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Productive performance, eggshell quality, and eggshell ultrastructure of laying hens fed diets supplemented with organic trace minerals
Productive performance, eggshell quality, and eggshell ultrastructure of laying hens fed diets supplemented with organic trace minerals C. Stefanello, T. C. Santos,1 A. E. Murakami, E. N. Martins, and T. C. Carneiro Department of Animal Science, State University of Maringá, Av. Colombo 5790, Maringá, PR 87020-900, Paraná, Brazil intake, feed conversion, specific weight, and Haugh unit of eggs. However, there was a quadratic effect (P < 0.05) of the levels of trace mineral supplementation on average egg weight and egg mass; the results did not differ regarding the source used. The increase in the levels of supplementation of Mn, Zn, and Cu provided a linear increase (P < 0.05) in the breaking strength and the percentage of eggshell. There was a linear decrease (P < 0.05) in the egg loss and the number of mammillary buttons in the shell. The best results were obtained using diets supplemented with trace minerals from an organic source because these diets provided lower egg loss, higher thickness, and increased strength of the shell. Structurally, organic Mn, Zn, and Cu provided higher thickness of the palisade layer and lower mammillary density. The trace mineral supplementation improved the structural characteristics and the quality of the eggshells.
Key words: copper, manganese, proteinate, scanning electron microscope, zinc 2014 Poultry Science 93:104–113 http://dx.doi.org/10.3382/ps.2013-03190
INTRODUCTION
sulfates, carbonates, and phosphates. However, organic mineral sources have emerged on the market with the prospect of being more easily absorbed and retained by birds, thereby reducing the excretion of trace minerals that potentially pollute the environment (Zamani et al., 2005). The organic minerals were conceptualized by AAFCo (2001), such as metal ions, chemically link with an organic molecule, forming unique structures and providing stability with high mineral bioavailability. However, the practical results of organic trace mineral supplementation in poultry diets remain controversial, due to the differences between sources and supplementation levels used (Vieira, 2008). Trace minerals are essential in the diet of laying hens because they participate in the biochemical processes necessary for normal growth and development, including bone and eggshell formation (Richards et al., 2010). Zinc is a cofactor of the enzyme carbonic anhydrase inhibitors that are involved in the formation of the eggshell (Robinson and King, 1963). Manganese acts as activator of the enzymes that are involved in the syn-
Improved understanding of the factors that affect the performance and quality of the eggshells produced by commercial laying hens is essential for the production of the highest quality eggs because defects in shell quality can cause significant losses to the commercial egg industry. Between 10 and 15% of laying eggs are lost before and during the collection process due to problems related to the shell quality (Roland, 1988; Coutts et al., 2007). Therefore, different strategies, especially mineral nutrition and supplementation, have been considered to improve eggshell quality (Nys, 2001; Roberts, 2004). Most mineral sources used in diets for laying hens are derived from inorganic compounds such as oxides,
©2014 Poultry Science Association Inc. Received March 18, 2013. Accepted october 12, 2013. 1 Corresponding author: [email protected]
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ABSTRACT This study was carried out with the purpose of evaluating the effect of supplementing hens’ diets with trace minerals from inorganic or organic sources on the productive performance, eggshell quality, and eggshell ultrastructure of laying hens. Three hundred sixty Hy-Line W36 laying hens between 47 to 62 wk of age were used and distributed in a completely randomized experimental design with 9 treatments, 5 replicates, and 8 birds for each experimental unit. The treatments consisted of a control diet without supplementation of the trace minerals Mn, Zn, and Cu; 4 supplementation levels of these trace minerals from an inorganic source; and the same levels of supplementation from an organic source (proteinates). The supplementation levels in milligrams per kilogram for Mn, Zn, and Cu, were, respectively, 35-30-05, 65-60-10, 9590-15, and 125-120-20. There was no effect of supplementation of trace minerals on the rate of posture, feed
HEN DIETS SUPPLEMENTED WITH ORGANIC TRACE MINERALS
eggshells. These studies would establish greater insight into the mechanisms of action of the trace minerals and their participation in the eggshell ultrastructure and subsequent resistance to cracking. The objective of the study was to evaluate the effects of dietary supplementation of inorganic and organic source trace minerals on the productive performance, the quality of the shell, and the eggshell ultrastructure of laying hens between 47 and 62 wk of age.
MATERIALS AND METHODS Birds The experiment was conducted in the Experimental Farm of Iguatemi of Universidade Estadual de Maringá, Paraná, Brazil. Three hundred sixty Hy-Line W36 laying hens, 47 to 62 wk of age, were used. Hens were housed in collective laying cages (1 m × 0.50 m), divided in 4 subunits (8 hens). The experimental unit was the cage of 8 hens. Feed and water were provided ad libitum, and birds were standardized by weight and egg production before starting the experiment, (i.e., birds were housed with an average weight within an SD of 5% from the average of all birds). Only laying hens that were producing eggs were housed. Birds were subjected to 14 d of adjustment to the experimental diets. The lighting program was 17 h of light per day. Average minimum and maximum temperature, as monitored on a daily basis inside the shed, were 17.4 and 26.5°C, respectively, and the RH averages recorded were 37.6% (minimum) and 84.6% (maximum).
Experimental Diets The experimental design was completely randomized with 9 treatments, 5 replicates and 8 birds per experimental unit. The inorganic trace minerals sources were sulfate of Mn (MnSO4 with 31% of Mn) and Zn (ZnSO4 with 94% of Zn), and oxide of Cu (CuO with 24% of Cu). The trace minerals sources used were proteinate of Zn (150,000 mg of Zn/kg of product), proteinate of Mn (150,000 mg of Mn/kg of product), and proteinate of Cu (100,000 mg of Cu/kg of product), all products Bioplex (Alltech, Lexington, KY). The treatments are shown in Table 1. The control diet (without addition of Mn, Zn, and Cu) was supplemented with trace minerals. The experimental diets were formulated for today’s industry standards for the egg production phase, according to Rostagno et al. (2005), including the trace mineral proportion. The recommended levels corresponded to treatments 3 and 7 (65 mg/kg of Mn; 60 mg/kg of Zn, and 10 mg/kg of Cu). The other treatments have been reduced or increased the trace minerals by the same level to 30 mg of Zn, 30 mg of Mn, and 5 of Cu mg per treatment.
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thesis of glycosaminoglycans and glycoproteins, which contribute to the formation of the organic matrix of the shell. Copper is an integral part of the lysyl oxidase enzyme that is important in the formation of collagen present in the eggshell membrane (Leeson and Summers, 2001). The eggshell is structurally composed by organic and inorganic components. The organic component of the shell consists of the shell membranes, mammillary buttons or tips, the matrix of the shell, and the cuticle (Solomon, 1991). The inorganic component of the eggshell consists of calcium carbonate crystals. The eggshell layers, the inside to the outside, are composed by mammillary buttons layer (internal), palisade layer (intermediate), and crystal surface layer (external; Roberts, 2004). The eggshell structure partly results from a competition for space during crystal growth between adjacent sites of nucleation (Rodriguez-Navarro et al., 2002). Additionally, the interaction between crystal and organic matrix components resulting in anisotropy of the crystal growth as shown by Hernandez-Hernandez et al. (2008). Trace minerals can affect the quality of the shell as a result of either their catalytic properties as components of the enzymes involved in the synthesis of the shell or by direct interaction with the crystals of calcium during shell formation (Fernandes et al., 2008). However, the results reported in the literature show that supplementation of organic minerals is still controversial. Mabe et al. (2003) found that when assessing supplemental sources, organic and inorganic Zn, Mn, and Cu had similar effects on egg quality. Zamani et al. (2005) reported that the basal diet supplemented with Mn and Zn in combination from inorganic sources had a positive effect on the percentage of eggshell. The trace elements Zn, Mn, and Cu influence the organic matrix of eggshells and therefore can influence the mechanical properties of the eggshell. RodriguezNavarro et al. (2002) stated that membranes that compose the organic matrix may provide a network of fibrous reinforcement within the shell that can contribute to the resistance to breaking of the egg. To Solomon (1991), the issues involved in the resistance of eggshell are very complex, possibly reflecting the interaction between its organic and inorganic components. Thus, evaluation of eggshell ultrastructure allows a greater understanding of the organizat ion and reinforces the view that the mechanical properties of the eggs cannot be defined by simple measurement of shell thickness when working with trace minerals (Nys et al., 2004). According to Bain (1992) and Ruiz and Lunam (2000), the palisade layer provides the characteristic of stiffness and thus resistance of shell. In this case, a reduction in the shell’s relative thickness could compromise its strength, leading to a higher incidence of disruptions. Thus, further studies are warranted using organic mineral sources to observe how the trace minerals can influence the structural organization of
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Stefanello et al. Table 1. Supplemented trace minerals in the experimental diets (mg/kg) Treatment
Source of trace minerals
T1 T2 T3 T4 T5 T6 T7 T8 T9
Inorganic1 Inorganic Inorganic Inorganic Organic2 Organic Organic Organic
(without supplement) (35-30-5) (65-60-10) (95-90-15) (125-120-20) (35-30-5) (65-60-10) (95-90-15) (125-120-20)
Manganese
Zinc
Copper
0 35 65 95 125 35 65 95 125
0 30 60 90 120 30 60 90 120
0 5 10 15 20 5 10 15 20
1Inorganic 2Organic
source (Nucleopar Animal Nutrition Ltda., Mandaguari, Brazil). source, proteinate of trace minerals (Bioplex Alltech, Lexington, KY).
