Copper requirements of broiler breeder hens

Copper requirements of broiler breeder hens Abstract One-hundred-twenty Cobb 500 hens, 20 wk of age, were randomly allocated into individual cages with the objective of estimating Cu requirements. After being fed a Cu deficient diet for 4 wk, hens were fed diets with graded increments of supplemental Cu (0.0; 3.5; 7.0; 10.5; 14; and 17.5 ppm) from Cu sulfate (CuSO4 5H2O), totaling 2.67; 5.82; 9.38; 12.92; 16.83; and 20.19 ppm analyzed Cu in feeds for 20 weeks. Estimations of Cu requirements were done using exponential asymptotic (EA), broken line quadratic (BLQ), and quadratic polynomial (QP) models. Obtained Cu requirements for hen d egg production and total settable eggs per hen were 6.2, 7.3, and 12.9 ppm and 8.1, 9.0, and 13.4 ppm, respectively, using EA, BLQ, and QP models. The QP model was the only one having a fit for total eggs per hen with 13.1 ppm Cu as a requirement. Hemoglobin, hematocrit, and serum Cu from hens had requirements estimated as 13.9, 11.3, and 18.5, ppm; 14.6, 13.0, and 19.0 ppm; and 16.2, 14.6, and 14.2 ppm, respectively, for EA, BLQ, and QP models. Hatching chick hemoglobin was not affected by dietary Cu, whereas requirements estimated for hatching chick hematocrit and body weight and length were 10.2, 12.3, and 13.3 ppm using EA, BLQ, and QP models; and 6.8 and 7.1 ppm, and 12.9 and 13.9 ppm Cu using EA and BLQ models, respectively. Maximum responses for egg weight, yolk Cu content, and eggshell membrane thickness were 14.9, 12.7, and 15.1 ppm; 15.0, 16.3, and 15.7 ppm; and 7.3, 7.8, and 14.0 ppm Cu, respectively, for EA, BLQ, and QP models. Yolk and albumen percentage were adjusted only with the QP model and had requirements estimated at 11.0 ppm and 11.3 ppm, respectively, whereas eggshell mammillary layer was maximized with 10.6, 10.1, and 14.4 ppm Cu using EA, BLQ, and QP models, respectively. The average of all Cu requirement estimates obtained in the present study was 12.5 ppm Cu. INTRODUCTION Copper (Cu) is an essential trace mineral for poultry that has many roles in metabolism, most of them related to enzyme function (Richards et al., 2010; Karimi et al., 2011). A primary function of Cu is related to its role in Fe oxidation, as part of ceruloplasmin (Cp), an essential step in Fe absorption and hemoglobin (Hb) synthesis (Chen et al., 1994; Reeves et al., 2005; Chen et al., 2006). Needed for the adequate functioning of reproductive functions, Cu is a precursor of β-monooxygenase, which catalyzes the hydroxylation of dopamine to norepinephrine, needed for the production of the gonadotropin-releasing hormone (Roychoudhury et al., 2016). Important to support successful chick production, the maturity of the eggshell membrane depends on adequate intake of Cu because of its role in the setup of collagen crosslinks by being part of lysyl oxidase (Rucker and Murray, 1978; Opsahl et al., 1982). The yolk is the largest deposit of Cu in the egg, and, therefore, it is its main source to the embryo during incubation (Richards, 1997). Yolk Cu is bound to lipovitellin and phosvitin (Kozlowski et al., 1988). A riboflavin binding protein (RBP) is also involved in the transport and storage of Cu (Smith et al., 2008), but in contrast to phosvitin, which presents a voluminous capability to store positive charged metals (Samaraweera et al., 2011), the binding to RBP is specific to Cu and occurs in a 1:1 molar ratio (Hall et al., 2013). Therefore, Cu deficiency negatively affects embryo development with resulting gross structural and biochemical abnormalities (Roychoudhury et al., 2016). Because of the tight involvement of Cu in collagen synthesis, an adequate supply of this mineral is also essential for embryo bone development and, therefore, for the independent feed seeking immediately after hatching. A considerable number of reports have been published on the supplementation of Cu for broilers (Schmidt et al., 2005; Pang and Applegate, 2007; Karimi et al., 2011; Kim et al., 2011) and laying hens (Pekel et al., 2012; Kim et al., 2016); however, few studies have been conducted with broiler breeders. Due to the lack of supporting literature, recommendations for Cu supplementation presented in the most popularly referred tables of requirements for chickens are, therefore, likely to be imprecise or outdated (NRC, 1994; Cobb-Vantress, 2013; Aviagen, 2017; Rostagno et al., 2017). The concentration of Cu in feed ingredients is variable, and its bioavailability from different feed ingredients is largely unknown (NRC, 1994; Aoyagi and Baker, 1995). Concerns with environmental pollution have been leading to the surge of a series of regulations limiting the concentrations of micro minerals in animal feeds (López-Alonso, 2012). Since micro minerals are low in cost when compared to many other nutrients, a possibility of excessive Cu being present in poultry feeds exists, leading to a concentration in excreta that can be seen as an environmental contaminant (Pesti and Bakalli, 1996; Ewing et al., 1998; Brainer et al., 2003). The European Commission (EC) has recently established a maximum allowance of 25 ppm total Cu in poultry feeds (EFSA, 2016). On the other hand, intensive farming has led to a decrease in Cu contents in plant feedstuffs in the last century (Klevay, 2016), which therefore, raises uncertainties about its contents in those ingredients and, therefore, as a reliable supply source of Cu for poultry. Since Cu requirements are low and supplementation is usual, its deficiency in poultry is uncommon (Zhao et al., 2010). To the authors’ knowledge, comprehensive Cu requirement studies have not been conducted with broiler breeder hens in recent years. The objective of the present study was to assess the Cu requirements of broiler breeder hens using Cu sulfate, the most common Cu supplement utilized worldwide. Evaluated responses were related to productive performance as well as with eggshell quality, blood constituents, and quality hatching chicks of breeders. MATERIALS AND METHODS All procedures utilized in the present study were approved by the Ethics and Research Committee of the Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Bird Husbandry One-hundred-twenty Cobb 500 broiler breeder hens and 30 Cobb breeder males, 20 wk of age, were obtained from a commercial breeder farm (BRF, Arroio do Meio, RS, Brazil). Hens were individually placed in cages (0.33 m length x 0.46 m deep x 0.40 m height), whereas the males were placed in 3 collective floor pens (2.0 × 1.5 m each, 10 males in each) for semen collection. Cages are electrostatically painted and have one stainless steel nipple drinker per cage, whereas feeders were plastic. Temperature control, lighting, and feeding programs followed Cobb-Vantress (2016) recommendations. Hens were inseminated weekly utilizing freshly collected semen diluted at the ratio of 3 parts of physiological solution to 1 of semen. This was done with 0.1 mL of the dilution using a 1 mL syringe directly into the oviduct. Experimental Diets The study was composed of pre-experimental (depletion) and experimental phases. Immediately after placement, hens at 20 wk of age were fed a Cu deficient diet for 4 wk (2.67 ± 0.329 ppm Cu); 15.4% CP, 2,760 Kcal/kg AMEn; 3.20% Ca; and 0.45% non-phytate P. (Table 1). Hens were 24 wk of age at the beginning of the experimental phase, and birds were weighed and randomly distributed into individual cages. The Cu deficient diet was supplemented with increased levels of laboratory grade Cu sulfate pentahydrate (CuSO4 5H2O) (Sigma Aldrich, St. Louis, MO) at the expense of the mineral mix diluent at levels of 3.5, 7.0, 10.5, 14.0, and 17.5 ppm Cu. Resulting analyzed Cu in the feeding treatments were of 5.82 ± 0.69; 9.38 ± 1.86; 12.92 ± 0.20; 16.83 ± 0.05; and 20.19 ± 1.15 ppm, respectively. Each one of 6 dietary treatments was replicated 20 times with one hen being the experimental unit. The experiment lasted from 25 to 44 wk of age, which for the matter of collecting and analyzing data were divided into 5 periods of 28 days. The experiment was a 6 × 5 factorial comprising 6 Cu supplementation levels and 5 periods. Table 1. Composition of Cu-deficient diet provided to breeder hens from 20 to 44 wk of age. Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  1Calcium carbonate, phosphoric acid, sodium bicarbonate, and potassium chloride were Lab grade and had trace amounts of Cu (4.58; 1.80; 0.26; 3.08 ppm). 2Mineral and vitamin premix supplied the following per kilogram of diet: Zn, 110 mg; Mn, 120 mg, Fe, 50 mg; Se, 0.3 mg and I, 2 mg (all laboratory grade); vitamin A, 12,000 IU; vitamin D3, 3,000 IU; vitamin E, 100 IU; vitamin C, 50 mg; vitamin K3, 6 mg; vitamin B12, 40 μg; thiamine, 3.5 mg; riboflavin, 16 mg; vitamin B6, 6 mg; niacin, 40 mg; pantothenic acid, 25 mg; folic acid, 4 mg; biotin, 0.3 mg; BHT, 100 mg. 3Values were from a pooled sample of batches. View Large Table 1. Composition of Cu-deficient diet provided to breeder hens from 20 to 44 wk of age. Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  1Calcium carbonate, phosphoric acid, sodium bicarbonate, and potassium chloride were Lab grade and had trace amounts of Cu (4.58; 1.80; 0.26; 3.08 ppm). 2Mineral and vitamin premix supplied the following per kilogram of diet: Zn, 110 mg; Mn, 120 mg, Fe, 50 mg; Se, 0.3 mg and I, 2 mg (all laboratory grade); vitamin A, 12,000 IU; vitamin D3, 3,000 IU; vitamin E, 100 IU; vitamin C, 50 mg; vitamin K3, 6 mg; vitamin B12, 40 μg; thiamine, 3.5 mg; riboflavin, 16 mg; vitamin B6, 6 mg; niacin, 40 mg; pantothenic acid, 25 mg; folic acid, 4 mg; biotin, 0.3 mg; BHT, 100 mg. 3Values were from a pooled sample of batches. View Large All ingredients utilized during the study were from the same batch and remained under normal storage conditions, i.e., in a cool, dry, well-ventilated warehouse in 50 kg bags and on pallets to prevent feed from being in direct contact with damp floors until experimental diets were mixed. Analyses of Cu in ingredients and feeds were performed using inductive coupled plasma atomic emission spectroscopy (ICP—Spectro Flamme, Spectro Analytical Instruments, Kleve, Germany) (Anderson, 1999). Breeder hen feeds were provided daily as recommended by Cobb-Vantress (2016). Feed and Cu intake per hen per d and per period is shown in Table 2. Analysis of Cu in drinking water was done using atomic absorption (ZEEnit 650 P, Analytik Jena, Jena, Germany). Averaged duplicate analyzed Cu in water was <0.007 ± 0.002 ppm and, therefore, it was not considered a significant dietary source of the mineral. Males were fed a corn-soy-wheat bran mash diet that met Cobb-Vantress (2013) recommendations. Table 2. Supplemented, calculated, and analyzed Cu concentrations in the experimental diets, feed intake, and Cu intake per hen d in each period. Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  1Analyzed Cu was from 2 pooled samples from all batches. View Large Table 2. Supplemented, calculated, and analyzed Cu concentrations in the experimental diets, feed intake, and Cu intake per hen d in each period. Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  1Analyzed Cu was from 2 pooled samples from all batches. View Large Hen Performance Measurements Egg collection as well as their classification as hatchable or not (broken and deformed) were performed daily. In the last 3 d of each period, the hatchable eggs were weighed and grouped in 3 replicates per treatment and incubated after a 7-day storage in an environmentally controlled room at 18°C and 75% relative humidity (RH). A single-stage incubator (Avicomave, Iracemápolis, SP, Brazil) set at 37.5°C and 65% RH was used until 18 d; eggs were afterwards transferred to a hatcher set to 36.6°C and 80% RH. Hatchability and hatchability of fertile eggs were expressed as percentage of hatching chicks to the total eggs set and fertile eggs, respectively. Hen Blood Measurements Hematocrit (Ht) and Hb, serum Cu concentrations, and Cp activity were obtained from blood samples pooled from 3 broiler breeder hens randomly selected from each treatment per period. Birds were bled only once throughout the study. Blood obtained was partially transferred to 0.5 mL test tubes containing EDTA for Ht and Hb analyzes. Determination of Ht was done using micro capillaries containing blood centrifuged for 5 min at 15,650 to 18,510 × g. Concentration of Hb was determined using the cyanmethemoglobin method as described by Crosby et al. (1954). Blood (3 mL) also was centrifuged to obtain serum, which was transferred to Eppendorf tubes. A portion of the serum was used for Cu concentration analysis (Meret and Henkin, 1971), whereas the remaining was used for analysis of Cp (Schosinsky et al., 1974). Hatching Chick Measurements All hatched chicks were individually weighed, and the distance from the tip of the beak to the end of the middle toe (third toe) was used to determine chick length (Molenaar et al., 2008). Hb and Ht concentration was determined with 15 chicks hatched per treatment in each period using the same methodology as with the hens. Chick blood samples were obtained from the jugular vein after sacrifice by cervical dislocation. Egg Analysis Eggs from 10 replications were collected in the last 3 d of each 28-day period, totaling 30 eggs per treatment. One replication as a pool of 3 yolks from the same treatment was lyophilized, and a total of 10 replicates was collected per treatment per period. Yolk Cu content was quantified using ICP, as it was done with ingredients and feeds. Specific gravity was determined using saline solutions with concentrations ranging from 1.065 to 1.095 g/cm3 in intervals of 0.005 units (Novikoff and Gutteridge, 1949). Shell weight was obtained after washing and drying at 105°C overnight, whereas shell thickness was measured using a micrometer (Model IP65, Mitutoyo Corp., Kawasaky, Japan) in the apical, equatorial, and basal regions with these values being averaged for statistical analysis. In addition, 3 eggs with an average weight ± 10% SD per treatment obtained in the last 3 d of each period (age 28, 32, 36, 40, and 44 wk) were used in the analysis of eggshell ultrastructure using scanning electron microscopy (King and Robinson, 1972). In preparation for this analysis, eggshells were gently broken into 3 sections (0.5 to 1 cm2) in the apical, equatorial, and basal regions, totaling 270 pieces. Samples were then transversely mounted on aluminum stubs using carbon tape. These were gold metallized at 35 nm (BAL-TEC SCD050 Sputter Coater, Capovani Brothers Inc., Scotia, NY) for 3 min and were subsequently examined in the scanning electron microscope (JEOL JSM 5800, GenTech, Arcade, NY) with a 20 kW acceleration voltage and at magnification of 200×. Mammillary and palisade layers identifications were done according to the descriptions of Dennis et al. (1996). The digital microscopy images were uploaded to the Image-Pro Plus analyzer (Media Cybernetics, Rockville, MD). Averages of eggshell layer thickness were estimated from 5 measurements (μm) done in each photograph. Statistical Analysis Data were submitted to the normalcy of variance test (Shapiro and Wilk, 1965), and data not presenting normal distribution were subjected to transformation in order to stabilize variances using the arcsine square root percentage (z = asin (sqrt (y+ 0.5))) (Ahrens et al., 1990). Data were analyzed using the PROC MIXED of SAS (2011) with the repeated statement included in the statistical model. The covariance structure used was the variable components, which showed the best fit based on the Akaike criteria (Littell et al., 1998), except for eggshell thickness, which was better fitted with the Toeplizt covariance (Wolfinger, 1993). Total egg production and settable egg production per hen at 44 wk were analyzed using the general linear models (PROC GLM). Means were compared using the Tukey-Kramer test, and differences were considered significant at P < 0.05 (Tukey, 1991). Estimates of Cu requirements were done using 3 different models: exponential asymptotic (EA), broken line quadratic (BLQ), and quadratic polynomial (QP) (Robbins et al., 1979). The EA model (Y = β1 + β2 × (1- EXP (-β3 × (Cu—β4)))) had Y as the dependent variable as a function of dietary level of Cu; β1 estimated the relative response to the diet containing the lowest Cu (deficient diet); β2 estimated the difference between the minimum and the maximum response obtained with Cu supplementation; β3 was the curve slope coefficient; and β4 was the Cu level of the deficient diet. The maximum response for Cu was defined as Cu = (ln (0.05)/-β3) + β4 for 95% of the requirement. The BLQ model (Y = β1 + β2 × (β3 - Cu)2) had (β3 - Cu) = 0 for Cu > β3 with Y as the dependent variable as a function of the dietary level of Cu, β1 the value of the dependent variable at the plateau, and β2 as the slope of the line. The Cu level at the break point (β3) was considered the one providing maximum responses. The QP model (Y = β1 + β2 × Cu + β3 × (Cu)2) had Y as the dependent variable and as a function of dietary level of Cu; β1 as the intercept; β2 as the linear coefficient; and β3 as the quadratic coefficient. The maximum response for Cu was defined as Cu = –β2 ÷ (2 × β3). The coefficient of determination (R2) was used to assess the goodness of fit for the different models. RESULTS Formulated and analyzed diets had similar Cu contents (Table 2). Analyses of Cu in the dietary treatments were conducted on samples from 2 pools of the 5 mixed batches utilized throughout the study and averaged 5.82 ± 0.69; 9.38 ± 1.86; 12.92 ± 0.20; 16.83 ± 0.05; and 20.19 ± 1.15 ppm. There were no interactions between dietary Cu and period for any response; therefore, responses are presented as main effects of dietary Cu and period throughout the text. During the experimental phase, period affected (P < 0.05) almost all studied variables (Tables 3 and 4) with the exception of egg hatchability, hatchability of fertile eggs, hen Ht, and Cp activity of breeders (P > 0.05). As expected, egg production decreased after peak (29 to 32 wk) (P < 0.05). In parallel, breeder Hb decreased as hens aged to 44 wk (P < 0.05), whereas serum Cu peaked in the period of 33 to 36 wk and decreased afterwards (P < 0.05). Hatching chick Hb was higher, and body length was longer (P < 0.05) when chicks were obtained from eggs laid by hens after 40 wk of age, while the chick Ht and chick weight increased (P < 0.05) from eggs laid from hens after 33 wk of age (Table 3). Egg weight was higher (P < 0.05) in the period of 40 to 44 wk, while specific gravity, thickness of eggshell membrane, and yolk Cu concentration were lower in the same period (P < 0.05). Yolk percentage increased, and albumen percentage decreased (P < 0.05) in the 3 last periods (33 to 44 wk), whereas eggshell thickness palisade layer decreased from 33 to 36 wk (P < 0.05), and, on other hand, eggshell mammillary layer thickness was highest from 29 to 36 wk (P < 0.05; Table 4). Table 3. Response of broiler breeder hens to increased dietary Cu.   Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533    Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Eggs produced as a percentage of total live hens at the moment of measurement. 3Total settable eggs at the end of the experiment. 4Total eggs at the end of the experiment. 5Hematocrit. 6Hemoglobin. 7Ceruloplasmin. View Large Table 3. Response of broiler breeder hens to increased dietary Cu.   Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533    Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Eggs produced as a percentage of total live hens at the moment of measurement. 3Total settable eggs at the end of the experiment. 4Total eggs at the end of the experiment. 5Hematocrit. 6Hemoglobin. 7Ceruloplasmin. View Large Table 4. Broiler breeder hen egg characteristics as affected by increased dietary Cu.               Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999                Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). eAll egg analyzes were affected by the period (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Dry matter Cu concentration. View Large Table 4. Broiler breeder hen egg characteristics as affected by increased dietary Cu.               Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999                Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). eAll egg analyzes were affected by the period (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Dry matter Cu concentration. View Large Dietary Cu affected all responses with the exception of hatchability, hatchability of fertile eggs, breeder Cp activity, Hb of hatching chicks, eggshell percentage, specific gravity, and thickness of eggshell palisade layer (P > 0.05; Tables 3 and 4). Providing breeders dietary increases of Cu affected (P < 0.05) hen d egg production as well as total settable eggs per hen and, therefore, impacted the total number of eggs per hen at the end of the experiment at 44 weeks. Breeder Hb increased (P < 0.05) when they were fed diets with 9.38 ppm Cu or greater as compared to the non-supplemented diet. Breeder Ht, serum Cu, hatching chick Ht, and body weight and length of hatching chicks increased (P < 0.05) as dietary Cu reached 12.92 ppm. Egg weight and yolk percentage were significantly increased when dietary Cu was 9.38 ppm (P < 0.05). However, egg weight increased in the next levels, whereas yolk percentage decreased (P < 0.05). The yolk Cu concentration was highest (P < 0.05) when dietary Cu was 12.92 ppm or above. Scanning electronic microscopy photographs (Figure 1) showed that dietary Cu deficiency led to a less stable eggshell ultrastructure compared with the level above 5.82 ppm dietary Cu (P < 0.05). The Cu deficient diet (2.67 ppm Cu) led to decreased eggshell membrane thickness, whereas the eggshell mammillary layer was affected by dietary Cu (P < 0.05) only in the period between 29 and 32 weeks. Figure 1. View largeDownload slide Scanning electron cross-sections of eggshells from broiler breeder hens fed a Cu-deficient diet (2.67 ppm) (A), and diets with 5.82 ppm (B), 9.38 ppm (C), 12.92 ppm (D), 16.83 ppm (E), and 20.19 ppm (F) Cu (200x). * Membrane. ** Mammillary layer. ***Palisade layer. Figure 1. View largeDownload slide Scanning electron cross-sections of eggshells from broiler breeder hens fed a Cu-deficient diet (2.67 ppm) (A), and diets with 5.82 ppm (B), 9.38 ppm (C), 12.92 ppm (D), 16.83 ppm (E), and 20.19 ppm (F) Cu (200x). * Membrane. ** Mammillary layer. ***Palisade layer. Requirements of Cu were determined using EA, BLQ, and QP regression models. These are shown in Tables 5 and 6 in ppm as well as in mg per hen day. A sharp increase in egg production was observed at the first Cu level supplemented (5.82 ppm), while the maximum responses were obtained with 6.2 ppm (0.89 mg/hen/d), 7.3 ppm (1.04 mg/hen/d), and 12.9 ppm (1.84 mg/hen/d) provided by EA, BLQ, and QP models, respectively. The requirements of Cu estimated for total settable eggs per hen were 8.1 ppm (1.16 mg/hen/d), 9.0 ppm (1.29 mg/hen/d), and 13.4 ppm (1.91 mg/hen/d) Cu. Table 5. Requirements of Cu estimated for diverse broiler breeder responses adjusted with exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16    Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Table 5. Requirements of Cu estimated for diverse broiler breeder responses adjusted with exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16    Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Table 6. Cu requirements for performance and blood measurements of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 5. 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Table 6. Cu requirements for performance and blood measurements of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 5. 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Breeder hen requirements of Cu for Hb were estimated as 13.9 ppm (1.98 mg/hen/d), 11.3 ppm (1.61 mg/hen/d), and 18.5 ppm (2.63 mg/hen/d) using EA, BLQ, and QP models, respectively, whereas the requirement for Ht was estimated at 15.6 ppm (2.08 mg/hen/d), 13.0 ppm (1.86 mg/hen/d), and 19.0 ppm (2.71 mg/hen/d), using EA, BLQ, and QP models, respectively. In parallel, serum Cu concentration was maximized at 16.2 ppm (2.31 mg/hen/d), 14.6 ppm (2.08 mg/hen/d), and 14.2 ppm (2.03 mg/hen/d), using EA, BLQ, and QP models, respectively. The requirements of Cu for hatching chick Ht and body weight and length were estimated at 10.2, 12.3, and 13.3 ppm (1.45, 1.76, and 1.90 mg/hen/d) using EA, BLQ, and QP models; and 6.8 and 7.1 ppm (0.97 and 1.01 mg/hen/d) and 12.9 and 13.9 ppm (1.84 and 1.98 mg/hen/d) Cu using EA and BLQ models, respectively. The R squared obtained using the different tested models for hatching chicks Ht, weight, and length were very low. It was probably due to the high variation of the data among the different evaluation periods. Estimations of Cu requirements for egg component measures are shown in Tables 7 and 8 (ppm and mg per hen d, respectively). All models were well fitted in estimating Cu requirements for analysis done with eggs, except for egg yolk and albumen percentage, which did not fit for EA and BLQ models. The Cu requirement for percent yolk and albumen were 11.0 ppm (1.57 mg/hen/d) and 11.3 ppm (1.61 mg/hen/d) for percent yolk and albumen, respectively, using the QP model. The requirement of Cu estimated for egg weight was 14.9 ppm (2.13 mg/hen/d), 12.7 ppm (1.81 mg/hen/d), and 15.1 ppm (2.15 mg/hen/d), using EA, BLQ, and QP models, respectively. Dietary Cu requirements to maximize the Cu content in the yolk were 15.0 ppm (2.14 mg/hen/d), 16.3 ppm (2.33 mg/hen/d), and 15.7 ppm (2.24 mg/hen/d) using EA, BLQ, and QP models, respectively. Eggshell membrane was thickest when dietary Cu was fed at 7.3 ppm (1.04 mg/hen/d), 7.9 ppm (1.12 mg/hen/d), and 14.0 ppm (1.99 mg/hen/d) Cu, using EA, BLQ, and QP models, respectively. Table 7. Requirements of Cu (ppm) estimated for egg characteristics exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42    Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large Table 7. Requirements of Cu (ppm) estimated for egg characteristics exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42    Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large Table 8. Cu requirements for egg analyses of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 7. 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large Table 8. Cu requirements for egg analyses of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 7. 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large DISCUSSION In the present study, broiler breeder hens fed a non-supplemented Cu diet demonstrated signals of deficiency throughout most evaluated responses. Since Cu is necessary for adequate activities of a great number of metalloenzymes, biochemical reactions are not carried out properly during Cu deficiency (Kaya et al., 2006; Scheiber et al., 2014; Cao et al., 2016). The lack of interaction between dietary Cu and period is an indication that Cu requirements do not change as hens age from 25 to 44 weeks. However, values vary within the same database depending on the model used to estimate its requirements (Robbins et al., 1979). Prediction of requirements do not have one particular true model; therefore, results of experiments should be the ones to dictate the model choice (Vedenov and Pesti, 2008). Non-linear models, such as BLQ and EA, have been thought to be advantageous compared to linear models, due to the fact that biological systems rarely work in perfect linearity (Pesti et al., 2009). In the present study, adequate fit was sometimes not found for one or more models and, in those cases, they were not included in this section. The EA and BLQ models fitted better and estimated lower requirement values for most responses, whereas the QP model provided the best fit only for total egg production, yolk, and albumen percentage and estimated higher requirements values. Requirements of Cu for hen d egg production varied between 6.2 and 12.9 ppm total Cu in the diet, which represents a dietary intake of Cu from 0.89 mg/hen/d to 1.84 mg/hen/day. Requirement for total egg production at the end of wk 44 was higher (13.1 ppm or 1.87 mg/hen/d) than for egg production per hen day. Traditionally the tables of requirements for poultry do not provide Cu requirements for broiler breeders. Instead, supplemental levels are suggested, which range from 10 to 15 ppm, intending to provide safety margins in feed formulation (Mondal et al., 2010). Data from the present study indicate that the usually suggested supplemental Cu levels (NRC, 1994; Cobb-Vantress, 2013; Aviagen, 2017; Rostagno et al., 2017) are excessive. A safety margin is generally used when it comes to micronutrients due to numerous factors that can influence the nutritional requirements (Applegate and Angel, 2014), including an overall scarcity of research. The maximum concentration of 25 ppm Cu in the diet as established by the EC (EFSA, 2016), therefore, seems to be very safe in attending breeder hen needs, since it represents about 2 times what has been estimated as an average requirement in the present research. A non-supplemented broiler breeder hen diet formulated with corn, soy, and wheat bran has an average of 8 ppm total Cu and, therefore, is expected to provide Cu in amounts close to the requirements determined in the present study. Dietary Cu fed to hens impacted their blood Hb and Ht. A decrease in the Ht can be related to depression in the phospholipid synthesis (Kaya et al., 2006) and in the platelet-mediated hemostasis in dietary Cu deficiency (Schuschke et al., 1994). A reduction in Ht was reported by Baumgartner et al. (1978) when hens were fed a Cu deficient diet. Effects of dietary Cu also were observed in Hb, a response that also was previously reported in poultry (Samanta et al., 2011; Mroczek-Sosnowska et al., 2013). In the present study, Ht and Hb were maximized when hens were fed 12.6 and 13.8 ppm Cu (average of the EA and BLQ models, which presented high R-squares). Kubena et al. (1972) also observed the same tendency when diets were tested without Cu supplementation (3 ppm) or were supplemented at 2.2 and 9.9 ppm. It has been earlier reported that Cu facilitates Hb formation in anemic chicks (Elvehjem and Hart, 1929), and Cp-stimulated iron uptake requires Cp ferroxidase activity and utilizes a novel, trivalent cation-specific transport pathway (Attieh et al., 1999; Lenartowicz et al., 2015; Linder, 2016). Hephaestin and Cp are Cu-dependent enzymes that can modify blood Hb values (Reeves et al., 2005; Ha et al., 2016). In the present