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METABOLISM AND NUTRITION

Copper requirements of broiler breeder hens

E. Berwanger,∗ S. L. Vieira,∗,1 C. R. Angel,† L. Kindlein,‡ A. N. Mayer,∗ M. A. Ebbing,∗ and M. Lopes∗

∗Department of Animal Science, Federal University of Rio Grande do Sul, Av. Bento Goncalves, 7712, PortoAlegre, RS, Brazil, 91540-000; †Department of Animal and Avian Sciences, University of Maryland, College Park20742; and ‡Department of Preventive Veterinary Medicine, Federal University of Rio Grande do Sul, Av. Bento

Goncalves, 8834, Porto Alegre, RS, Brazil, 91540-000

ABSTRACT One-hundred-twenty Cobb 500 hens, 20wk of age, were randomly allocated into individualcages with the objective of estimating Cu requirements.After being fed a Cu deficient diet for 4 wk, hens werefed 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. Es-timations of Cu requirements were done using expo-nential asymptotic (EA), broken line quadratic (BLQ),and quadratic polynomial (QP) models. Obtained Curequirements for hen d egg production and total set-table eggs per hen were 6.2, 7.3, and 12.9 ppm and8.1, 9.0, and 13.4 ppm, respectively, using EA, BLQ,and QP models. The QP model was the only one hav-ing a fit for total eggs per hen with 13.1 ppm Cu asa requirement. Hemoglobin, hematocrit, and serum Cufrom 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, andQP models. Hatching chick hemoglobin was not af-fected by dietary Cu, whereas requirements estimatedfor hatching chick hematocrit and body weight andlength 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 and13.9 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, and15.1 ppm; 15.0, 16.3, and 15.7 ppm; and 7.3, 7.8,and 14.0 ppm Cu, respectively, for EA, BLQ, and QPmodels. Yolk and albumen percentage were adjustedonly with the QP model and had requirements esti-mated at 11.0 ppm and 11.3 ppm, respectively, whereaseggshell mammillary layer was maximized with 10.6,10.1, and 14.4 ppm Cu using EA, BLQ, and QP mod-els, respectively. The average of all Cu requirement es-timates obtained in the present study was 12.5 ppmCu.

Key words: broiler breeder, broiler chick, copper, mineral2018 Poultry Science 97:2785–2797

http://dx.doi.org/10.3382/ps/pex437

INTRODUCTION

Copper (Cu) is an essential trace mineral for poultrythat has many roles in metabolism, most of them re-lated to enzyme function (Richards et al., 2010; Karimiet al., 2011). A primary function of Cu is relatedto 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 function-ing of reproductive functions, Cu is a precursor of β-monooxygenase, which catalyzes the hydroxylation ofdopamine to norepinephrine, needed for the produc-tion of the gonadotropin-releasing hormone (Roychoud-hury et al., 2016). Important to support successful chickproduction, the maturity of the eggshell membrane de-

C© 2018 Poultry Science Association Inc.Received August 7, 2017.1Corresponding author: [email protected]

pends on adequate intake of Cu because of its role inthe setup of collagen crosslinks by being part of lysyloxidase (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 dur-ing incubation (Richards, 1997). Yolk Cu is bound tolipovitellin and phosvitin (Kozlowski et al., 1988). A ri-boflavin binding protein (RBP) is also involved in thetransport and storage of Cu (Smith et al., 2008), but incontrast to phosvitin, which presents a voluminous ca-pability to store positive charged metals (Samaraweeraet al., 2011), the binding to RBP is specific to Cu andoccurs in a 1:1 molar ratio (Hall et al., 2013). There-fore, Cu deficiency negatively affects embryo develop-ment with resulting gross structural and biochemicalabnormalities (Roychoudhury et al., 2016). Because ofthe tight involvement of Cu in collagen synthesis, an ad-equate supply of this mineral is also essential for embryobone development and, therefore, for the independentfeed seeking immediately after hatching.

2785

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2786 BERWANGER ET AL.