Table 2. Composition and calculated analysis of the basal diets Item Ingredient (%) Corn (8.8% CP) Soybean meal (45% CP) Dicalcium phosphate Limestone Soybean oil Salt dl-Methionine (98%) l-Lysine HCl (78%) Vitamin and mineral premix1 Antioxidant2 Calculated composition (% or as shown) ME (kcal/kg) CP Calcium Available phosphorus Digestible Lys Digestible Met + Cys Digestible Thr Digestible Trp Analyzed value3 (mg/kg) Manganese Zinc Copper
Value 64.00 22.40 1.49 9.06 2.12 0.35 0.15 0.03 0.40 0.01 2,880 15.50 3.90 0.36 0.71 0.64 0.52 0.16 37.10 24.20 5.65
1Mineral and vitamin supplement, Nucleopar Animal Nutrition Ltda. (content per kg of diet): vitamin A, 2,550 IU/g; vitamin E, 2,083.33 mg; vitamin D3, 500 IU/g; vitamin K3, 650 mg; vitamin B1, 408.33 mg; vitamin B12, 2,500 µg; vitamin B2, 1,000 mg; vitamin B6, 412.5 mg; Ac. folic, 66.67 mg; biotin, 8.33 mg; choline, 70,000 mg; Ac. pantothenic, 2,375 mg; methionine, 226,875 mg; niacin, 5,308.33 mg; iron, 12,500 mg; iodine, 258.33 mg; selenium 75 mg; cobalt, 83.33 mg; antioxidant, 1,250 mg. 2Butyrate hydroxytoluene. 3According to AOAC Method 990.10 (AOAC, 1990).
Performance and Eggshell Quality Daily feed intake (g/bird per d), feed conversion (kg of feed/dozen eggs and kg of feed/kg of eggs), egg production (%), and egg loss (%) were recorded at 28-d intervals. Daily egg mass was calculated by multiplying the laying rate (%) by the average weight of eggs (g) and divided by 100. The means for each variable was considered as the sum of eggs (n = 8 birds). The egg loss (%) was considered as eggs that were broken, cracked, porous, or thin shell. At the end of each 28-d time period, for 3 consecutive days in a row, the following parameters were assessed: average egg weight, albumen height, specific weight, percentage, and thickness of the shell. All intact eggs from each experimental unit were identified and weighed individually in a precision scale (0.01 g), and were submitted afterward to a specific weight test using the flotation method in saline solution. Six saline solutions were prepared, ranging in density from 1.070 to 1.090 g/cm3 with a variation of 0.004 g/cm3 for each solution. The saline solution densities were measured using densimeter oil. Subsequent to the completion of the specific weight test, a sample of 3 eggs per experimental unit was used for the determination of the albumen height. Measurements in millimeters (mm) were related to the weight of the egg, thereby determining the Haugh unit: HU log 100 (H + 7.57 − 1.7 W0.37), H = where albumen height (mm) and W = egg weight (g). The shells were washed and dried at room temperature for 72 h and were weighed using a precision digital scale (0.001 g). The shell mass was obtained by calculating the weight of the dry shell divided by the overall of total egg weight. After weighing the shells, the shell thickness was measured at 3 points in the central region of each shell using a digital micrometer (Mitutoyo Sul Americana, São Paulo, Brazil). Three intact eggs per experimental unit (n = 135) were used on d 28 of each cycle for the measurement of the resistance force of the shell. The resistance, kilogram-force, was obtained using a texturometer TA.XT2 Texture Analyzer with a delrin probe with a 5-mm diameter (Texture Technologies Corp. and Stable Micro Systems Ltd., Hamilton, MA).
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The percentage composition and experimental diet calculated are listed in Table 2. The trace minerals Mn, Zn, and Cu were supplemented in accordance with the respective treatment (i.e., inorganic and organic sources). Other trace minerals (Fe, I, Co, and Se) were supplemented in the inorganic form and in equal amounts for all treatments. The basal diet was mixed in a vertical mixer and then the experimental diets were obtained by mixing with the appropriate amounts of minerals from the different sources. The amount of Zn, Mn, and Cu was analyzed in the ingredients and in the basal diet.
HEN DIETS SUPPLEMENTED WITH ORGANIC TRACE MINERALS
Eggshell Ultrastructure
Statistical Analysis Statistical analysis was performed by the SAS statistical program (SAS Institute, 2009). The effects of supplementation levels and source were analyzed using regression. To this end, the supplementation levels of Mn, Zn, and Cu were, respectively, level 1 (35-30-5), level 2 (65-60-10), level 3 (95-90-15), and level 4 (125120-20). These levels were used because the increase in the supplementation of trace minerals in each level was equivalent (30 mg of Mn/kg, 30 mg of Zn/kg, and 5 mg of Cu/kg).
RESULTS AND DISCUSSION The results of the regression analysis of the performance variables and quality of eggs of laying hens fed diets supplemented with increasing levels of Mn, Zn, and Cu are represented in Table 3. There was no interaction (P > 0.05) between the mineral source and level of supplementation. There was also no interaction between the production cycles and the mineral source, and between cycles and levels of supplementation. There was no effect of supplementation of Mn, Zn, and Cu trace minerals on the laying production of eggs,
feed intake, feed conversion (kg/dz and kg/kg), specific weight, and the Haugh unit for eggs. The results of analysis of specific weight and Haugh unit supported studies by Dale and Strong (1998) and Saldanha et al. (2009), which did not observe an effect of supplementation of trace minerals. Several other previous studies did not report the effects of organic trace minerals on egg production, feed intake, and feed conversion (Fernandes et al., 2008; Maciel et al., 2010). The egg weight had a quadratic effect (P < 0.05) on Mn, Cu, and Zn levels, independent of source. Eggshell thickness also increased to quadratic (P < 0.05), depending on the level of supplementation of trace minerals. There was an observed effect of the mineral source used, specifically, that there was greater shell thickness when the mineral came from an organic source. Possibly this quadratic increase in the eggshell thickness due to the increasing supplementation of trace minerals, regardless of source, may have influenced the egg weight in which a quadratic increase was also observed. Mineral proteinates are obtained by means of hydrolysis of a protein source such that the resulting hydrolyses contain a mixture of amino acids and small peptides with chains of different lengths. In this way stable chelates are formed that protect trace elements against chemical reactions taking place in the course of digestion. This protection maintains the solubility of these substances during their passage through the gastrointestinal tract to the sites of absorption (Close, 1998). Such absorption would explain the apparent decrease in interaction between mineral forms shown in various reports as well as allowing inorganic and organic forms to be used together to advantage. Greater stability during digestion, along with absorption and transport via peptide and amino acid routes, results in higher biological availability (Solomon, 1991; Nys et al., 2004). However, despite the increased absorption of organic trace mineral source, in this study, no effects were observed on the performance characteristics, as reported in other researches (Fernandes et al., 2008; Swiatkiewicz and Koreleski, 2008; Saldanha et al., 2009; Maciel et al., 2010), but it was possible to observe a direct effect of the mineral supplementation on the eggshell formation. With respect to the eggshell, increased levels of supplementation of Mn, Zn, and Cu resulted in a linear reduction (P < 0.05) of the percentage of egg loss, and a linear increase (P < 0.05) of shell strength, demonstrating a better quality in the eggshell formation. The effect of Zn and Mn supplementation has also been observed by Zamani et al. (2005), who observed that supplementing 150 mg of Zn/kg and 90 mg of Mn/kg improved shell thickness, the index of stiffness, and resistance to breakage. On the other hand, Swiatkiewicz and Koreleski (2008) evaluated the addition of Zn and Mn from inorganic and organic sources for layers between 35 and 70 wk of age and did not observe changes in percentage and shell thickness of the eggs. Maciel et al. (2010) did not observe an effect of the use of Zn, Mn, and Cu on the shell thickness of the eggs.
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On the last day of the experiment, the birds were 62 wk old, and 2 eggs per experimental unit were collected randomly. The eggs were broken and washed, and sample peels of the equatorial region were separated and saved. The membranes of the shell were removed by immersion of samples in a solution of 6% sodium hypochlorite, 4.12% sodium chloride, and 0.15% sodium hydroxide. Afterward, the shells were washed in water and dried at room temperature for at least 48 h according to the methodology described by Radwan et al. (2010). Two samples of the shell of each egg (0.5 cm2) were prepared for scanning electron microscope analysis using the Shimadzu SS-550 Superscan (Shimadzu Corporation, EVISA, Kyoto, Japan). One sample was used for the analysis of the inner surface of the shell, and the other was used for the assessment of the transversal surface. The samples were glued on an aluminum support (stub), metalized with gold, and analyzed in the electron microscope. For the inner surface analysis, the number of mammillary buttons/mm2 and 3 scanned images were obtained for each sample using 50× magnification. For the analysis of the cross-section of the shell, the variables measured were the palisade layer thickness (µm), the mammillary button thickness, and the total thickness (palisade + mammillary). The percentage of the palisade layer thickness and the percentage of the mammillary button thickness were obtained by calculating the ratio of the thickness of each layer to the total thickness measure.