study, however, hen Cp activity when a deficient Cu diet was fed (2.67 ppm Cu) was not affected. Others have found the opposite, with 3.44 ppm Cu leading to decreases in Cp (Kaya et al., 2006). Concentration of Cu in the serum has been improved as dietary Cu increases (Zanetti et al., 1991; Schmidt et al. 2005), and maximum serum Cu level obtained in the present study was obtained at 15.0 ppm dietary Cu (2.14 mg/hen/d). The lack of significant differences in the Cp activity in response to dietary Cu increase found in the present study and changes in the Cu contents in Hb, as well as in the serum observed, indicate that blood parameters are more sensitive to dietary Cu than Cp and that Cp is highly preserved. Average Cu requirements using the 3 models for Hb, Ht, and serum Cu were similar (14.6, 15.5, and 15.0 ppm or 2.07, 2.21, and 2.14 mg/hen/d, respectively). These values were higher than those needed to maximize egg production. Blood Cu has been shown to change in relation to growth rate and age (Abdel-Mageed and Oehme, 1990), which may explain the differences in the amount of Cu in the serum among periods in this study. Deficiencies observed with the lowest Cu contents in the diets fed in the present study resulted in decreases in yolk Cu concentration, as well as in Ht of hatching chicks. Contents of Cu in eggs are dependent on the dietary supply to breeder hens (Kim et al., 2016) and, therefore, deficiencies or excesses affect egg quality and subsequent performance of the progeny (Whitehead et al., 1985). In the present study, it seemed that the deposition of Cu in the yolk had a non-linear behavior, reaching maximum Cu depositions at 15.7 ppm dietary Cu (or 2.24 mg/hen/d). Egg mineral constituents are transferred from the hen, which originate from the feed. Copper deprivation impairs eggshell quality, as it was observed by scanning electronic microscopy in the present study. Eggshells are composed of ultrastructural layers divided into shell membranes, mammillary knobs, palisade, and a cuticle (Arias et al., 1993; Hunton, 2005). Activation of lysyl oxidase by Cu is necessary for the collagen synthesis in the eggshell membrane (Leach et al., 1981). This enzyme is responsible for oxidative deamination of the lysine side chains, which form crosslinks and thus confer characteristics of insolubility, flexibility, and structure for the deposition of other egg components (Linder and Hazegh-Azam, 1996; Akagawa et al., 1999). In the present study, the thickening of the eggshell membrane provided by Cu intake did not increase the total thickness of the shell or palisade layer, or of the mammillary layer in most periods. There was an increase in thickness of the eggshell mammillary layer only at 29 to 32 wk using 11.7 ppm Cu (or 1.77 mg/hen/d), but there was no change in the thickness of the palisade layer at any time. Thus, the amount of Cu that is deposited in the eggshell must be influenced by other factors. Duan et al. (2016) observed that the thickness of the mammillary layer was affected by mammillary density and might be regulated by some protein interactions, which can be responsible for nucleation sites formation. Although the membrane thickness does not regulate the amount of mineral deposition in the other eggshell layers, it appears to be important for structural organization. Therefore, a well-structured outer shell membrane is necessary for correct mineralization and consequently to produce resistant eggshells (Nys et al., 1999; Nys et al., 2004). Any modification of the eggshell membranes by Cu deficiency occurs due to inhibition of fiber formation or crosslinking, characterized by an abnormal distribution of the eggshell membrane fibers (Mabe et al., 2003), and consequently altering its mechanical properties (Hincke et al., 2012). The lowest thickness of the eggshell membrane observed using low Cu levels in the present study may have resulted in an increased occurrence of unsettable eggs. The requirement of 7.6 ppm Cu (1.08 mg/hen/d) for the maximum eggshell membrane thickness obtained in this experiment was similar to the requirement of the maximum total settable eggs of 8.6 ppm Cu (1.22 mg/hen/d) using the mean EA and BLQ models. Results obtained indicate that Cu most likely plays an important role in thickening of the eggshell membrane, and, therefore, it is expected to affect eggshell breaking resistance. Some studies suggest that the dry eggshell membrane weight decreases in eggs with eggshell deformation, as well as the membrane strength having a positive correlation with eggshell breaking strength (Britton, 1977; Essary et al., 1977). In addition, adequately structured eggshell membranes provide the proper formation of the air chamber immediately after oviposition, which is dependent on separation between the inner and the outer shell membranes (Vieira, 2007). 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Copper requirements of broiler breeder hens

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

Abstract One-hundred-twenty Cobb 500 hens, 20 wk of age, were randomly allocated into individual cages with the objective of estimating Cu requirements. After being fed a Cu deficient diet for 4 wk, hens were fed diets with graded increments of supplemental Cu (0.0; 3.5; 7.0; 10.5; 14; and 17.5 ppm) from Cu sulfate (CuSO4 5H2O), totaling 2.67; 5.82; 9.38; 12.92; 16.83; and 20.19 ppm analyzed Cu in feeds for 20 weeks. Estimations of Cu requirements were done using exponential asymptotic (EA), broken line quadratic (BLQ), and quadratic polynomial (QP) models. Obtained Cu requirements for hen d egg production and total settable eggs per hen were 6.2, 7.3, and 12.9 ppm and 8.1, 9.0, and 13.4 ppm, respectively, using EA, BLQ, and QP models. The QP model was the only one having a fit for total eggs per hen with 13.1 ppm Cu as a requirement. Hemoglobin, hematocrit, and serum Cu from hens had requirements estimated as 13.9, 11.3, and 18.5, ppm; 14.6, 13.0, and 19.0 ppm; and 16.2, 14.6, and 14.2 ppm, respectively, for EA, BLQ, and QP models. Hatching chick hemoglobin was not affected by dietary Cu, whereas requirements estimated for hatching chick hematocrit and body weight and length were 10.2, 12.3, and 13.3 ppm using EA, BLQ, and QP models; and 6.8 and 7.1 ppm, and 12.9 and 13.9 ppm Cu using EA and BLQ models, respectively. Maximum responses for egg weight, yolk Cu content, and eggshell membrane thickness were 14.9, 12.7, and 15.1 ppm; 15.0, 16.3, and 15.7 ppm; and 7.3, 7.8, and 14.0 ppm Cu, respectively, for EA, BLQ, and QP models. Yolk and albumen percentage were adjusted only with the QP model and had requirements estimated at 11.0 ppm and 11.3 ppm, respectively, whereas eggshell mammillary layer was maximized with 10.6, 10.1, and 14.4 ppm Cu using EA, BLQ, and QP models, respectively. The average of all Cu requirement estimates obtained in the present study was 12.5 ppm Cu. INTRODUCTION Copper (Cu) is an essential trace mineral for poultry that has many roles in metabolism, most of them related to enzyme function (Richards et al., 2010; Karimi et al., 2011). A primary function of Cu is related to its role in Fe oxidation, as part of ceruloplasmin (Cp), an essential step in Fe absorption and hemoglobin (Hb) synthesis (Chen et al., 1994; Reeves et al., 2005; Chen et al., 2006). Needed for the adequate functioning of reproductive functions, Cu is a precursor of β-monooxygenase, which catalyzes the hydroxylation of dopamine to norepinephrine, needed for the production of the gonadotropin-releasing hormone (Roychoudhury et al., 2016). Important to support successful chick production, the maturity of the eggshell membrane depends on adequate intake of Cu because of its role in the setup of collagen crosslinks by being part of lysyl oxidase (Rucker and Murray, 1978; Opsahl et al., 1982). The yolk is the largest deposit of Cu in the egg, and, therefore, it is its main source to the embryo during incubation (Richards, 1997). Yolk Cu is bound to lipovitellin and phosvitin (Kozlowski et al., 1988). A riboflavin binding protein (RBP) is also involved in the transport and storage of Cu (Smith et al., 2008), but in contrast to phosvitin, which presents a voluminous capability to store positive charged metals (Samaraweera et al., 2011), the binding to RBP is specific to Cu and occurs in a 1:1 molar ratio (Hall et al., 2013). Therefore, Cu deficiency negatively affects embryo development with resulting gross structural and biochemical abnormalities (Roychoudhury et al., 2016). Because of the tight involvement of Cu in collagen synthesis, an adequate supply of this mineral is also essential for embryo bone development and, therefore, for the independent feed seeking immediately after hatching. A considerable number of reports have been published on the supplementation of Cu for broilers (Schmidt et al., 2005; Pang and Applegate, 2007; Karimi et al., 2011; Kim et al., 2011) and laying hens (Pekel et al., 2012; Kim et al., 2016); however, few studies have been conducted with broiler breeders. Due to the lack of supporting literature, recommendations for Cu supplementation presented in the most popularly referred tables of requirements for chickens are, therefore, likely to be imprecise or outdated (NRC, 1994; Cobb-Vantress, 2013; Aviagen, 2017; Rostagno et al., 2017). The concentration of Cu in feed ingredients is variable, and its bioavailability from different feed ingredients is largely unknown (NRC, 1994; Aoyagi and Baker, 1995). Concerns with environmental pollution have been leading to the surge of a series of regulations limiting the concentrations of micro minerals in animal feeds (López-Alonso, 2012). Since micro minerals are low in cost when compared to many other nutrients, a possibility of excessive Cu being present in poultry feeds exists, leading to a concentration in excreta that can be seen as an environmental contaminant (Pesti and Bakalli, 1996; Ewing et al., 1998; Brainer et al., 2003). The European Commission (EC) has recently established a maximum allowance of 25 ppm total Cu in poultry feeds (EFSA, 2016). On the other hand, intensive farming has led to a decrease in Cu contents in plant feedstuffs in the last century (Klevay, 2016), which therefore, raises