A considerable number of reports have been pub-lished 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 stud-ies have been conducted with broiler breeders. Due tothe lack of supporting literature, recommendations forCu supplementation presented in the most popularly re-ferred 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 islargely unknown (NRC, 1994; Aoyagi and Baker, 1995).

Concerns with environmental pollution have beenleading to the surge of a series of regulations limitingthe concentrations of micro minerals in animal feeds(Lopez-Alonso, 2012). Since micro minerals are low incost when compared to many other nutrients, a possibil-ity of excessive Cu being present in poultry feeds exists,leading to a concentration in excreta that can be seen asan environmental contaminant (Pesti and Bakalli, 1996;Ewing et al., 1998; Brainer et al., 2003). The EuropeanCommission (EC) has recently established a maximumallowance of 25 ppm total Cu in poultry feeds (EFSA,2016). On the other hand, intensive farming has ledto a decrease in Cu contents in plant feedstuffs in thelast century (Klevay, 2016), which therefore, raises un-certainties about its contents in those ingredients and,therefore, as a reliable supply source of Cu for poultry.Since Cu requirements are low and supplementation isusual, its deficiency in poultry is uncommon (Zhao etal., 2010).

To the authors’ knowledge, comprehensive Cu re-quirement studies have not been conducted with broilerbreeder hens in recent years. The objective of thepresent study was to assess the Cu requirements ofbroiler breeder hens using Cu sulfate, the most com-mon Cu supplement utilized worldwide. Evaluated re-sponses were related to productive performance as wellas with eggshell quality, blood constituents, and qualityhatching chicks of breeders.

MATERIALS AND METHODS

All procedures utilized in the present study were ap-proved by the Ethics and Research Committee of theFederal University of Rio Grande do Sul, Porto Alegre,RS, Brazil.

Bird Husbandry

One-hundred-twenty Cobb 500 broiler breeder hensand 30 Cobb breeder males, 20 wk of age, were ob-tained from a commercial breeder farm (BRF, Arroiodo Meio, RS, Brazil). Hens were individually placed incages (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 collec-

Table 1. Composition of Cu-deficient diet provided to breederhens from 20 to 44 wk of age.

Ingredient, % as-is1 Deficient diet

Rice, polished and broken, 8.0% CP 40.98Corn, 7.8% CP 27.64Soy protein isolate, 89% CP 10.06Calcium carbonate 7.75Oat hulls 8.98Soybean oil 1.00Phosphoric acid, 85% P 1.93Potassium carbonate 0.72Sodium bicarbonate 0.24Potassium chloride 0.21Choline chloride 0.16DL-methionine, 99% 0.16L-threonine 98.5% 0.05Vitamin and mineral mix2 0.10L-lysine HCl, 78% 0.02Total 100.00

Calculated nutrient composition, % or as shownAMEn, kcal/kg 2,760CP 15.40Ca 3.20Available P 0.45Na 0.20Cu, ppmCalculated 2.38Analyzed3 2.67 ± 0.33

Choline, mg/kg 1,500

1Calcium carbonate, phosphoric acid, sodium bicarbonate, and potas-sium 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 ofdiet: Zn, 110 mg; Mn, 120 mg, Fe, 50 mg; Se, 0.3 mg and I, 2 mg (alllaboratory grade); vitamin A, 12,000 IU; vitamin D3, 3,000 IU; vitaminE, 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.

tion. Cages are electrostatically painted and have onestainless steel nipple drinker per cage, whereas feederswere plastic. Temperature control, lighting, and feedingprograms followed Cobb-Vantress (2016) recommenda-tions. Hens were inseminated weekly utilizing freshlycollected semen diluted at the ratio of 3 parts of phys-iological solution to 1 of semen. This was done with0.1 mL of the dilution using a 1 mL syringe directlyinto the oviduct.