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EP (%)
1.40 1.39 1.40 1.40 1.40 0.003 NS NS
1.40 1.39 1.39 1.40 1.40 0.004
1.40
FC (kg/dz)
1.75 1.74 1.74 1.73 1.74 0.002 NS NS
1.74 1.73 1.74 1.73 1.74 0.009
1.74
FC (kg/kg)
1.21 0.79 0.49 0.22 0.68 0.109 L* NS
1.55 1.09 0.59 0.32 0.89 0.116
1.57
Egg loss (%)
1.08 1.08 1.08 1.08 1.08 0.000 NS NS r2 0.85 0.83 0.57 0.66 0.87 0.97 0.85 0.91
1.08 1.08 1.08 1.08 1.08 0.000
1.08
Specific weight
Shell (%) 8.61 8.65 8.64 8.73 8.76 8.70 0.015 8.65 8.70 8.75 8.80 8.73 0.017 L* NS
Haugh unit 88.6 88.7 88.8 88.9 89.0 88.9 0.063 88.6 89.0 88.9 88.9 88.8 0.084 NS NS
0.36 0.37 0.38 0.38 0.37 0.002 Q* NS P 0.0001 0.0001 0.0003 0.0001 0.0002 0.0001 0.0001 0.0001
0.36 0.36 0.37 0.37 0.37 0.002
0.35
Thickness (mm)
3.57 3.74 3.99 4.12 3.85 0.049 L* NS
3.56 3.67 3.93 3.97 3.78 0.042
3.39
Shell strength (kgf)
= linear effect and Q* = quadratic effect (P < 0.05); EP = egg production (%); FC = feed conversion; shell = percentage of shell; kgf = kilogram-force; M = trace minerals (2 sources).
57.6 57.2 57.5 57.4 57.2 0.122 Q* NS
56.8 57.3 57.3 57.4 57.2 0.140
56.5
Egg mass (g)
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1L*
67.3 67.8 67.0 67.2 67.4 0.118 Q* NS
102.9 102.8 102.8 102.6 102.8 0.061
66.3 67.0 67.3 67.3 67.0 0.132
102.8 103.1 102.8 102.6 102.8 0.070
NS NS
66.2
Egg weight (g)
102.5
Feed intake (g/bird per d)
Without supplementation T1 (0-0-0) 85.0 Inorganic trace minerals (IM) T2 (35-30-5) 85.7 T3 (65-60-10) 85.9 T4 (95-90-15) 85.4 T5 (125-120-20) 85.7 Mean 85.7 SEM 0.201 Organic trace minerals (OM) T6 (35-30-5) 84.9 T7 (65-60-10) 85.8 T8 (95-90-15) 85.9 T9 (125-120-20) 85.6 Mean 85.6 SEM 0.146 Regression analysis Supplement level NS Level × source NS Regression equation Egg loss = 1.543 – 0.345IM Egg loss = 1.543 – 0.343OM Shell % = 8.606 + 0.037IM Shell % = 8.606 + 0.047IM Shell thickness = 0.352 + 0.007IM – 0.0004IM2 Shell thickness = 0.352 + 0.008OM – 0.0004OM2 Shell strength = 3.453 + 0.130IM Shell strength = 3.453 + 0.164OM
Supplementation Mn-Zn-Cu (mg/kg)
Table 3. Mean values of performance and eggshell quality of laying hens 47 to 62 wk of age relative to the level of supplementation and source of trace minerals (Mn, Zn, and Cu)1
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Table 4. Mean values obtained by electron microscopy of the eggshell of laying hens at 62 wk of age relative to the level of supplementation and source of trace minerals (Mn, Zn, and Cu)1 Supplementation Mn-Zn-Cu (mg/kg)
Mammillary (µm)
Thickness (µm)
Palisade (%)
Mammillary (%)
Mammillary buttons/mm2
231
66.1
297
77.7
22.7
263
240 265 267 270 260 3.648
64.3 63.6 62.9 63.6 63.6 0.638
304 329 330 334 324 3.624
78.9 80.6 81.0 80.9 80.3 0.304
21.1 19.4 19.0 19.1 19.6 0.304
192 176 170 162 175 8.772
248 266 277 278 267 3.964
67.9 65.1 64.5 64.4 65.5 0.930
316 331 342 343 333 3.968
78.5 80.3 81.1 81.2 80.3 0.357
21.5 19.7 18.8 18.8 19.7 0.357
177 167 161 149 163 9.520
Q* NS
NS NS
Q* NS
Q* NS
Q* NS r2 0.86 0.79 0.75 0.87 0.54 0.54
L* NS P 0.0029 0.0219 0.0393 0.0061 0.0001 0.0001
1L* = linear effect (P < 0.05); Q* = quadratic effect (P < 0.05); M = trace minerals; T = shell thickness; PAL = palisade layer; MB = number of mammillary buttons/mm2.
Increased eggshell strength was obtained using trace minerals, from either organic or inorganic sources, which may also be related to the decrease in egg loss. Increased dietary trace minerals should be linked with the formation of the eggshell membrane. Particularly, the Cu can improve shell membrane and the Zn and Mn participate in both the organic and inorganic chemistry of shells, resulting in eggs with better quality shells. This effect has also been observed by Maciel et
al. (2010), who observed less egg loss when the layers were given feed supplemented with 50 mg/kg of Zn, Mn, or Cu in organic form. The reduction in the egg loss and the increased resistance of shells are desirable features that have economic importance in a commercial laying segment. In this way, the results of this study are consistent with those obtained by Ludeen (2001), who noted improvement in resistance to breakage of eggs from chickens aged 40 to
Figure 1. Scanning electron microscopy of the cross section of the eggshell of laying hens at 62 wk of age. Palisade layer (pa), mammillary (ma), and vertical view of the mammillary buttons (mb). A) Treatment without addition of Mn, Zn, and Cu; B) supplementation of 35-30-05 mg/kg of Mn-Zn-Cu from an inorganic source; C) supplementation of 125-120-20 mg/kg of Mn-Zn-Cu from an organic source. Scale bar: 100 µm, 54 × 19 mm.
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Without supplementation T1 (0-0-0) Inorganic trace minerals (IM) T2 (35-30-5) T3 (65-60-10) T4 (95-90-15) T5 (125-120-20) Mean SEM Organic trace minerals (OM) T6 (35-30-5) T7 (65-60-10) T8 (95-90-15) T9 (125-120-20) Mean SEM Regression analysis Supplement level Level × source Regression equation T = 296.7 + 18.41IM – 2.128IM2 T = 296.7 + 23.03OM – 2.827OM2 PAL = 228.6 + 20.52IM – 2.497IM2 PAL = 228.6 + 23.24IM – 2.710IM2 MB = 237.34 – 22.30IM MB = 237.34 – 24.31OM
Palisade (µm)
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60 wk that had received proteinates of Zn and Mn. Paik (2001) also reported that organic Zn and Mn improved the eggshell strength and that there was influence of Zn in the synthesis of the enzyme carbonic anhydrase, which is essential for the formation of the shell. Results of the regression analysis of the eggshell ultrastructure of laying hens 62 wk of age fed diets supplemented with increasing levels and 2 sources of trace minerals are depicted in Table 4. There was no interaction (P > 0.05) between the source and the level of supplementation. Supplementation of Mn, Zn, and Cu in commercial laying hen diets did not affect (P > 0.05) the thickness of the mammillary layer of the eggshell. Increasing quantities of the trace minerals Mn, Zn, and Cu gave a quadratic increase (P < 0.05) in the
total shell thickness and the palisade layer and a linear reduction (P < 0.05) in the number of mammillary buttons. The best results for a lower number of mammillary tips by millimeter squared of eggshell were obtained when supplemented with increased levels of trace minerals, as illustrated in Figures 1, 2, and 3. Obtaining measurements of eggshell ultrastructure by scanning electron microscopy has allowed comparisons between the results obtained from different sources and the results using different mineral supplementation levels of Mn, Cu, and Zn. Increasing the palisade layer thickness of eggshells is important because, as the study of Radwan et al. (2010) notes, the strength of the shell depends on the thickness of the palisade layer and the organization of calcite crystals in this layer. Solomon
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Figure 2. Scanning electron microscopy of the inner surface of the eggshell of laying hens at 62 wk of age. Note the different distribution of mammillary buttons (mb) that became larger as levels of trace mineral supplementation increase in the diet. A) Treatment without addition of Mn, Zn, and Cu, control diet or negative control (neg); B) supplementation of 65-60-10 mg/kg of Mn-Zn-Cu from an inorganic source; C) supplementation of 65-60-10 mg/kg of Mn-Zn-Cu from an organic source; D) supplementation of 125-120-20 mg/kg of Mn-Zn-Cu from an organic source. IM: inorganic mineral, OM: organic mineral. Scale bar: 200 µm, 127 × 108 mm.
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(1991) suggested that the organization of the columns of the palisade layer is one of the major determinants of the rigidity of the shell and therefore the strength, and shell resistance of the eggs. Traditionally, the resistance of the eggshell has been considered to be almost entirely dependent on the properties of the inorganic components (Bain, 1992; El-Safty, 2004). The strength of the shell is directly related to its thickness, and the palisade layer comprises approximately two-thirds of the total thickness of the shell (Fathi et al., 2007). Therefore, it is likely that changes in the palisade layer thickness will affect the eggshell resistance, with diets supplemented with Mn, Zn, and Cu improving the quality of the eggshell
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produced, especially when these trace minerals are supplied in the form of proteinates. The properties of the eggshell depend on the microstructure and chemical composition, which can vary through the thickness of the shell (Rodriguez-Navarro et al., 2002; Nys et al., 2004; Bain et al., 2006). It is thought that the supplementation of Mn, Zn, and Cu participate in the formation of eggshells by influencing the thickness via their action as enablers of important enzymes, such as carbonic anhydrase (Nys et al., 2004; Zamani et al., 2005). The activity of carbonic anhydrase and the effects caused by trace minerals are important because the number of organic components and their concentration Downloaded from http://ps.oxfordjournals.org/ at University of Massachusetts/Amherst on May 9, 2014
Figure 3. Scanning electron microscopy of the inner surface of the eggshell of laying hens at 62 wk of age with greater magnification. The mammillary buttons (mb) are wider and together with each other depending on the levels of trace minerals that were supplemented in the diet. Control diet or negative control (neg). IM: inorganic mineral, OM: organic mineral. Scale bar: 50 µm, 150 × 136 mm.