uncertainties about its contents in those ingredients and, therefore, as a reliable supply source of Cu for poultry. Since Cu requirements are low and supplementation is usual, its deficiency in poultry is uncommon (Zhao et al., 2010). To the authors’ knowledge, comprehensive Cu requirement studies have not been conducted with broiler breeder hens in recent years. The objective of the present study was to assess the Cu requirements of broiler breeder hens using Cu sulfate, the most common Cu supplement utilized worldwide. Evaluated responses were related to productive performance as well as with eggshell quality, blood constituents, and quality hatching chicks of breeders. MATERIALS AND METHODS All procedures utilized in the present study were approved by the Ethics and Research Committee of the Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Bird Husbandry One-hundred-twenty Cobb 500 broiler breeder hens and 30 Cobb breeder males, 20 wk of age, were obtained from a commercial breeder farm (BRF, Arroio do Meio, RS, Brazil). Hens were individually placed in cages (0.33 m length x 0.46 m deep x 0.40 m height), whereas the males were placed in 3 collective floor pens (2.0 × 1.5 m each, 10 males in each) for semen collection. Cages are electrostatically painted and have one stainless steel nipple drinker per cage, whereas feeders were plastic. Temperature control, lighting, and feeding programs followed Cobb-Vantress (2016) recommendations. Hens were inseminated weekly utilizing freshly collected semen diluted at the ratio of 3 parts of physiological solution to 1 of semen. This was done with 0.1 mL of the dilution using a 1 mL syringe directly into the oviduct. Experimental Diets The study was composed of pre-experimental (depletion) and experimental phases. Immediately after placement, hens at 20 wk of age were fed a Cu deficient diet for 4 wk (2.67 ± 0.329 ppm Cu); 15.4% CP, 2,760 Kcal/kg AMEn; 3.20% Ca; and 0.45% non-phytate P. (Table 1). Hens were 24 wk of age at the beginning of the experimental phase, and birds were weighed and randomly distributed into individual cages. The Cu deficient diet was supplemented with increased levels of laboratory grade Cu sulfate pentahydrate (CuSO4 5H2O) (Sigma Aldrich, St. Louis, MO) at the expense of the mineral mix diluent at levels of 3.5, 7.0, 10.5, 14.0, and 17.5 ppm Cu. Resulting analyzed Cu in the feeding treatments were of 5.82 ± 0.69; 9.38 ± 1.86; 12.92 ± 0.20; 16.83 ± 0.05; and 20.19 ± 1.15 ppm, respectively. Each one of 6 dietary treatments was replicated 20 times with one hen being the experimental unit. The experiment lasted from 25 to 44 wk of age, which for the matter of collecting and analyzing data were divided into 5 periods of 28 days. The experiment was a 6 × 5 factorial comprising 6 Cu supplementation levels and 5 periods. Table 1. Composition of Cu-deficient diet provided to breeder hens from 20 to 44 wk of age. Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  1Calcium carbonate, phosphoric acid, sodium bicarbonate, and potassium chloride were Lab grade and had trace amounts of Cu (4.58; 1.80; 0.26; 3.08 ppm). 2Mineral and vitamin premix supplied the following per kilogram of diet: Zn, 110 mg; Mn, 120 mg, Fe, 50 mg; Se, 0.3 mg and I, 2 mg (all laboratory grade); vitamin A, 12,000 IU; vitamin D3, 3,000 IU; vitamin E, 100 IU; vitamin C, 50 mg; vitamin K3, 6 mg; vitamin B12, 40 μg; thiamine, 3.5 mg; riboflavin, 16 mg; vitamin B6, 6 mg; niacin, 40 mg; pantothenic acid, 25 mg; folic acid, 4 mg; biotin, 0.3 mg; BHT, 100 mg. 3Values were from a pooled sample of batches. View Large Table 1. Composition of Cu-deficient diet provided to breeder hens from 20 to 44 wk of age. Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  Ingredient, % as-is1  Deficient diet  Rice, polished and broken, 8.0% CP  40.98  Corn, 7.8% CP  27.64  Soy protein isolate, 89% CP  10.06  Calcium carbonate  7.75  Oat hulls  8.98  Soybean oil  1.00  Phosphoric acid, 85% P  1.93  Potassium carbonate  0.72  Sodium bicarbonate  0.24  Potassium chloride  0.21  Choline chloride  0.16  DL-methionine, 99%  0.16  L-threonine 98.5%  0.05  Vitamin and mineral mix2  0.10  L-lysine HCl, 78%  0.02  Total  100.00  Calculated nutrient composition, % or as shown   AMEn, kcal/kg  2,760   CP  15.40   Ca  3.20   Available P  0.45   Na  0.20   Cu, ppm     Calculated  2.38   Analyzed3  2.67 ± 0.33  Choline, mg/kg  1,500  1Calcium carbonate, phosphoric acid, sodium bicarbonate, and potassium chloride were Lab grade and had trace amounts of Cu (4.58; 1.80; 0.26; 3.08 ppm). 2Mineral and vitamin premix supplied the following per kilogram of diet: Zn, 110 mg; Mn, 120 mg, Fe, 50 mg; Se, 0.3 mg and I, 2 mg (all laboratory grade); vitamin A, 12,000 IU; vitamin D3, 3,000 IU; vitamin E, 100 IU; vitamin C, 50 mg; vitamin K3, 6 mg; vitamin B12, 40 μg; thiamine, 3.5 mg; riboflavin, 16 mg; vitamin B6, 6 mg; niacin, 40 mg; pantothenic acid, 25 mg; folic acid, 4 mg; biotin, 0.3 mg; BHT, 100 mg. 3Values were from a pooled sample of batches. View Large All ingredients utilized during the study were from the same batch and remained under normal storage conditions, i.e., in a cool, dry, well-ventilated warehouse in 50 kg bags and on pallets to prevent feed from being in direct contact with damp floors until experimental diets were mixed. Analyses of Cu in ingredients and feeds were performed using inductive coupled plasma atomic emission spectroscopy (ICP—Spectro Flamme, Spectro Analytical Instruments, Kleve, Germany) (Anderson, 1999). Breeder hen feeds were provided daily as recommended by Cobb-Vantress (2016). Feed and Cu intake per hen per d and per period is shown in Table 2. Analysis of Cu in drinking water was done using atomic absorption (ZEEnit 650 P, Analytik Jena, Jena, Germany). Averaged duplicate analyzed Cu in water was <0.007 ± 0.002 ppm and, therefore, it was not considered a significant dietary source of the mineral. Males were fed a corn-soy-wheat bran mash diet that met Cobb-Vantress (2013) recommendations. Table 2. Supplemented, calculated, and analyzed Cu concentrations in the experimental diets, feed intake, and Cu intake per hen d in each period. Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  1Analyzed Cu was from 2 pooled samples from all batches. View Large Table 2. Supplemented, calculated, and analyzed Cu concentrations in the experimental diets, feed intake, and Cu intake per hen d in each period. Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  Supplemented Cu, ppm  Total dietary Cu, ppm  Periods, wk  Average        25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44    Calculated  Analyzed1  Cu Intake, mg/hen/d  0.0  2.38  2.67 ± 0.329  0.32  0.40  0.40  0.39  0.38  0.38  3.5  5.88  5.82 ± 0.695  0.70  0.88  0.88  0.85  0.83  0.83  7.0  9.38  9.38 ± 1.865  1.13  1.42  1.42  1.38  1.34  1.34  10.5  12.88  12.92 ± 0.200  1.56  1.95  1.95  1.90  1.84  1.84  14.0  16.38  16.83 ± 0.054  2.04  2.55  2.54  2.47  2.40  2.40  17.5  19.88  20.19 ± 1.151  2.44  3.05  3.05  2.96  2.88  2.88  Cu intake, mg/hen/d      1.37  1.71  1.71  1.66  1.61  1.61  Feed Intake, g/hen/d      121.0  151.3  151.0  146.8  142.8  142.6  1Analyzed Cu was from 2 pooled samples from all batches. View Large Hen Performance Measurements Egg collection as well as their classification as hatchable or not (broken and deformed) were performed daily. In the last 3 d of each period, the hatchable eggs were weighed and grouped in 3 replicates per treatment and incubated after a 7-day storage in an environmentally controlled room at 18°C and 75% relative humidity (RH). A single-stage incubator (Avicomave, Iracemápolis, SP, Brazil) set at 37.5°C and 65% RH was used until 18 d; eggs were afterwards transferred to a hatcher set to 36.6°C and 80% RH. Hatchability and hatchability of fertile eggs were expressed as percentage of hatching chicks to the total eggs set and fertile eggs, respectively. Hen Blood Measurements Hematocrit (Ht) and Hb, serum Cu concentrations, and Cp activity were obtained from blood samples pooled from 3 broiler breeder hens randomly selected from each treatment per period. Birds were bled only once throughout the study. Blood obtained was partially transferred to 0.5 mL test tubes containing EDTA for Ht and Hb analyzes. Determination of Ht was done using micro capillaries containing blood centrifuged for 5 min at 15,650 to 18,510 × g. Concentration of Hb was determined using the cyanmethemoglobin method as described by Crosby et al. (1954). Blood (3 mL) also was centrifuged to obtain serum, which was transferred to Eppendorf tubes. A portion of the serum was used for Cu concentration analysis (Meret and Henkin, 1971), whereas the remaining was used for analysis of Cp (Schosinsky et al., 1974). Hatching Chick Measurements All hatched chicks were individually weighed, and the distance from the tip of the beak to the end of the middle toe (third toe) was used to determine chick length (Molenaar et al., 2008). Hb and Ht concentration was determined with 15 chicks hatched per treatment in each period using the same methodology as with the hens. Chick blood samples were obtained from the jugular vein after sacrifice by cervical dislocation. Egg Analysis Eggs from 10 replications were collected in the last 3 d of each 28-day period, totaling 30 eggs per treatment. One replication as a pool of 3 yolks from the same treatment was lyophilized, and a total of 10 replicates was collected per treatment per period. Yolk Cu content was quantified using ICP, as it was done with ingredients and feeds. Specific gravity was determined using saline solutions with concentrations ranging from 1.065 to 1.095 g/cm3 in intervals of 0.005 units (Novikoff and Gutteridge, 1949). Shell weight was obtained after washing and drying at 105°C overnight, whereas shell thickness was measured using a micrometer (Model IP65, Mitutoyo Corp., Kawasaky, Japan) in the apical, equatorial, and basal regions with these values being averaged for statistical analysis. In addition, 3 eggs with an average weight ± 10% SD per treatment obtained in the last 3 d of each period (age 28, 32, 36, 40, and 44 wk) were used in the analysis of eggshell ultrastructure using scanning electron microscopy (King and Robinson, 