D ow nloaded from https://academ ic.oup.com /ps/article-abstract/96/11/4017/4157254 by U niversidade Estadual Paulista J� lio de M esquita Filho user on 24 June 2019 4024 SGAVIOLI ET AL. 0.09% of glucosamine increased phosphorus concentra- tion in the tibia. According to the present study, the inclusion of glu- cosamine in the broiler feed was efficient in promot- ing bone development of birds. It is known that glu- cosamine sulfate can help the synovial fluid become more resistant and elastic. This leads to reinforcement of the locomotor system since some of the glucosamine’s anti-inflammatory action might be associated with the stimulus of biosynthesis of proteoglycans (Vaughan- Scott and Taylor, 1997). It has been suggested that the newly synthesized proteoglycans can stabilize cell membranes, resulting in an anti-inflammatory effect (Sweetman, 2002). Chondrocytes are highly specialized and their main function is to produce and maintain biomechanical properties of the cartilage, by the synthesis of the components of the extracellular matrix (Wollenweber et al., 2006). According to the results shown in Table 3, the number of articular cartilage chondrocytes is influ- enced by the addition of chondroitin and glucosamine in the broiler diet. The addition of glucosamine and chondroitin were ideal for maximizing the number of chondrocytes. Chondroitin sulfate (GAG mono sulfate) has been investigated in biochemical studies due to its role in the physiology of joint cartilage. Beale et al. (1990) de- scribed its chondroitin-stimulating properties from the increase in the proteoglycan synthesis by chondrocytes, and Diaz et al. (1996) described its chondroprotective properties through the enhanced inhibitory activity of cartilage-degrading enzymes. 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