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production of eggs, feed intake, and feed conversion. However, supplementation of Mn, Zn, and Cu did improve quality characteristics and ultrastructure of eggshells. Levels equal to or above 65-60-10 mg/kg, for Mn, Zn, and Cu, respectively, resulted in less egg loss and increased strength of shell.
REFERENCES AAFCO (Association of American Feed Control Officials). 2001. Official Publication. Assoc. Am. Feed Control Off., Atlanta, GA. AOAC. 1990. Official Methods of Analysis. 15th ed. Assoc. Off. Anal. Chem., Arlington, VA. Bain, M. M. 1992. Eggshell strength: A relationship between the mechanism of failure and the ultrastructural organization of the mammillary layer. Br. Poult. Sci. 33:303–319. Bain, M. M., N. Macleod, R. Thomson, and J. W. Hancock. 2006. Microcracks in eggs. Poult. Sci. 85:2001–2008. Close, W. H. 1998. New developments in the use of trace mineral proteinates to improve pig performance and reduce environmental impact. Pages 51–68 in European Lecture Tour. Alltech Inc. Technical Publications, Nicholasville, KY. Coutts, J. A., G. C. Wilson, and S. Fernandez. 2007. Optimum Egg Quality. 5M Publishing, Sheffield, UK. Dale, N., and C. F. Strong Jr. 1998. Inability to demonstrate an effect of eggshell 49 on shell quality in older laying hens. J. Appl. Poult. Res. 7:219–224. El-Safty, S. A. 2004. Stepwise regression analysis for ultrastructural measurements of eggshell quality in two local breeds of chicken. Egypt. Poult. Sci. 24:189–203. Fathi, M. M., A. Zein El-Dein, S. A. El-Safty, and L. M. Radwan. 2007. Using scanning electron microscopy to detect the ultrastructural variations in eggshell quality of Fayoumi and Dandarawi chicken breeds. Int. J. Poult. Sci. 6:236–241. Fernandes, J. I. M., A. E. Murakami, M. I. Sakamoto, L. M. G. Souza, A. Malaguido, and E. N. Martins. 2008. Effects of organic mineral dietary supplementation on production performance and egg quality of white layers. Braz. J. Poult. Sci. 10:9–65. Gautron, J., M. T. Hincke, and Y. Nys. 1997. Precursor matrix proteins in the uterine fluid change with stages of eggshell formation in hens. Connect. Tissue Res. 36:195–210. Hernandez-Hernandez, A., J. Gomez-Morales, A. B. Rodriguez-Navarro, J. Gautron, Y. Nys, and J. M. Garcia-Ruiz. 2008. Identification of some active proteins in the process of hen eggshell formation. Cryst. Growth Des. 8:4330–4339. Hincke, M. T., Y. C. Chien, L. C. Gerstenfeld, and M. D. McKee. 2008. Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell. J. Histochem. Cytochem. 56:467–476. Leeson, S., and J. D. Summers. 2001. Nutrition of the Chicken. 4th ed. University Books, Guelph, ON, Canada. Ludeen, T. 2001. Mineral proteinates may have positive effect on shell quality. Feedstuffs 73:10–15. Mabe, I., C. Rapp, M. M. Bain, and Y. Nys. 2003. Supplementation of a corn-soybean meal diet with manganese, copper, and zinc from organic or inorganic sources improves eggshell quality in aged laying hens. Poult. Sci. 82:1903–1913. Maciel, M. P., E. P. Saraiva, E. F. Aguiar, P. A. Ribeiro, D. P. Passos, and J. B. Silva. 2010. Effect of using organic microminerals on performance and external quality of eggs of commercial laying hens at the end of laying. R. Bras. Zootec. 39:344–348. Nys, Y. 2001. Recent developments in layer nutrition for optimizing shell quality. 13th European Symposium on Poultry Nutrition, Blankenberge (BEL), 2001. WPSA, Belgium Branch. Nys, Y., J. Gautron, J. M. Garcia-Ruiz, and M. T. Hincke. 2004. Avian eggshell mineralization: Biochemical and functional characterization of matrix proteins. C. R. Palevol 3:549–562. Paik, I. 2001. Application of chelated minerals in animal production. Asian-australas. J. Anim. Sci. 14:191–198. Radwan, L. M., A. Galal, M. M. Fathi, and A. Zein El-Dein. 2010. Mechanical and ultrastructural properties of eggshell in two Egyptian native breeds of chicken. Int. J. Poult. Sci. 9:77–81.
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change in the uterine fluid along the different stages of eggshell deposition happen in a well-defined way (Gautron et al., 1997). Also, these organic components are secreted at specific times and locations in the oviduct and incorporated at specific substructural regions of the eggshell. For instance, sulfate proteoglycans are the main components of the mammillary buttons or tips and osteopontin has been found in eggshell associated with particular crystallographic faces of calcite in the palisade region (Hincke et al., 2008); the proteoglycans are influenced by the presence of manganese, and as a result, there can be an eggshell formation that is thicker and more resistant to breakage. The knowledge of the layers that form the eggshell is important because the specific nucleation sites on the outer surface of the outer shell membrane attracts calcium salts and so initiate the formation of the mammillary layer in that region of the oviduct termed the tubular shell gland and may be influenced by the enzymatic activity, which has trace minerals as cofactors Mn, Zn, and Cu. The gross morphology of the palisade layer has not lent itself to extensive investigation. Each palisade column grows from one mammillary buttons and as the calcification mechanism proceeds adjacent columns fuse, which provides greater resistance of the shell (Solomon, 2010). This study found that in the treatment group that was not supplemented with trace minerals, and at the lowest level of supplementation, there was clutter on the distribution of mammillary buttons on the inner surface of the shell, and this observation was made in the shell that had a higher density of mammillary buttons. This finding supports the study of Van Toledo et al. (1982), who observed that shell with a higher density of mammillary buttons, cracks, and scratches have clutter on their inner surface and that these shells were also less resistant. Such evidence supports the notion that not only do the thickness and the percentage of shell influence resistance but also that the palisade layer thickness and the number of mammillary buttons present in the shell influence shell resistance. Evaluating the inner surface of the eggshell peelings of eggs with low resistance, it is also possible to observe clutter and various structural changes that are not typically observed in shells of high resistance (Solomon, 1991). This author has concluded that these structural changes may act as nucleation sites of breakage, thereby contributing to the relative weakness of these shells. Contrary to this study, Rodriguez-Navarro et al. (2002) did not observe any significant change in density or areola or surface deformities, which could explain the differences in the mechanical properties of the eggshells. Based on this study, it was also possible to infer that the mineral supplementation exerted an influence on the formation of the palisade layer, with supplementation resulting in larger mammillary buttons on the inner surface and improved shell ultrastructure. In conclusion, the supplementation of Mn, Zn, and Cu did not affect key performance variables, such as
HEN DIETS SUPPLEMENTED WITH ORGANIC TRACE MINERALS
Saldanha, E. S. P. B., E. A. Garcia, C. C. Pizzolante, A. B. G. Faittarone, A. Sechinato, A. B. Molino, and C. Laganá. 2009. Effect of organic mineral supplementation on the egg quality of semi-heavy layers in their second cycle of lay. Braz. J. Poult. Sci. 11:215–229. SAS Institute. 2009. SAS/STAT User’s Guide: Release 9.2 ed. SAS Inst. Inc., Cary, NC. Solomon, S. E. 1991. Egg and Eggshell Quality. Wolfe, London, UK. Solomon, S. E. 2010. The eggshell: Strength, structure and function. Br. Poult. Sci. 51:52–59. Swiatkiewicz, S., and J. Koreleski. 2008. The effect of zinc and manganese source in the diet for laying hens on eggshell and bones quality. Vet. Med. 53:555–563. Van Toledo, B., A. H. Parsons, and G. F. Combs. 1982. Role of ultrastructure in determining eggshell strength. Poult. Sci. 61:569–572. Vieira, S. L. 2008. Chelated minerals for poultry. Braz. J. Poult. Sci. 10:73–79. Zamani, A., H. R. Rahmani, and J. Pourreza. 2005. Supplementation of a corn-soybean meal diet with manganese and zinc improves eggshell quality in laying hens. Pak. J. Biol. Sci. 8:1311–1317.
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Richards, J. D., J. Z. Zhao, R. J. Harrel, C. A. Atwell, and J. J. Dibner. 2010. Trace mineral nutrition in poultry and swine. Asianaustralas. J. Anim. Sci. 23:1527–1534. Roberts, J. R. 2004. Factors affecting egg internal quality and egg shell quality in laying hens. Jpn. Poult. Sci. 41:161–177. Robinson, D. S., and N. R. King. 1963. Carbonic anhydrase and formation of the hen’s egg shell. Nature 199:497–498. Rodriguez-Navarro, A., O. Kalin, Y. Nys, and J. M. Garcia-Ruiz. 2002. Influence of the microstructure on the shell strength of eggs laid by hens of different ages. Br. Poult. Sci. 43:395–403. Roland, D. A. 1988. Eggshell breakage: Incidence and economic impact. Poult. Sci. 67:1801–1803. Rostagno, H. S., L. F. T. Albino, J. L. Donzele, P. C. Gomes, R. F. Oliveira, D. C. Lopes, A. S. Ferreira, S. L. T. Barreto, and P. F. Euclides. 2005. Tabelas brasileiras para aves e suínos. Composição de alimentos e exigências nutricionais. 2nd ed. UFV, Viçosa, MG, Brazil. Ruiz, J., and C. A. Lunam. 2000. Ultrastructural analysis of the eggshell: Contribution of the individual calcified layers and the cuticle to hatchability and egg viability in broiler breeders. Br. Poult. Sci. 41:584–592.