1972). In preparation for this analysis, eggshells were gently broken into 3 sections (0.5 to 1 cm2) in the apical, equatorial, and basal regions, totaling 270 pieces. Samples were then transversely mounted on aluminum stubs using carbon tape. These were gold metallized at 35 nm (BAL-TEC SCD050 Sputter Coater, Capovani Brothers Inc., Scotia, NY) for 3 min and were subsequently examined in the scanning electron microscope (JEOL JSM 5800, GenTech, Arcade, NY) with a 20 kW acceleration voltage and at magnification of 200×. Mammillary and palisade layers identifications were done according to the descriptions of Dennis et al. (1996). The digital microscopy images were uploaded to the Image-Pro Plus analyzer (Media Cybernetics, Rockville, MD). Averages of eggshell layer thickness were estimated from 5 measurements (μm) done in each photograph. Statistical Analysis Data were submitted to the normalcy of variance test (Shapiro and Wilk, 1965), and data not presenting normal distribution were subjected to transformation in order to stabilize variances using the arcsine square root percentage (z = asin (sqrt (y+ 0.5))) (Ahrens et al., 1990). Data were analyzed using the PROC MIXED of SAS (2011) with the repeated statement included in the statistical model. The covariance structure used was the variable components, which showed the best fit based on the Akaike criteria (Littell et al., 1998), except for eggshell thickness, which was better fitted with the Toeplizt covariance (Wolfinger, 1993). Total egg production and settable egg production per hen at 44 wk were analyzed using the general linear models (PROC GLM). Means were compared using the Tukey-Kramer test, and differences were considered significant at P < 0.05 (Tukey, 1991). Estimates of Cu requirements were done using 3 different models: exponential asymptotic (EA), broken line quadratic (BLQ), and quadratic polynomial (QP) (Robbins et al., 1979). The EA model (Y = β1 + β2 × (1- EXP (-β3 × (Cu—β4)))) had Y as the dependent variable as a function of dietary level of Cu; β1 estimated the relative response to the diet containing the lowest Cu (deficient diet); β2 estimated the difference between the minimum and the maximum response obtained with Cu supplementation; β3 was the curve slope coefficient; and β4 was the Cu level of the deficient diet. The maximum response for Cu was defined as Cu = (ln (0.05)/-β3) + β4 for 95% of the requirement. The BLQ model (Y = β1 + β2 × (β3 - Cu)2) had (β3 - Cu) = 0 for Cu > β3 with Y as the dependent variable as a function of the dietary level of Cu, β1 the value of the dependent variable at the plateau, and β2 as the slope of the line. The Cu level at the break point (β3) was considered the one providing maximum responses. The QP model (Y = β1 + β2 × Cu + β3 × (Cu)2) had Y as the dependent variable and as a function of dietary level of Cu; β1 as the intercept; β2 as the linear coefficient; and β3 as the quadratic coefficient. The maximum response for Cu was defined as Cu = –β2 ÷ (2 × β3). The coefficient of determination (R2) was used to assess the goodness of fit for the different models. RESULTS Formulated and analyzed diets had similar Cu contents (Table 2). Analyses of Cu in the dietary treatments were conducted on samples from 2 pools of the 5 mixed batches utilized throughout the study and averaged 5.82 ± 0.69; 9.38 ± 1.86; 12.92 ± 0.20; 16.83 ± 0.05; and 20.19 ± 1.15 ppm. There were no interactions between dietary Cu and period for any response; therefore, responses are presented as main effects of dietary Cu and period throughout the text. During the experimental phase, period affected (P < 0.05) almost all studied variables (Tables 3 and 4) with the exception of egg hatchability, hatchability of fertile eggs, hen Ht, and Cp activity of breeders (P > 0.05). As expected, egg production decreased after peak (29 to 32 wk) (P < 0.05). In parallel, breeder Hb decreased as hens aged to 44 wk (P < 0.05), whereas serum Cu peaked in the period of 33 to 36 wk and decreased afterwards (P < 0.05). Hatching chick Hb was higher, and body length was longer (P < 0.05) when chicks were obtained from eggs laid by hens after 40 wk of age, while the chick Ht and chick weight increased (P < 0.05) from eggs laid from hens after 33 wk of age (Table 3). Egg weight was higher (P < 0.05) in the period of 40 to 44 wk, while specific gravity, thickness of eggshell membrane, and yolk Cu concentration were lower in the same period (P < 0.05). Yolk percentage increased, and albumen percentage decreased (P < 0.05) in the 3 last periods (33 to 44 wk), whereas eggshell thickness palisade layer decreased from 33 to 36 wk (P < 0.05), and, on other hand, eggshell mammillary layer thickness was highest from 29 to 36 wk (P < 0.05; Table 4). Table 3. Response of broiler breeder hens to increased dietary Cu.   Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533    Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Eggs produced as a percentage of total live hens at the moment of measurement. 3Total settable eggs at the end of the experiment. 4Total eggs at the end of the experiment. 5Hematocrit. 6Hemoglobin. 7Ceruloplasmin. View Large Table 3. Response of broiler breeder hens to increased dietary Cu.   Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533    Eggs  Breeder  Hatching chick    Hen day production2, %  Total settable/hen3  Total/hen4  Hatchability, %  Hatchability of fertile, %  Ht,5%  Hb6, g/dL  Cp7, moles/min/L  Serum Cu, mg/L  Hb, g/dL  Ht, %  Body weight, g  Body length, cm  Cu, ppm1 (mg/day)                            2.67 (0.38)  67.7b  70b  95b  80.4  81.9  26.4b  6.26c  8.63  0.226b  6.60  27.18b  44.8b  18.4c  5.82 (0.83)  73.7a  80a,b  104a  85.2  85.2  28.1a,b  6.85b  10.39  0.244a,b  6.62  27.97a,b  45.9a,b  18.6a,b,c  9.38 (1.34)  75.4a  87a  105a  84.4  84.4  28.3a,b  7.07a,b  11.26  0.265a,b  6.62  28.20a,b  45.9a,b  18.5b,c  12.92 (1.84)  74.8a  85a  103a  83.5  83.5  29.1a  7.10a,b  11.09  0.282a  6.59  29.01a  46.6a  18.7a  16.83 (2.40)  74.1a  81a,b  104a  83.1  83.0  28.4a,b  7.05a,b  12.08  0.279a  6.63  28.24a,b  45.7a,b  18.6a,b  20.19 (2.88)  73.2a  82a,b  103a  81.5  81.4  29.8a  7.38a  12.52  0.259a,b  6.40  28.16a,b  46.3a  18.7a,b  Period, wk                            25 to 28  65.4c  −  –  83.1  83.1  27.6  7.10a,b  11.93  0.216d  6.36b,c  27.61b  42.6c  17.9d  29 to 32  81.5a  −  –  84.6  84.6  28.8  7.32a  10.96  0.228c,d  6.25c  27.53b  45.0b  18.4c  33 to 36  80.2a  −  –  83.3  83.3  27.0  6.80b,c  9.06  0.321a  6.64b  28.21a,b  46.1b  18.7b  37 to 40  69.8b  –  –  83.2  83.1  29.0  6.88b,c  10.88  0.269b  6.39b,c  28.95a  47.7a  18.8b  41 to 44  68.8b  −  –  80.8  82.1  28.9  6.66c  12.12  0.261b,c  7.23a  28.33a,b  48.6a  19.0a  SEM  0.3736  1.3489  0.5362  0.5601  0.5446  0.2354  0.0636  0.4626  0.0061  0.0367  0.1312  0.1400  0.0248  Prob                            Level  <.0001  0.0032  < .0001  0.2237  0.4709  0.0013  <.0001  0.2238  0.0057  0.3032  0.0055  0.0018  <.0001  Period  <.0001  –  –  0.4276  0.7815  0.0509  0.0006  0.2948  <.0001  <.0001  0.0039  <.0001  <.0001  Level x Period  0.7479  –  –  0.9999  0.999  0.9629  0.4758  0.8871  0.6393  0.5161  0.475  0.4075  0.4533  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Eggs produced as a percentage of total live hens at the moment of measurement. 3Total settable eggs at the end of the experiment. 4Total eggs at the end of the experiment. 5Hematocrit. 6Hemoglobin. 7Ceruloplasmin. View Large Table 4. Broiler breeder hen egg characteristics as affected by increased dietary Cu.               Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999                Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). eAll egg analyzes were affected by the period (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Dry matter Cu concentration. View Large Table 4. Broiler breeder hen egg characteristics as affected by increased dietary Cu.               Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999                Eggshell    Egg weight, g  Yolk, %2  Albumen, %3  Eggshell, %  Yolk Cu, ppm4  Specific gravity, g/cm3  Palisade layer, μm  Mammillary layer, μm  Membrane, μm  Thickness, μm  Cu, ppm1 (mg/d)  2.67 (0.38)  62.0c  29.4b  61.7a  8.90  1.30b  1.082  241.8  92.6b  58.4b  418.9  5.82 (0.83)  62.7b,c  29.6b  61.4a  9.01  1.80a,b  1.082  240.8  106.5ª  69.9a,b  424.0  9.38 (1.34)  64.3a  30.4a  60.6b  8.97  1.79a,b  1.082  256.7  113.6ª  72.9ª  426.8  12.92 (1.84)  64.4a  30.0a,b  61.1a,b  8.93  2.04a  1.084  244.3  116.8ª  73.0ª  427.0  16.83 (2.40)  63.6a,b  29.5b  61.5a  8.95  2.25a  1.083  248.8  114.4ª  68.8a,b  426.0  20.19 (2.88)  64.2a  29.3b  61.7a  8.92  1.98a  1.082  239.4  111.2ª  72.6ª  425.7  Period, wk  25 to 28  54.9e  27.2c  63.7a  9.09a  -  1.085a  254.5ª  107.4b  72.4ª  438.1a  29 to 32  61.3d  28.9b  62.2b  8.92a,b  -  1.082b  244.7ª  117.1ª  75.7ª  437.4a  33 to 36  63.7c  30.8a  60.1c  9.09a  1.96a  1.081b,c  223.6b  121.1ª  68.1a,b  414.3b  37 to 40  67.4b  31.0a  60.3c  8.74b  2.14a  1.083a,b  251.0ª  102.5b,c  68.7a,b  421.5b  41 to 44  70.4a  30.7a  60.3c  8.90a,b  1.48b  1.080c  252.7ª  97.8c  61.5b  412.3b  SEM  0.2253  0.0858  0.0936  0.0293  0.0635  0.1755  2.2795  1.6642  1.2850  1.1680  P-value  Level  <.0001  <.0001  0.0001  0.9317  0.0002  0.0821  0.0859  <.0001  0.0059  0.2933  Period  <.0001  <.0001  <.0001  0.0005  <.0001  <.0001  <.0001  <.0001  0.0058  <.0001  Level x Period  0.1359  0.1814  0.4910  0.4286  0.3580  0.9684  0.2585  0.0188  0.8487  0.9999  a–dMeans within a column without a common superscript differ significantly by Tukey's test (P < 0.05). eAll egg analyzes were affected by the period (P < 0.05). 1Values are analyzed, and values between parentheses are Cu intake (mg/hen/d). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Dry matter Cu concentration. View Large Dietary Cu affected all responses with the exception of hatchability, hatchability of fertile eggs, breeder Cp activity, Hb of hatching chicks, eggshell percentage, specific gravity, and thickness of eggshell palisade layer (P > 0.05; Tables 3 and 4). Providing breeders dietary increases of Cu affected (P < 0.05) hen d egg production as well as total settable eggs per hen and, therefore, impacted the total number of eggs per hen at the end of the experiment at 44 weeks. Breeder Hb increased (P < 0.05) when they were fed diets with 9.38 ppm Cu or greater as compared to the non-supplemented diet. Breeder Ht, serum Cu, hatching chick Ht, and body weight and length of hatching chicks increased (P < 0.05) as dietary Cu reached 12.92 ppm. Egg weight and yolk percentage were significantly increased when dietary Cu was 9.38 ppm (P < 0.05). However, egg weight increased in the next levels, whereas yolk percentage decreased (P < 0.05). The yolk Cu concentration was highest (P < 0.05) when dietary Cu was 12.92 ppm or above. Scanning electronic microscopy photographs (Figure 1) showed that dietary Cu deficiency led to a less stable eggshell ultrastructure compared with the level above 5.82 ppm dietary Cu (P < 0.05). The Cu deficient diet (2.67 ppm Cu) led to decreased eggshell membrane thickness, whereas the eggshell mammillary layer was affected by dietary Cu (P < 0.05) only in the period between 29 and 32 weeks. Figure 1. View largeDownload slide Scanning electron cross-sections of eggshells from broiler breeder hens fed a Cu-deficient diet (2.67 ppm) (A), and diets with 5.82 ppm (B), 9.38 ppm (C), 12.92 ppm (D), 16.83 ppm (E), and 20.19 ppm (F) Cu (200x). * Membrane. ** Mammillary layer. ***Palisade layer. Figure 1. View largeDownload slide Scanning electron cross-sections of eggshells from broiler breeder hens fed a Cu-deficient diet (2.67 ppm) (A), and diets with 5.82 ppm (B), 9.38 ppm (C), 12.92 ppm (D), 16.83 ppm (E), and 20.19 ppm (F) Cu (200x). * Membrane. ** Mammillary layer. ***Palisade layer. Requirements of Cu were determined using EA, BLQ, and QP regression models. These are shown in Tables 5 and 6 in ppm as well as in mg per hen day. A sharp increase in egg production was observed at the first Cu level supplemented (5.82 ppm), while the maximum responses were obtained with 6.2 ppm (0.89 mg/hen/d), 7.3 ppm (1.04 mg/hen/d), and 12.9 ppm (1.84 mg/hen/d) provided by EA, BLQ, and QP models, respectively. The requirements of Cu estimated for total settable eggs per hen were 8.1 ppm (1.16 mg/hen/d), 9.0 ppm (1.29 mg/hen/d), and 13.4 ppm (1.91 mg/hen/d) Cu. Table 5. Requirements of Cu estimated for diverse broiler breeder responses adjusted with exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16    Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Table 5. Requirements of Cu estimated for diverse broiler breeder responses adjusted with exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16    Model  Regression equations1  R2  Prob  Requirement, ppm  Hen d egg production2  EA  Y = 68.1333 + 6.6151 × (1 −EXP (−0.8394 × (x −2.67)))  0.43  <.0001  6.23    BLQ  Y = 74.7817 −0.3121 × (7.2833 −x)2  0.43  <.0001  7.28    QP  Y = 64.85864 + 1.76914x −0.06864x2  0.41  <.0001  12.89  Total settable eggs/hen3  EA  Y = 69.7485 + 13.915 × (1 −EXP (−0.5460 × (x −2.67)))  0.13  0.0006  8.15    BLQ  Y = 83.8056 −0.3454 × (9.0286 −x)2  0.13  0.0005  9.03    QP  Y = 63.23373 + 3.41788x −0.12732x2  0.11  0.0009  13.42  Total eggs/hen4  QP  Y = 92.69186 + 1.96035x −0.07453x2  0.21  <.0001  13.15    EA  Y = 6.2875 + 0.9345 × (1 −EXP (−0.2672 × (x −2.67)))  0.29  <.0001  13.88  Hen Hb5, g/dl  BLQ  Y = 7.1721 −0.0118 × (11.32 −x)2  0.28  <.0001  11.32    QP  Y = 6.08503 + 0.12756x −0.00345x2  0.25  <.0001  18.48  Hen Ht6, %  EA  Y = 26.4371 + 2.7027 × (1 −EXP (−0.2516 × (x −2.67)))  0.19  0.0001  14.58    BLQ  Y = 29.0243 −0.0236 × (13.0201 −x)2  0.19  0.0002  13.02    QP  Y = 25.83414 + 0.35998x −0.00946x2  0.16  0.0002  19.03  Serum Cu, mg/L  EA  Y = 0.2264 + 0.0527 × (1 −EXP (−0.2210 × (x −2.67)))  0.10  0.0136  16.22    BLQ  Y = 0.2721 −0.0004 × (14.5797 −x)2  0.11  0.0101  14.58    QP  Y = 0.18865 + 0.01239x −0.00043x2  0.10  0.0055  14.25  Hatching chick Ht, %  EA  Y = 27.1525 + 1.2731 × (1−EXP (−0.3976 × (x −2.67)))  0.03  0.0028  10.20    BLQ  Y = 28.4538 −0.0133 × (12.3415 −x)2  0.03  0.0027  12.34    QP  Y = 26.34112 + 0.34919x −0.0131x2  0.03  0.0009  13.33  Hatching chick weight, g  EA  Y = 45.0314 + 1.0776 × (1−EXP (−0.7251 × (x −2.67)))  0.01  0.0365  6.80    BLQ  Y = 46.1032 −0.0549 × (7.089 −x)2  0.01  0.0368  7.09    QP  Y = 44.71787 + 0.2136x −0.00743x2  0.00  0.0741  14.37  Hatching chick length, cm  EA  Y = 18.4315 + 0.2379 × (1 −EXP (−0.2916 × (x −2.67)))  0.02  0.0039  12.94    BLQ  Y = 18.6696 −0.00181 × (13.8886 −x)2  0.02  0.0036  13.89    QP  Y = 18.32994 + 0.04699x −0.00155x2  0.01  0.0039  15.16  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Table 6. Cu requirements for performance and blood measurements of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 5. 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Table 6. Cu requirements for performance and blood measurements of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Hen d egg production2  EA  0.75  0.94  0.94  0.91  0.89  0.89    BLQ  0.88  1.10  1.10  1.07  1.04  1.04    QP  1.56  1.95  1.95  1.89  1.84  1.84  Total settable eggs/hen3  EA  0.99  1.23  1.23  1.20  1.16  1.16    BLQ  1.09  1.37  1.36  1.33  1.29  1.29    QP  1.62  2.03  2.03  1.97  1.92  1.91  Total eggs/hen4  QP  1.59  1.99  1.99  1.93  1.88  1.87  Hen Hb5, g/dl  EA  1.68  2.10  2.10  2.04  1.98  1.98    BLQ  1.37  1.71  1.71  1.66  1.62  1.61    QP  2.24  2.80  2.79  2.71  2.64  2.63  Hen Ht6, %  EA  1.76  2.21  2.20  2.14  2.08  2.08    BLQ  1.58  1.97  1.97  1.91  1.86  1.86    QP  2.30  2.88  2.87  2.79  2.72  2.71  Serum Cu, mg/L  EA  1.96  2.45  2.45  2.38  2.32  2.31    BLQ  1.76  2.21  2.20  2.14  2.08  2.08    QP  1.72  2.16  2.15  2.09  2.03  2.03  Hatching chick Ht, %  EA  1.23  1.54  1.54  1.50  1.46  1.45    BLQ  1.49  1.87  1.86  1.81  1.76  1.76    QP  1.61  2.02  2.01  1.96  1.90  1.90  Hatching chick weight, g  EA  0.82  1.03  1.03  1.00  0.97  0.97    BLQ  0.86  1.07  1.07  1.04  1.01  1.01    QP  1.74  2.17  2.17  2.11  2.05  2.05  Hatching chick length, cm  EA  1.57  1.96  1.95  1.90  1.85  1.84    BLQ  1.68  2.10  2.10  2.04  1.98  1.98    QP  1.83  2.29  2.29  2.22  2.16  2.16  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 5. 2Eggs produced as a percentage of total live hens. 3Total settable egg produced by live hens at the end of the experiment. 4Total eggs produced by live hens at the end of the experiment. 5Hemoglobin. 6Hematocrit. View Large Breeder hen requirements of Cu for Hb were estimated as 13.9 ppm (1.98 mg/hen/d), 11.3 ppm (1.61 mg/hen/d), and 18.5 ppm (2.63 mg/hen/d) using EA, BLQ, and QP models, respectively, whereas the requirement for Ht was estimated at 15.6 ppm (2.08 mg/hen/d), 13.0 ppm (1.86 mg/hen/d), and 19.0 ppm (2.71 mg/hen/d), using EA, BLQ, and QP models, respectively. In parallel, serum Cu concentration was maximized at 16.2 ppm (2.31 mg/hen/d), 14.6 ppm (2.08 mg/hen/d), and 14.2 ppm (2.03 mg/hen/d), using EA, BLQ, and QP models, respectively. The requirements of Cu for hatching chick Ht and body weight and length were estimated at 10.2, 12.3, and 13.3 ppm (1.45, 1.76, and 1.90 mg/hen/d) using EA, BLQ, and QP models; and 6.8 and 7.1 ppm (0.97 and 1.01 mg/hen/d) and 12.9 and 13.9 ppm (1.84 and 1.98 mg/hen/d) Cu using EA and BLQ models, respectively. The R squared obtained using the different tested models for hatching chicks Ht, weight, and length were very low. It was probably due to the high variation of the data among the different evaluation periods. Estimations of Cu requirements for egg component measures are shown in Tables 7 and 8 (ppm and mg per hen d, respectively). All models were well fitted in estimating Cu requirements for analysis done with eggs, except for egg yolk and albumen percentage, which did not fit for EA and BLQ models. The Cu requirement for percent yolk and albumen were 11.0 ppm (1.57 mg/hen/d) and 11.3 ppm (1.61 mg/hen/d) for percent yolk and albumen, respectively, using the QP model. The requirement of Cu estimated for egg weight was 14.9 ppm (2.13 mg/hen/d), 12.7 ppm (1.81 mg/hen/d), and 15.1 ppm (2.15 mg/hen/d), using EA, BLQ, and QP models, respectively. Dietary Cu requirements to maximize the Cu content in the yolk were 15.0 ppm (2.14 mg/hen/d), 16.3 ppm (2.33 mg/hen/d), and 15.7 ppm (2.24 mg/hen/d) using EA, BLQ, and QP models, respectively. Eggshell membrane was thickest when dietary Cu was fed at 7.3 ppm (1.04 mg/hen/d), 7.9 ppm (1.12 mg/hen/d), and 14.0 ppm (1.99 mg/hen/d) Cu, using EA, BLQ, and QP models, respectively. Table 7. Requirements of Cu (ppm) estimated for egg characteristics exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42    Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large Table 7. Requirements of Cu (ppm) estimated for egg characteristics exponential asymptotic (EA), broken line with quadratic (BLQ), or quadratic polynomial (QP) models.   Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42    Model  Regression equation1  R2  Prob  Requirement, ppm  Egg weight, g  EA  Y = 61.881 + 2.2848 × (1 −EXP (−0.2442 × (x −2.67)))  0.12  <.0001  14.94    BLQ  Y = 64.0796 −0.021 × (12.7022 −x)2  0.13  <.0001  12.70    QP  Y = 61.03122 + 0.42536x −0.01408x2  0.11  <.0001  15.11  Yolk2, %  QP  Y = 28.7739 + 0.23388x −0.01062x2  0.09  <.0001  11.01  Albumen3, %  QP  Y = 62.37853 −0.23536x + 0.01044x2  0.06  0.0011  11.27  Yolk Cu, ppm  EA  Y = 1.2812 + 0.8156 × (1 −EXP (−0.2433 × (x −2.67)))  0.11  <.0001  14.98    BLQ  Y = 2.0983 −0.00409 × (16.3348 −x)2  0.11  <.0001  16.33    QP  Y = 0.94839 + 0.14915x −0.00474x2  0.10  <.0001  15.73  Eggshell membrane layer, μm  EA  Y = 58.6506 + 13.3153 × (1 −EXP (−0.6477 × (x −2.67)))  0.16  0.0006  7.29    BLQ  Y = 71.9917 −0.4945 × (7.8596 −x)2  0.16  0.0006  7.86    QP  Y = 54.11949 + 2.83491x −0.10146x2  0.11  0.0026  13.97  Eggshell mammillary  EA  Y = 75.3034 + 54.5649 × (1 −EXP (−0.3796 × (x −2.67)))  0.68  0.0003  10.56  Layer4, μm  BLQ  Y = 129 −0.9524 × (10.1504 −x)2  0.70  0.0002  10.15    QP  Y = 53.5817 + 11.40007x −0.39535x2  0.66  0.0005  14.42  1Regression equations obtained using the increasing analyzed Cu in the diets (5.82; 9.38; 12.92; 16.83, and 20.19 ppm). 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large Table 8. Cu requirements for egg analyses of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 7. 