Experimental Diets

The study was composed of pre-experimental (de-pletion) and experimental phases. Immediately afterplacement, hens at 20 wk of age were fed a Cu de-ficient 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 atthe beginning of the experimental phase, and birdswere weighed and randomly distributed into individ-ual cages. The Cu deficient diet was supplemented withincreased levels of laboratory grade Cu sulfate pen-tahydrate (CuSO4 5H2O) (Sigma Aldrich, St. Louis,MO) at the expense of the mineral mix diluent atlevels of 3.5, 7.0, 10.5, 14.0, and 17.5 ppm Cu. Re-sulting analyzed Cu in the feeding treatments were of


COPPER AND BROILER BREEDERS 2787

Table 2. Supplemented, calculated, and analyzed Cu concentrations in the experimental diets, feed intake, and Cu intake per hen din 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 44Calculated Analyzed1 Cu Intake, mg/hen/d

0.0 2.38 2.67 ± 0.329 0.32 0.40 0.40 0.39 0.38 0.383.5 5.88 5.82 ± 0.695 0.70 0.88 0.88 0.85 0.83 0.837.0 9.38 9.38 ± 1.865 1.13 1.42 1.42 1.38 1.34 1.3410.5 12.88 12.92 ± 0.200 1.56 1.95 1.95 1.90 1.84 1.8414.0 16.38 16.83 ± 0.054 2.04 2.55 2.54 2.47 2.40 2.4017.5 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 1.61Feed 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.

5.82 ± 0.69; 9.38 ± 1.86; 12.92 ± 0.20; 16.83 ± 0.05; and20.19 ± 1.15 ppm, respectively. Each one of 6 dietarytreatments was replicated 20 times with one hen beingthe experimental unit. The experiment lasted from 25to 44 wk of age, which for the matter of collecting andanalyzing data were divided into 5 periods of 28 days.The experiment was a 6 × 5 factorial comprising 6 Cusupplementation levels and 5 periods.

All ingredients utilized during the study were fromthe same batch and remained under normal storage con-ditions, i.e., in a cool, dry, well-ventilated warehouse in50 kg bags and on pallets to prevent feed from beingin direct contact with damp floors until experimentaldiets were mixed. Analyses of Cu in ingredients andfeeds were performed using inductive coupled plasmaatomic emission spectroscopy (ICP—Spectro Flamme,Spectro Analytical Instruments, Kleve, Germany) (An-derson, 1999). Breeder hen feeds were provided dailyas recommended by Cobb-Vantress (2016). Feed andCu intake per hen per d and per period is shown inTable 2. Analysis of Cu in drinking water was done us-ing atomic absorption (ZEEnit 650 P, Analytik Jena,Jena, Germany). Averaged duplicate analyzed Cu inwater was <0.007 ± 0.002 ppm and, therefore, it wasnot considered a significant dietary source of the min-eral. Males were fed a corn-soy-wheat bran mash dietthat met Cobb-Vantress (2013) recommendations.

Hen Performance Measurements

Egg collection as well as their classification as hatch-able or not (broken and deformed) were performeddaily. In the last 3 d of each period, the hatchable eggswere weighed and grouped in 3 replicates per treat-ment and incubated after a 7-day storage in an envi-ronmentally controlled room at 18◦C and 75% relativehumidity (RH). A single-stage incubator (Avicomave,Iracemapolis, SP, Brazil) set at 37.5◦C and 65% RHwas used until 18 d; eggs were afterwards transferred toa hatcher set to 36.6◦C and 80% RH. Hatchability andhatchability of fertile eggs were expressed as percentageof 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 samplespooled from 3 broiler breeder hens randomly selectedfrom each treatment per period. Birds were bled onlyonce throughout the study. Blood obtained was par-tially transferred to 0.5 mL test tubes containing EDTAfor Ht and Hb analyzes. Determination of Ht was doneusing micro capillaries containing blood centrifuged for5 min at 15,650 to 18,510 × g. Concentration of Hbwas determined using the cyanmethemoglobin methodas described by Crosby et al. (1954). Blood (3 mL)also was centrifuged to obtain serum, which was trans-ferred to Eppendorf tubes. A portion of the serum wasused for Cu concentration analysis (Meret and Henkin,1971), whereas the remaining was used for analysis ofCp (Schosinsky et al., 1974).