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Key words: copper, manganese, proteinate, scanning electron microscope, zinc 2014 Poultry Science 93:104–113 http://dx.doi.org/10.3382/ps.2013-03190
INTRODUCTION
sulfates, carbonates, and phosphates. However, organic mineral sources have emerged on the market with the prospect of being more easily absorbed and retained by birds, thereby reducing the excretion of trace minerals that potentially pollute the environment (Zamani et al., 2005). The organic minerals were conceptualized by AAFCo (2001), such as metal ions, chemically link with an organic molecule, forming unique structures and providing stability with high mineral bioavailability. However, the practical results of organic trace mineral supplementation in poultry diets remain controversial, due to the differences between sources and supplementation levels used (Vieira, 2008). Trace minerals are essential in the diet of laying hens because they participate in the biochemical processes necessary for normal growth and development, including bone and eggshell formation (Richards et al., 2010). Zinc is a cofactor of the enzyme carbonic anhydrase inhibitors that are involved in the formation of the eggshell (Robinson and King, 1963). Manganese acts as activator of the enzymes that are involved in the syn-
Improved understanding of the factors that affect the performance and quality of the eggshells produced by commercial laying hens is essential for the production of the highest quality eggs because defects in shell quality can cause significant losses to the commercial egg industry. Between 10 and 15% of laying eggs are lost before and during the collection process due to problems related to the shell quality (Roland, 1988; Coutts et al., 2007). Therefore, different strategies, especially mineral nutrition and supplementation, have been considered to improve eggshell quality (Nys, 2001; Roberts, 2004). Most mineral sources used in diets for laying hens are derived from inorganic compounds such as oxides,
©2014 Poultry Science Association Inc. Received March 18, 2013. Accepted october 12, 2013. 1 Corresponding author: [email protected]
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ABSTRACT This study was carried out with the purpose of evaluating the effect of supplementing hens’ diets with trace minerals from inorganic or organic sources on the productive performance, eggshell quality, and eggshell ultrastructure of laying hens. Three hundred sixty Hy-Line W36 laying hens between 47 to 62 wk of age were used and distributed in a completely randomized experimental design with 9 treatments, 5 replicates, and 8 birds for each experimental unit. The treatments consisted of a control diet without supplementation of the trace minerals Mn, Zn, and Cu; 4 supplementation levels of these trace minerals from an inorganic source; and the same levels of supplementation from an organic source (proteinates). The supplementation levels in milligrams per kilogram for Mn, Zn, and Cu, were, respectively, 35-30-05, 65-60-10, 9590-15, and 125-120-20. There was no effect of supplementation of trace minerals on the rate of posture, feed
HEN DIETS SUPPLEMENTED WITH ORGANIC TRACE MINERALS
eggshells. These studies would establish greater insight into the mechanisms of action of the trace minerals and their participation in the eggshell ultrastructure and subsequent resistance to cracking. The objective of the study was to evaluate the effects of dietary supplementation of inorganic and organic source trace minerals on the productive performance, the quality of the shell, and the eggshell ultrastructure of laying hens between 47 and 62 wk of age.
MATERIALS AND METHODS Birds The experiment was conducted in the Experimental Farm of Iguatemi of Universidade Estadual de Maringá, Paraná, Brazil. Three hundred sixty Hy-Line W36 laying hens, 47 to 62 wk of age, were used. Hens were housed in collective laying cages (1 m × 0.50 m), divided in 4 subunits (8 hens). The experimental unit was the cage of 8 hens. Feed and water were provided ad libitum, and birds were standardized by weight and egg production before starting the experiment, (i.e., birds were housed with an average weight within an SD of 5% from the average of all birds). Only laying hens that were producing eggs were housed. Birds were subjected to 14 d of adjustment to the experimental diets. The lighting program was 17 h of light per day. Average minimum and maximum temperature, as monitored on a daily basis inside the shed, were 17.4 and 26.5°C, respectively, and the RH averages recorded were 37.6% (minimum) and 84.6% (maximum).
Experimental Diets The experimental design was completely randomized with 9 treatments, 5 replicates and 8 birds per experimental unit. The inorganic trace minerals sources were sulfate of Mn (MnSO4 with 31% of Mn) and Zn (ZnSO4 with 94% of Zn), and oxide of Cu (CuO with 24% of Cu). The trace minerals sources used were proteinate of Zn (150,000 mg of Zn/kg of product), proteinate of Mn (150,000 mg of Mn/kg of product), and proteinate of Cu (100,000 mg of Cu/kg of product), all products Bioplex (Alltech, Lexington, KY). The treatments are shown in Table 1. The control diet (without addition of Mn, Zn, and Cu) was supplemented with trace minerals. The experimental diets were formulated for today’s industry standards for the egg production phase, according to Rostagno et al. (2005), including the trace mineral proportion. The recommended levels corresponded to treatments 3 and 7 (65 mg/kg of Mn; 60 mg/kg of Zn, and 10 mg/kg of Cu). The other treatments have been reduced or increased the trace minerals by the same level to 30 mg of Zn, 30 mg of Mn, and 5 of Cu mg per treatment.
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thesis of glycosaminoglycans and glycoproteins, which contribute to the formation of the organic matrix of the shell. Copper is an integral part of the lysyl oxidase enzyme that is important in the formation of collagen present in the eggshell membrane (Leeson and Summers, 2001). The eggshell is structurally composed by organic and inorganic components. The organic component of the shell consists of the shell membranes, mammillary buttons or tips, the matrix of the shell, and the cuticle (Solomon, 1991). The inorganic component of the eggshell consists of calcium carbonate crystals. The eggshell layers, the inside to the outside, are composed by mammillary buttons layer (internal), palisade layer (intermediate), and crystal surface layer (external; Roberts, 2004). The eggshell structure partly results from a competition for space during crystal growth between adjacent sites of nucleation (Rodriguez-Navarro et al., 2002). Additionally, the interaction between crystal and organic matrix components resulting in anisotropy of the crystal growth as shown by Hernandez-Hernandez et al. (2008). Trace minerals can affect the quality of the shell as a result of either their catalytic properties as components of the enzymes involved in the synthesis of the shell or by direct interaction with the crystals of calcium during shell formation (Fernandes et al., 2008). However, the results reported in the literature show that supplementation of organic minerals is still controversial. Mabe et al. (2003) found that when assessing supplemental sources, organic and inorganic Zn, Mn, and Cu had similar effects on egg quality. Zamani et al. (2005) reported that the basal diet supplemented with Mn and Zn in combination from inorganic sources had a positive effect on the percentage of eggshell. The trace elements Zn, Mn, and Cu influence the organic matrix of eggshells and therefore can influence the mechanical properties of the eggshell. RodriguezNavarro et al. (2002) stated that membranes that compose the organic matrix may provide a network of fibrous reinforcement within the shell that can contribute to the resistance to breaking of the egg. To Solomon (1991), the issues involved in the resistance of eggshell are very complex, possibly reflecting the interaction between its organic and inorganic components. Thus, evaluation of eggshell ultrastructure allows a greater understanding of the organizat ion and reinforces the view that the mechanical properties of the eggs cannot be defined by simple measurement of shell thickness when working with trace minerals (Nys et al., 2004). According to Bain (1992) and Ruiz and Lunam (2000), the palisade layer provides the characteristic of stiffness and thus resistance of shell. In this case, a reduction in the shell’s relative thickness could compromise its strength, leading to a higher incidence of disruptions. Thus, further studies are warranted using organic mineral sources to observe how the trace minerals can influence the structural organization of
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Stefanello et al. Table 1. Supplemented trace minerals in the experimental diets (mg/kg) Treatment
Source of trace minerals
T1 T2 T3 T4 T5 T6 T7 T8 T9
Inorganic1 Inorganic Inorganic Inorganic Organic2 Organic Organic Organic
(without supplement) (35-30-5) (65-60-10) (95-90-15) (125-120-20) (35-30-5) (65-60-10) (95-90-15) (125-120-20)
Manganese
Zinc
Copper
0 35 65 95 125 35 65 95 125
0 30 60 90 120 30 60 90 120
0 5 10 15 20 5 10 15 20
1Inorganic 2Organic
source (Nucleopar Animal Nutrition Ltda., Mandaguari, Brazil). source, proteinate of trace minerals (Bioplex Alltech, Lexington, KY).