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large Table 8. Cu requirements for egg analyses of breeders per period in mg/hen/d.     Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18      Cu requirements, mg/hen/d1      Periods, wk  Average    Model  25 to 28  29 to 32  33 to 36  37 to 40  41 to 44  25 to 44  Egg weight, g  EA  1.81  2.26  2.26  2.19  2.13  2.13    BLQ  1.54  1.92  1.92  1.86  1.81  1.81    QP  1.83  2.29  2.28  2.22  2.16  2.15  Yolk, %2  QP  1.33  1.67  1.66  1.62  1.57  1.57  Albumen, %3  QP  1.36  1.70  1.70  1.65  1.61  1.61  Yolk Cu, ppm  EA  1.81  2.27  2.26  2.20  2.14  2.14    BLQ  1.98  2.47  2.47  2.40  2.33  2.33    QP  1.90  2.38  2.38  2.31  2.25  2.24  Eggshell membrane layer, μm  EA  0.88  1.10  1.10  1.07  1.04  1.04    BLQ  0.95  1.19  1.19  1.15  1.12  1.12    QP  1.69  2.11  2.11  2.05  1.99  1.99  Eggshell mammillary layer4, μm  EA  –  1.60  –  –  –  1.60    BLQ  –  1.54  –  –  –  1.54    QP  –  2.18  –  –  –  2.18  1Values obtained using feed intake of Table 1 and Cu requirements (ppm) of Table 7. 2Percentage of yolk in relation to egg weight. 3Percentage of albumen in relation to egg weight. 4Obtained with eggs laid from 29 to 32 weeks. View Large DISCUSSION In the present study, broiler breeder hens fed a non-supplemented Cu diet demonstrated signals of deficiency throughout most evaluated responses. Since Cu is necessary for adequate activities of a great number of metalloenzymes, biochemical reactions are not carried out properly during Cu deficiency (Kaya et al., 2006; Scheiber et al., 2014; Cao et al., 2016). The lack of interaction between dietary Cu and period is an indication that Cu requirements do not change as hens age from 25 to 44 weeks. However, values vary within the same database depending on the model used to estimate its requirements (Robbins et al., 1979). Prediction of requirements do not have one particular true model; therefore, results of experiments should be the ones to dictate the model choice (Vedenov and Pesti, 2008). Non-linear models, such as BLQ and EA, have been thought to be advantageous compared to linear models, due to the fact that biological systems rarely work in perfect linearity (Pesti et al., 2009). In the present study, adequate fit was sometimes not found for one or more models and, in those cases, they were not included in this section. The EA and BLQ models fitted better and estimated lower requirement values for most responses, whereas the QP model provided the best fit only for total egg production, yolk, and albumen percentage and estimated higher requirements values. Requirements of Cu for hen d egg production varied between 6.2 and 12.9 ppm total Cu in the diet, which represents a dietary intake of Cu from 0.89 mg/hen/d to 1.84 mg/hen/day. Requirement for total egg production at the end of wk 44 was higher (13.1 ppm or 1.87 mg/hen/d) than for egg production per hen day. Traditionally the tables of requirements for poultry do not provide Cu requirements for broiler breeders. Instead, supplemental levels are suggested, which range from 10 to 15 ppm, intending to provide safety margins in feed formulation (Mondal et al., 2010). Data from the present study indicate that the usually suggested supplemental Cu levels (NRC, 1994; Cobb-Vantress, 2013; Aviagen, 2017; Rostagno et al., 2017) are excessive. A safety margin is generally used when it comes to micronutrients due to numerous factors that can influence the nutritional requirements (Applegate and Angel, 2014), including an overall scarcity of research. The maximum concentration of 25 ppm Cu in the diet as established by the EC (EFSA, 2016), therefore, seems to be very safe in attending breeder hen needs, since it represents about 2 times what has been estimated as an average requirement in the present research. A non-supplemented broiler breeder hen diet formulated with corn, soy, and wheat bran has an average of 8 ppm total Cu and, therefore, is expected to provide Cu in amounts close to the requirements determined in the present study. Dietary Cu fed to hens impacted their blood Hb and Ht. A decrease in the Ht can be related to depression in the phospholipid synthesis (Kaya et al., 2006) and in the platelet-mediated hemostasis in dietary Cu deficiency (Schuschke et al., 1994). A reduction in Ht was reported by Baumgartner et al. (1978) when hens were fed a Cu deficient diet. Effects of dietary Cu also were observed in Hb, a response that also was previously reported in poultry (Samanta et al., 2011; Mroczek-Sosnowska et al., 2013). In the present study, Ht and Hb were maximized when hens were fed 12.6 and 13.8 ppm Cu (average of the EA and BLQ models, which presented high R-squares). Kubena et al. (1972) also observed the same tendency when diets were tested without Cu supplementation (3 ppm) or were supplemented at 2.2 and 9.9 ppm. It has been earlier reported that Cu facilitates Hb formation in anemic chicks (Elvehjem and Hart, 1929), and Cp-stimulated iron uptake requires Cp ferroxidase activity and utilizes a novel, trivalent cation-specific transport pathway (Attieh et al., 1999; Lenartowicz et al., 2015; Linder, 2016). Hephaestin and Cp are Cu-dependent enzymes that can modify blood Hb values (Reeves et al., 2005; Ha et al., 2016). In the present study, however, hen Cp activity when a deficient Cu diet was fed (2.67 ppm Cu) was not affected. Others have found the opposite, with 3.44 ppm Cu leading to decreases in Cp (Kaya et al., 2006). Concentration of Cu in the serum has been improved as dietary Cu increases (Zanetti et al., 1991; Schmidt et al. 2005), and maximum serum Cu level obtained in the present study was obtained at 15.0 ppm dietary Cu (2.14 mg/hen/d). The lack of significant differences in the Cp activity in response to dietary Cu increase found in the present study and changes in the Cu contents in Hb, as well as in the serum observed, indicate that blood parameters are more sensitive to dietary Cu than Cp and that Cp is highly preserved. Average Cu requirements using the 3 models for Hb, Ht, and serum Cu were similar (14.6, 15.5, and 15.0 ppm or 2.07, 2.21, and 2.14 mg/hen/d, respectively). These values were higher than those needed to maximize egg production. Blood Cu has been shown to change in relation to growth rate and age (Abdel-Mageed and Oehme, 1990), which may explain the differences in the amount of Cu in the serum among periods in this study. Deficiencies observed with the lowest Cu contents in the diets fed in the present study resulted in decreases in yolk Cu concentration, as well as in Ht of hatching chicks. Contents of Cu in eggs are dependent on the dietary supply to breeder hens (Kim et al., 2016) and, therefore, deficiencies or excesses affect egg quality and subsequent performance of the progeny (Whitehead et al., 1985). In the present study, it seemed that the deposition of Cu in the yolk had a non-linear behavior, reaching maximum Cu depositions at 15.7 ppm dietary Cu (or 2.24 mg/hen/d). Egg mineral constituents are transferred from the hen, which originate from the feed. Copper deprivation impairs eggshell quality, as it was observed by scanning electronic microscopy in the present study. Eggshells are composed of ultrastructural layers divided into shell membranes, mammillary knobs, palisade, and a cuticle (Arias et al., 1993; Hunton, 2005). Activation of lysyl oxidase by Cu is necessary for the collagen synthesis in the eggshell membrane (Leach et al., 1981). This enzyme is responsible for oxidative deamination of the lysine side chains, which form crosslinks and thus confer characteristics of insolubility, flexibility, and structure for the deposition of other egg components (Linder and Hazegh-Azam, 1996; Akagawa et al., 1999). In the present study, the thickening of the eggshell membrane provided by Cu intake did not increase the total thickness of the shell or palisade layer, or of the mammillary layer in most periods. There was an increase in thickness of the eggshell mammillary layer only at 29 to 32 wk using 11.7 ppm Cu (or 1.77 mg/hen/d), but there was no change in the thickness of the palisade layer at any time. Thus, the amount of Cu that is deposited in the eggshell must be influenced by other factors. Duan et al. (2016) observed that the thickness of the mammillary layer was affected by mammillary density and might be regulated by some protein interactions, which can be responsible for nucleation sites formation. Although the membrane thickness does not regulate the amount of mineral deposition in the other eggshell layers, it appears to be important for structural organization. Therefore, a well-structured outer shell membrane is necessary for correct mineralization and consequently to produce resistant eggshells (Nys et al., 1999; Nys et al., 2004). Any modification of the eggshell membranes by Cu deficiency occurs due to inhibition of fiber formation or crosslinking, characterized by an abnormal distribution of the eggshell membrane fibers (Mabe et al., 2003), and consequently altering its mechanical properties (Hincke et al., 2012). The lowest thickness of the eggshell membrane observed using low Cu levels in the present study may have resulted in an increased occurrence of unsettable eggs. The requirement of 7.6 ppm Cu (1.08 mg/hen/d) for the maximum eggshell membrane thickness obtained in this experiment was similar to the requirement of the maximum total settable eggs of 8.6 ppm Cu (1.22 mg/hen/d) using the mean EA and BLQ models. Results obtained indicate that Cu most likely plays an important role in thickening of the eggshell membrane, and, therefore, it is expected to affect eggshell breaking resistance. Some studies suggest that the dry eggshell membrane weight decreases in eggs with eggshell deformation, as well as the membrane strength having a positive correlation with eggshell breaking strength (Britton, 1977; Essary et al., 1977). In addition, adequately structured eggshell membranes provide the proper formation of the air chamber immediately after oviposition, which is dependent on separation between the inner and the outer shell membranes (Vieira, 2007). 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Published: May 15, 2018

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