Hatching Chick Measurements

All hatched chicks were individually weighed, and thedistance from the tip of the beak to the end of the mid-dle toe (third toe) was used to determine chick length(Molenaar et al., 2008). Hb and Ht concentration wasdetermined with 15 chicks hatched per treatment ineach period using the same methodology as with thehens. Chick blood samples were obtained from the jugu-lar vein after sacrifice by cervical dislocation.

Egg Analysis

Eggs from 10 replications were collected in the last3 d of each 28-day period, totaling 30 eggs per treat-ment. One replication as a pool of 3 yolks from the sametreatment was lyophilized, and a total of 10 replicateswas collected per treatment per period. Yolk Cu contentwas quantified using ICP, as it was done with ingredi-ents and feeds. Specific gravity was determined usingsaline solutions with concentrations ranging from 1.065to 1.095 g/cm3 in intervals of 0.005 units (Novikoffand Gutteridge, 1949). Shell weight was obtained afterwashing and drying at 105◦C overnight, whereas shell



thickness was measured using a micrometer (ModelIP65, Mitutoyo Corp., Kawasaky, Japan) in the apical,equatorial, and basal regions with these values being av-eraged for statistical analysis. In addition, 3 eggs withan average weight ± 10% SD per treatment obtained inthe last 3 d of each period (age 28, 32, 36, 40, and 44wk) were used in the analysis of eggshell ultrastructureusing scanning electron microscopy (King and Robin-son, 1972). In preparation for this analysis, eggshellswere gently broken into 3 sections (0.5 to 1 cm2) inthe apical, equatorial, and basal regions, totaling 270pieces. Samples were then transversely mounted on alu-minum stubs using carbon tape. These were gold met-allized at 35 nm (BAL-TEC SCD050 Sputter Coater,Capovani Brothers Inc., Scotia, NY) for 3 min and weresubsequently examined in the scanning electron micro-scope (JEOL JSM 5800, GenTech, Arcade, NY) witha 20 kW acceleration voltage and at magnification of200×. Mammillary and palisade layers identificationswere done according to the descriptions of Dennis et al.(1996). The digital microscopy images were uploadedto the Image-Pro Plus analyzer (Media Cybernetics,Rockville, MD). Averages of eggshell layer thicknesswere estimated from 5 measurements (μm) done in eachphotograph.

Statistical Analysis

Data were submitted to the normalcy of variance test(Shapiro and Wilk, 1965), and data not presenting nor-mal distribution were subjected to transformation in or-der to stabilize variances using the arcsine square rootpercentage (z = asin (sqrt (y+ 0.5))) (Ahrens et al.,1990). Data were analyzed using the PROC MIXEDof SAS (2011) with the repeated statement includedin the statistical model. The covariance structure usedwas the variable components, which showed the best fitbased on the Akaike criteria (Littell et al., 1998), exceptfor eggshell thickness, which was better fitted with theToeplizt covariance (Wolfinger, 1993). Total egg pro-duction and settable egg production per hen at 44 wkwere analyzed using the general linear models (PROCGLM). Means were compared using the Tukey-Kramertest, and differences were considered significant at P <0.05 (Tukey, 1991).

Estimates of Cu requirements were done using 3 dif-ferent models: exponential asymptotic (EA), brokenline 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 depen-dent variable as a function of dietary level of Cu; β1estimated the relative response to the diet containingthe lowest Cu (deficient diet); β2 estimated the differ-ence between the minimum and the maximum responseobtained with Cu supplementation; β3 was the curveslope coefficient; and β4 was the Cu level of the defi-cient diet. The maximum response for Cu was definedas Cu = (ln (0.05)/-β3) + β4 for 95% of the require-

ment. The BLQ model (Y = β1 + β2 × (β3 - Cu)2)had (β3 - Cu) = 0 for Cu > β3 with Y as the depen-dent variable as a function of the dietary level of Cu, β1the value of the dependent variable at the plateau, andβ2 as the slope of the line. The Cu level at the breakpoint (β3) was considered the one providing maximumresponses. The QP model (Y = β1 + β2 × Cu + β3× (Cu)2) had Y as the dependent variable and as afunction of dietary level of Cu; β1 as the intercept; β2as the linear coefficient; and β3 as the quadratic coeffi-cient. 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 differentmodels.