Table 2. Composition and calculated analysis of the basal diets Item Ingredient (%) Corn (8.8% CP) Soybean meal (45% CP) Dicalcium phosphate Limestone Soybean oil Salt dl-Methionine (98%) l-Lysine HCl (78%) Vitamin and mineral premix1 Antioxidant2 Calculated composition (% or as shown) ME (kcal/kg) CP Calcium Available phosphorus Digestible Lys Digestible Met + Cys Digestible Thr Digestible Trp Analyzed value3 (mg/kg) Manganese Zinc Copper
Value 64.00 22.40 1.49 9.06 2.12 0.35 0.15 0.03 0.40 0.01 2,880 15.50 3.90 0.36 0.71 0.64 0.52 0.16 37.10 24.20 5.65
1Mineral and vitamin supplement, Nucleopar Animal Nutrition Ltda. (content per kg of diet): vitamin A, 2,550 IU/g; vitamin E, 2,083.33 mg; vitamin D3, 500 IU/g; vitamin K3, 650 mg; vitamin B1, 408.33 mg; vitamin B12, 2,500 µg; vitamin B2, 1,000 mg; vitamin B6, 412.5 mg; Ac. folic, 66.67 mg; biotin, 8.33 mg; choline, 70,000 mg; Ac. pantothenic, 2,375 mg; methionine, 226,875 mg; niacin, 5,308.33 mg; iron, 12,500 mg; iodine, 258.33 mg; selenium 75 mg; cobalt, 83.33 mg; antioxidant, 1,250 mg. 2Butyrate hydroxytoluene. 3According to AOAC Method 990.10 (AOAC, 1990).
Performance and Eggshell Quality Daily feed intake (g/bird per d), feed conversion (kg of feed/dozen eggs and kg of feed/kg of eggs), egg production (%), and egg loss (%) were recorded at 28-d intervals. Daily egg mass was calculated by multiplying the laying rate (%) by the average weight of eggs (g) and divided by 100. The means for each variable was considered as the sum of eggs (n = 8 birds). The egg loss (%) was considered as eggs that were broken, cracked, porous, or thin shell. At the end of each 28-d time period, for 3 consecutive days in a row, the following parameters were assessed: average egg weight, albumen height, specific weight, percentage, and thickness of the shell. All intact eggs from each experimental unit were identified and weighed individually in a precision scale (0.01 g), and were submitted afterward to a specific weight test using the flotation method in saline solution. Six saline solutions were prepared, ranging in density from 1.070 to 1.090 g/cm3 with a variation of 0.004 g/cm3 for each solution. The saline solution densities were measured using densimeter oil. Subsequent to the completion of the specific weight test, a sample of 3 eggs per experimental unit was used for the determination of the albumen height. Measurements in millimeters (mm) were related to the weight of the egg, thereby determining the Haugh unit: HU log 100 (H + 7.57 − 1.7 W0.37), H = where albumen height (mm) and W = egg weight (g). The shells were washed and dried at room temperature for 72 h and were weighed using a precision digital scale (0.001 g). The shell mass was obtained by calculating the weight of the dry shell divided by the overall of total egg weight. After weighing the shells, the shell thickness was measured at 3 points in the central region of each shell using a digital micrometer (Mitutoyo Sul Americana, São Paulo, Brazil). Three intact eggs per experimental unit (n = 135) were used on d 28 of each cycle for the measurement of the resistance force of the shell. The resistance, kilogram-force, was obtained using a texturometer TA.XT2 Texture Analyzer with a delrin probe with a 5-mm diameter (Texture Technologies Corp. and Stable Micro Systems Ltd., Hamilton, MA).
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The percentage composition and experimental diet calculated are listed in Table 2. The trace minerals Mn, Zn, and Cu were supplemented in accordance with the respective treatment (i.e., inorganic and organic sources). Other trace minerals (Fe, I, Co, and Se) were supplemented in the inorganic form and in equal amounts for all treatments. The basal diet was mixed in a vertical mixer and then the experimental diets were obtained by mixing with the appropriate amounts of minerals from the different sources. The amount of Zn, Mn, and Cu was analyzed in the ingredients and in the basal diet.
HEN DIETS SUPPLEMENTED WITH ORGANIC TRACE MINERALS
Eggshell Ultrastructure
Statistical Analysis Statistical analysis was performed by the SAS statistical program (SAS Institute, 2009). The effects of supplementation levels and source were analyzed using regression. To this end, the supplementation levels of Mn, Zn, and Cu were, respectively, level 1 (35-30-5), level 2 (65-60-10), level 3 (95-90-15), and level 4 (125120-20). These levels were used because the increase in the supplementation of trace minerals in each level was equivalent (30 mg of Mn/kg, 30 mg of Zn/kg, and 5 mg of Cu/kg).
RESULTS AND DISCUSSION The results of the regression analysis of the performance variables and quality of eggs of laying hens fed diets supplemented with increasing levels of Mn, Zn, and Cu are represented in Table 3. There was no interaction (P > 0.05) between the mineral source and level of supplementation. There was also no interaction between the production cycles and the mineral source, and between cycles and levels of supplementation. There was no effect of supplementation of Mn, Zn, and Cu trace minerals on the laying production of eggs,
feed intake, feed conversion (kg/dz and kg/kg), specific weight, and the Haugh unit for eggs. The results of analysis of specific weight and Haugh unit supported studies by Dale and Strong (1998) and Saldanha et al. (2009), which did not observe an effect of supplementation of trace minerals. Several other previous studies did not report the effects of organic trace minerals on egg production, feed intake, and feed conversion (Fernandes et al., 2008; Maciel et al., 2010). The egg weight had a quadratic effect (P < 0.05) on Mn, Cu, and Zn levels, independent of source. Eggshell thickness also increased to quadratic (P < 0.05), depending on the level of supplementation of trace minerals. There was an observed effect of the mineral source used, specifically, that there was greater shell thickness when the mineral came from an organic source. Possibly this quadratic increase in the eggshell thickness due to the increasing supplementation of trace minerals, regardless of source, may have influenced the egg weight in which a quadratic increase was also observed. Mineral proteinates are obtained by means of hydrolysis of a protein source such that the resulting hydrolyses contain a mixture of amino acids and small peptides with chains of different lengths. In this way stable chelates are formed that protect trace elements against chemical reactions taking place in the course of digestion. This protection maintains the solubility of these substances during their passage through the gastrointestinal tract to the sites of absorption (Close, 1998). Such absorption would explain the apparent decrease in interaction between mineral forms shown in various reports as well as allowing inorganic and organic forms to be used together to advantage. Greater stability during digestion, along with absorption and transport via peptide and amino acid routes, results in higher biological availability (Solomon, 1991; Nys et al., 2004). However, despite the increased absorption of organic trace mineral source, in this study, no effects were observed on the performance characteristics, as reported in other researches (Fernandes et al., 2008; Swiatkiewicz and Koreleski, 2008; Saldanha et al., 2009; Maciel et al., 2010), but it was possible to observe a direct effect of the mineral supplementation on the eggshell formation. With respect to the eggshell, increased levels of supplementation of Mn, Zn, and Cu resulted in a linear reduction (P < 0.05) of the percentage of egg loss, and a linear increase (P < 0.05) of shell strength, demonstrating a better quality in the eggshell formation. The effect of Zn and Mn supplementation has also been observed by Zamani et al. (2005), who observed that supplementing 150 mg of Zn/kg and 90 mg of Mn/kg improved shell thickness, the index of stiffness, and resistance to breakage. On the other hand, Swiatkiewicz and Koreleski (2008) evaluated the addition of Zn and Mn from inorganic and organic sources for layers between 35 and 70 wk of age and did not observe changes in percentage and shell thickness of the eggs. Maciel et al. (2010) did not observe an effect of the use of Zn, Mn, and Cu on the shell thickness of the eggs.
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On the last day of the experiment, the birds were 62 wk old, and 2 eggs per experimental unit were collected randomly. The eggs were broken and washed, and sample peels of the equatorial region were separated and saved. The membranes of the shell were removed by immersion of samples in a solution of 6% sodium hypochlorite, 4.12% sodium chloride, and 0.15% sodium hydroxide. Afterward, the shells were washed in water and dried at room temperature for at least 48 h according to the methodology described by Radwan et al. (2010). Two samples of the shell of each egg (0.5 cm2) were prepared for scanning electron microscope analysis using the Shimadzu SS-550 Superscan (Shimadzu Corporation, EVISA, Kyoto, Japan). One sample was used for the analysis of the inner surface of the shell, and the other was used for the assessment of the transversal surface. The samples were glued on an aluminum support (stub), metalized with gold, and analyzed in the electron microscope. For the inner surface analysis, the number of mammillary buttons/mm2 and 3 scanned images were obtained for each sample using 50× magnification. For the analysis of the cross-section of the shell, the variables measured were the palisade layer thickness (µm), the mammillary button thickness, and the total thickness (palisade + mammillary). The percentage of the palisade layer thickness and the percentage of the mammillary button thickness were obtained by calculating the ratio of the thickness of each layer to the total thickness measure.
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EP (%)
1.40 1.39 1.40 1.40 1.40 0.003 NS NS
1.40 1.39 1.39 1.40 1.40 0.004
1.40
FC (kg/dz)
1.75 1.74 1.74 1.73 1.74 0.002 NS NS
1.74 1.73 1.74 1.73 1.74 0.009
1.74
FC (kg/kg)
1.21 0.79 0.49 0.22 0.68 0.109 L* NS
1.55 1.09 0.59 0.32 0.89 0.116
1.57
Egg loss (%)
1.08 1.08 1.08 1.08 1.08 0.000 NS NS r2 0.85 0.83 0.57 0.66 0.87 0.97 0.85 0.91
1.08 1.08 1.08 1.08 1.08 0.000
1.08
Specific weight
Shell (%) 8.61 8.65 8.64 8.73 8.76 8.70 0.015 8.65 8.70 8.75 8.80 8.73 0.017 L* NS
Haugh unit 88.6 88.7 88.8 88.9 89.0 88.9 0.063 88.6 89.0 88.9 88.9 88.8 0.084 NS NS
0.36 0.37 0.38 0.38 0.37 0.002 Q* NS P 0.0001 0.0001 0.0003 0.0001 0.0002 0.0001 0.0001 0.0001
0.36 0.36 0.37 0.37 0.37 0.002
0.35
Thickness (mm)
3.57 3.74 3.99 4.12 3.85 0.049 L* NS
3.56 3.67 3.93 3.97 3.78 0.042
3.39
Shell strength (kgf)
= linear effect and Q* = quadratic effect (P < 0.05); EP = egg production (%); FC = feed conversion; shell = percentage of shell; kgf = kilogram-force; M = trace minerals (2 sources).
57.6 57.2 57.5 57.4 57.2 0.122 Q* NS
56.8 57.3 57.3 57.4 57.2 0.140
56.5
Egg mass (g)
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1L*
67.3 67.8 67.0 67.2 67.4 0.118 Q* NS
102.9 102.8 102.8 102.6 102.8 0.061
66.3 67.0 67.3 67.3 67.0 0.132
102.8 103.1 102.8 102.6 102.8 0.070
NS NS
66.2
Egg weight (g)
102.5
Feed intake (g/bird per d)
Without supplementation T1 (0-0-0) 85.0 Inorganic trace minerals (IM) T2 (35-30-5) 85.7 T3 (65-60-10) 85.9 T4 (95-90-15) 85.4 T5 (125-120-20) 85.7 Mean 85.7 SEM 0.201 Organic trace minerals (OM) T6 (35-30-5) 84.9 T7 (65-60-10) 85.8 T8 (95-90-15) 85.9 T9 (125-120-20) 85.6 Mean 85.6 SEM 0.146 Regression analysis Supplement level NS Level × source NS Regression equation Egg loss = 1.543 – 0.345IM Egg loss = 1.543 – 0.343OM Shell % = 8.606 + 0.037IM Shell % = 8.606 + 0.047IM Shell thickness = 0.352 + 0.007IM – 0.0004IM2 Shell thickness = 0.352 + 0.008OM – 0.0004OM2 Shell strength = 3.453 + 0.130IM Shell strength = 3.453 + 0.164OM
Supplementation Mn-Zn-Cu (mg/kg)
Table 3. Mean values of performance and eggshell quality of laying hens 47 to 62 wk of age relative to the level of supplementation and source of trace minerals (Mn, Zn, and Cu)1
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Table 4. Mean values obtained by electron microscopy of the eggshell of laying hens at 62 wk of age relative to the level of supplementation and source of trace minerals (Mn, Zn, and Cu)1 Supplementation Mn-Zn-Cu (mg/kg)
Mammillary (µm)
Thickness (µm)
Palisade (%)
Mammillary (%)
Mammillary buttons/mm2
231
66.1
297
77.7
22.7
263
240 265 267 270 260 3.648
64.3 63.6 62.9 63.6 63.6 0.638
304 329 330 334 324 3.624
78.9 80.6 81.0 80.9 80.3 0.304
21.1 19.4 19.0 19.1 19.6 0.304
192 176 170 162 175 8.772
248 266 277 278 267 3.964
67.9 65.1 64.5 64.4 65.5 0.930
316 331 342 343 333 3.968
78.5 80.3 81.1 81.2 80.3 0.357
21.5 19.7 18.8 18.8 19.7 0.357
177 167 161 149 163 9.520
Q* NS
NS NS
Q* NS
Q* NS
Q* NS r2 0.86 0.79 0.75 0.87 0.54 0.54
L* NS P 0.0029 0.0219 0.0393 0.0061 0.0001 0.0001
1L* = linear effect (P < 0.05); Q* = quadratic effect (P < 0.05); M = trace minerals; T = shell thickness; PAL = palisade layer; MB = number of mammillary buttons/mm2.
Increased eggshell strength was obtained using trace minerals, from either organic or inorganic sources, which may also be related to the decrease in egg loss. Increased dietary trace minerals should be linked with the formation of the eggshell membrane. Particularly, the Cu can improve shell membrane and the Zn and Mn participate in both the organic and inorganic chemistry of shells, resulting in eggs with better quality shells. This effect has also been observed by Maciel et
al. (2010), who observed less egg loss when the layers were given feed supplemented with 50 mg/kg of Zn, Mn, or Cu in organic form. The reduction in the egg loss and the increased resistance of shells are desirable features that have economic importance in a commercial laying segment. In this way, the results of this study are consistent with those obtained by Ludeen (2001), who noted improvement in resistance to breakage of eggs from chickens aged 40 to
Figure 1. Scanning electron microscopy of the cross section of the eggshell of laying hens at 62 wk of age. Palisade layer (pa), mammillary (ma), and vertical view of the mammillary buttons (mb). A) Treatment without addition of Mn, Zn, and Cu; B) supplementation of 35-30-05 mg/kg of Mn-Zn-Cu from an inorganic source; C) supplementation of 125-120-20 mg/kg of Mn-Zn-Cu from an organic source. Scale bar: 100 µm, 54 × 19 mm.
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Without supplementation T1 (0-0-0) Inorganic trace minerals (IM) T2 (35-30-5) T3 (65-60-10) T4 (95-90-15) T5 (125-120-20) Mean SEM Organic trace minerals (OM) T6 (35-30-5) T7 (65-60-10) T8 (95-90-15) T9 (125-120-20) Mean SEM Regression analysis Supplement level Level × source Regression equation T = 296.7 + 18.41IM – 2.128IM2 T = 296.7 + 23.03OM – 2.827OM2 PAL = 228.6 + 20.52IM – 2.497IM2 PAL = 228.6 + 23.24IM – 2.710IM2 MB = 237.34 – 22.30IM MB = 237.34 – 24.31OM
Palisade (µm)
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60 wk that had received proteinates of Zn and Mn. Paik (2001) also reported that organic Zn and Mn improved the eggshell strength and that there was influence of Zn in the synthesis of the enzyme carbonic anhydrase, which is essential for the formation of the shell. Results of the regression analysis of the eggshell ultrastructure of laying hens 62 wk of age fed diets supplemented with increasing levels and 2 sources of trace minerals are depicted in Table 4. There was no interaction (P > 0.05) between the source and the level of supplementation. Supplementation of Mn, Zn, and Cu in commercial laying hen diets did not affect (P > 0.05) the thickness of the mammillary layer of the eggshell. Increasing quantities of the trace minerals Mn, Zn, and Cu gave a quadratic increase (P < 0.05) in the
total shell thickness and the palisade layer and a linear reduction (P < 0.05) in the number of mammillary buttons. The best results for a lower number of mammillary tips by millimeter squared of eggshell were obtained when supplemented with increased levels of trace minerals, as illustrated in Figures 1, 2, and 3. Obtaining measurements of eggshell ultrastructure by scanning electron microscopy has allowed comparisons between the results obtained from different sources and the results using different mineral supplementation levels of Mn, Cu, and Zn. Increasing the palisade layer thickness of eggshells is important because, as the study of Radwan et al. (2010) notes, the strength of the shell depends on the thickness of the palisade layer and the organization of calcite crystals in this layer. Solomon
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Figure 2. Scanning electron microscopy of the inner surface of the eggshell of laying hens at 62 wk of age. Note the different distribution of mammillary buttons (mb) that became larger as levels of trace mineral supplementation increase in the diet. A) Treatment without addition of Mn, Zn, and Cu, control diet or negative control (neg); B) supplementation of 65-60-10 mg/kg of Mn-Zn-Cu from an inorganic source; C) supplementation of 65-60-10 mg/kg of Mn-Zn-Cu from an organic source; D) supplementation of 125-120-20 mg/kg of Mn-Zn-Cu from an organic source. IM: inorganic mineral, OM: organic mineral. Scale bar: 200 µm, 127 × 108 mm.
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(1991) suggested that the organization of the columns of the palisade layer is one of the major determinants of the rigidity of the shell and therefore the strength, and shell resistance of the eggs. Traditionally, the resistance of the eggshell has been considered to be almost entirely dependent on the properties of the inorganic components (Bain, 1992; El-Safty, 2004). The strength of the shell is directly related to its thickness, and the palisade layer comprises approximately two-thirds of the total thickness of the shell (Fathi et al., 2007). Therefore, it is likely that changes in the palisade layer thickness will affect the eggshell resistance, with diets supplemented with Mn, Zn, and Cu improving the quality of the eggshell
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produced, especially when these trace minerals are supplied in the form of proteinates. The properties of the eggshell depend on the microstructure and chemical composition, which can vary through the thickness of the shell (Rodriguez-Navarro et al., 2002; Nys et al., 2004; Bain et al., 2006). It is thought that the supplementation of Mn, Zn, and Cu participate in the formation of eggshells by influencing the thickness via their action as enablers of important enzymes, such as carbonic anhydrase (Nys et al., 2004; Zamani et al., 2005). The activity of carbonic anhydrase and the effects caused by trace minerals are important because the number of organic components and their concentration Downloaded from http://ps.oxfordjournals.org/ at University of Massachusetts/Amherst on May 9, 2014
Figure 3. Scanning electron microscopy of the inner surface of the eggshell of laying hens at 62 wk of age with greater magnification. The mammillary buttons (mb) are wider and together with each other depending on the levels of trace minerals that were supplemented in the diet. Control diet or negative control (neg). IM: inorganic mineral, OM: organic mineral. Scale bar: 50 µm, 150 × 136 mm.
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production of eggs, feed intake, and feed conversion. However, supplementation of Mn, Zn, and Cu did improve quality characteristics and ultrastructure of eggshells. Levels equal to or above 65-60-10 mg/kg, for Mn, Zn, and Cu, respectively, resulted in less egg loss and increased strength of shell.
REFERENCES AAFCO (Association of American Feed Control Officials). 2001. Official Publication. Assoc. Am. Feed Control Off., Atlanta, GA. AOAC. 1990. Official Methods of Analysis. 15th ed. Assoc. Off. Anal. Chem., Arlington, VA. Bain, M. M. 1992. Eggshell strength: A relationship between the mechanism of failure and the ultrastructural organization of the mammillary layer. Br. Poult. Sci. 33:303–319. Bain, M. M., N. Macleod, R. Thomson, and J. W. Hancock. 2006. Microcracks in eggs. Poult. Sci. 85:2001–2008. Close, W. H. 1998. New developments in the use of trace mineral proteinates to improve pig performance and reduce environmental impact. Pages 51–68 in European Lecture Tour. Alltech Inc. Technical Publications, Nicholasville, KY. Coutts, J. A., G. C. Wilson, and S. Fernandez. 2007. Optimum Egg Quality. 5M Publishing, Sheffield, UK. Dale, N., and C. F. Strong Jr. 1998. Inability to demonstrate an effect of eggshell 49 on shell quality in older laying hens. J. Appl. Poult. Res. 7:219–224. El-Safty, S. A. 2004. Stepwise regression analysis for ultrastructural measurements of eggshell quality in two local breeds of chicken. Egypt. Poult. Sci. 24:189–203. Fathi, M. M., A. Zein El-Dein, S. A. El-Safty, and L. M. Radwan. 2007. Using scanning electron microscopy to detect the ultrastructural variations in eggshell quality of Fayoumi and Dandarawi chicken breeds. Int. J. Poult. Sci. 6:236–241. Fernandes, J. I. M., A. E. Murakami, M. I. Sakamoto, L. M. G. Souza, A. Malaguido, and E. N. Martins. 2008. Effects of organic mineral dietary supplementation on production performance and egg quality of white layers. Braz. J. Poult. Sci. 10:9–65. Gautron, J., M. T. Hincke, and Y. Nys. 1997. Precursor matrix proteins in the uterine fluid change with stages of eggshell formation in hens. Connect. Tissue Res. 36:195–210. Hernandez-Hernandez, A., J. Gomez-Morales, A. B. Rodriguez-Navarro, J. Gautron, Y. Nys, and J. M. Garcia-Ruiz. 2008. Identification of some active proteins in the process of hen eggshell formation. Cryst. Growth Des. 8:4330–4339. Hincke, M. T., Y. C. Chien, L. C. Gerstenfeld, and M. D. McKee. 2008. Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell. J. Histochem. Cytochem. 56:467–476. Leeson, S., and J. D. Summers. 2001. Nutrition of the Chicken. 4th ed. University Books, Guelph, ON, Canada. Ludeen, T. 2001. Mineral proteinates may have positive effect on shell quality. Feedstuffs 73:10–15. Mabe, I., C. Rapp, M. M. Bain, and Y. Nys. 2003. Supplementation of a corn-soybean meal diet with manganese, copper, and zinc from organic or inorganic sources improves eggshell quality in aged laying hens. Poult. Sci. 82:1903–1913. Maciel, M. P., E. P. Saraiva, E. F. Aguiar, P. A. Ribeiro, D. P. Passos, and J. B. Silva. 2010. Effect of using organic microminerals on performance and external quality of eggs of commercial laying hens at the end of laying. R. Bras. Zootec. 39:344–348. Nys, Y. 2001. Recent developments in layer nutrition for optimizing shell quality. 13th European Symposium on Poultry Nutrition, Blankenberge (BEL), 2001. WPSA, Belgium Branch. Nys, Y., J. Gautron, J. M. Garcia-Ruiz, and M. T. Hincke. 2004. Avian eggshell mineralization: Biochemical and functional characterization of matrix proteins. C. R. Palevol 3:549–562. Paik, I. 2001. Application of chelated minerals in animal production. Asian-australas. J. Anim. Sci. 14:191–198. Radwan, L. M., A. Galal, M. M. Fathi, and A. Zein El-Dein. 2010. Mechanical and ultrastructural properties of eggshell in two Egyptian native breeds of chicken. Int. J. Poult. Sci. 9:77–81.
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change in the uterine fluid along the different stages of eggshell deposition happen in a well-defined way (Gautron et al., 1997). Also, these organic components are secreted at specific times and locations in the oviduct and incorporated at specific substructural regions of the eggshell. For instance, sulfate proteoglycans are the main components of the mammillary buttons or tips and osteopontin has been found in eggshell associated with particular crystallographic faces of calcite in the palisade region (Hincke et al., 2008); the proteoglycans are influenced by the presence of manganese, and as a result, there can be an eggshell formation that is thicker and more resistant to breakage. The knowledge of the layers that form the eggshell is important because the specific nucleation sites on the outer surface of the outer shell membrane attracts calcium salts and so initiate the formation of the mammillary layer in that region of the oviduct termed the tubular shell gland and may be influenced by the enzymatic activity, which has trace minerals as cofactors Mn, Zn, and Cu. The gross morphology of the palisade layer has not lent itself to extensive investigation. Each palisade column grows from one mammillary buttons and as the calcification mechanism proceeds adjacent columns fuse, which provides greater resistance of the shell (Solomon, 2010). This study found that in the treatment group that was not supplemented with trace minerals, and at the lowest level of supplementation, there was clutter on the distribution of mammillary buttons on the inner surface of the shell, and this observation was made in the shell that had a higher density of mammillary buttons. This finding supports the study of Van Toledo et al. (1982), who observed that shell with a higher density of mammillary buttons, cracks, and scratches have clutter on their inner surface and that these shells were also less resistant. Such evidence supports the notion that not only do the thickness and the percentage of shell influence resistance but also that the palisade layer thickness and the number of mammillary buttons present in the shell influence shell resistance. Evaluating the inner surface of the eggshell peelings of eggs with low resistance, it is also possible to observe clutter and various structural changes that are not typically observed in shells of high resistance (Solomon, 1991). This author has concluded that these structural changes may act as nucleation sites of breakage, thereby contributing to the relative weakness of these shells. Contrary to this study, Rodriguez-Navarro et al. (2002) did not observe any significant change in density or areola or surface deformities, which could explain the differences in the mechanical properties of the eggshells. Based on this study, it was also possible to infer that the mineral supplementation exerted an influence on the formation of the palisade layer, with supplementation resulting in larger mammillary buttons on the inner surface and improved shell ultrastructure. In conclusion, the supplementation of Mn, Zn, and Cu did not affect key performance variables, such as
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Saldanha, E. S. P. B., E. A. Garcia, C. C. Pizzolante, A. B. G. Faittarone, A. Sechinato, A. B. Molino, and C. Laganá. 2009. Effect of organic mineral supplementation on the egg quality of semi-heavy layers in their second cycle of lay. Braz. J. Poult. Sci. 11:215–229. SAS Institute. 2009. SAS/STAT User’s Guide: Release 9.2 ed. SAS Inst. Inc., Cary, NC. Solomon, S. E. 1991. Egg and Eggshell Quality. Wolfe, London, UK. Solomon, S. E. 2010. The eggshell: Strength, structure and function. Br. Poult. Sci. 51:52–59. Swiatkiewicz, S., and J. Koreleski. 2008. The effect of zinc and manganese source in the diet for laying hens on eggshell and bones quality. Vet. Med. 53:555–563. Van Toledo, B., A. H. Parsons, and G. F. Combs. 1982. Role of ultrastructure in determining eggshell strength. Poult. Sci. 61:569–572. Vieira, S. L. 2008. Chelated minerals for poultry. Braz. J. Poult. Sci. 10:73–79. Zamani, A., H. R. Rahmani, and J. Pourreza. 2005. Supplementation of a corn-soybean meal diet with manganese and zinc improves eggshell quality in laying hens. Pak. J. Biol. Sci. 8:1311–1317.
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Richards, J. D., J. Z. Zhao, R. J. Harrel, C. A. Atwell, and J. J. Dibner. 2010. Trace mineral nutrition in poultry and swine. Asianaustralas. J. Anim. Sci. 23:1527–1534. Roberts, J. R. 2004. Factors affecting egg internal quality and egg shell quality in laying hens. Jpn. Poult. Sci. 41:161–177. Robinson, D. S., and N. R. King. 1963. Carbonic anhydrase and formation of the hen’s egg shell. Nature 199:497–498. Rodriguez-Navarro, A., O. Kalin, Y. Nys, and J. M. Garcia-Ruiz. 2002. Influence of the microstructure on the shell strength of eggs laid by hens of different ages. Br. Poult. Sci. 43:395–403. Roland, D. A. 1988. Eggshell breakage: Incidence and economic impact. Poult. Sci. 67:1801–1803. Rostagno, H. S., L. F. T. Albino, J. L. Donzele, P. C. Gomes, R. F. Oliveira, D. C. Lopes, A. S. Ferreira, S. L. T. Barreto, and P. F. Euclides. 2005. Tabelas brasileiras para aves e suínos. Composição de alimentos e exigências nutricionais. 2nd ed. UFV, Viçosa, MG, Brazil. Ruiz, J., and C. A. Lunam. 2000. Ultrastructural analysis of the eggshell: Contribution of the individual calcified layers and the cuticle to hatchability and egg viability in broiler breeders. Br. Poult. Sci. 41:584–592.
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