RESULTS

Formulated and analyzed diets had similar Cu con-tents (Table 2). Analyses of Cu in the dietary treat-ments were conducted on samples from 2 pools of the5 mixed batches utilized throughout the study and av-eraged 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 andperiod for any response; therefore, responses are pre-sented as main effects of dietary Cu and period through-out the text. During the experimental phase, pe-riod 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 ofbreeders (P > 0.05). As expected, egg production de-creased 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 wkand decreased afterwards (P < 0.05). Hatching chickHb was higher, and body length was longer (P < 0.05)when chicks were obtained from eggs laid by hens after40 wk of age, while the chick Ht and chick weight in-creased (P < 0.05) from eggs laid from hens after 33 wkof age (Table 3). Egg weight was higher (P < 0.05) inthe period of 40 to 44 wk, while specific gravity, thick-ness of eggshell membrane, and yolk Cu concentrationwere lower in the same period (P < 0.05). Yolk per-centage increased, and albumen percentage decreased(P < 0.05) in the 3 last periods (33 to 44 wk), whereaseggshell thickness palisade layer decreased from 33to 36 wk (P < 0.05), and, on other hand, eggshellmammillary layer thickness was highest from 29 to 36wk (P < 0.05; Table 4).

Dietary Cu affected all responses with the exceptionof hatchability, hatchability of fertile eggs, breeder Cpactivity, Hb of hatching chicks, eggshell percentage, spe-cific gravity, and thickness of eggshell palisade layer(P > 0.05; Tables 3 and 4). Providing breeders dietaryincreases of Cu affected (P < 0.05) hen d egg produc-tion as well as total settable eggs per hen and, therefore,impacted the total number of eggs per hen at the endof the experiment at 44 weeks. Breeder Hb increased



Tab

le3.

Res

pons

eof

broi

ler

bree

der

hens

toin

crea

sed

diet

ary

Cu.

Egg

sB

reed

erH

atch

ing

chic

k

Hen

day

prod

ucti

on2 ,

%Tot

alse

ttab

le/

hen3

Tot

al/

hen4

Hat

chab

ility

,%

Hat

chab

ility

offe

rtile

,%

Ht,

5 %H

b6,g/

dL

Cp7

,m

oles

/m

in/L

Seru

mC

u,m

g/L

Hb,

g/dL

Ht,

%B

ody

wei

ght,

gB

ody

leng

th,cm

Cu,

ppm

1(m

g/da

y)

2.67

(0.3

8)67

.7b

70b

95b

80.4

81.9

26.4

b6.

26c

8.63

0.22

6b6.

6027

.18b

44.8

b18

.4c

5.82

(0.8

3)73

.7a

80a,

b10

4a85

.285

.228

.1a,

b6.

85b

10.3

90.

244a

,b6.

6227

.97a

,b45

.9a,

b18

.6a,

b,c

9.38

(1.3

4)75

.4a

87a

105a

84.4

84.4

28.3

a,b

7.07

a,b

11.2

60.

265a

,b6.

6228

.20a

,b45

.9a,

b18

.5b,

c

12.9

2(1

.84)

74.8

a85

a10

3a83

.583

.529

.1a

7.10

a,b

11.0

90.

282a

6.59

29.0

1a46

.6a

18.7

a

16.8

3(2

.40)

74.1

a81

a,b

104a

83.1

83.0

28.4

a,b

7.05

a,b

12.0

80.

279a

6.63

28.2

4a,b

45.7

a,b

18.6

a,b

20.1

9(2

.88)

73.2

a82

a,b

103a

81.5

81.4

29.8

a7.

38a

12.5

20.

259a

,b6.

4028

.16a

,b46

.3a

18.7

a,b

Per

iod,

wk

25to

2865

.4c

−–

83.1

83.1

27.6

7.10

a,b

11.9

30.

216d

6.36

b,c

27.6

1b42

.6c

17.9

d

29to

3281

.5a

−–

84.6

84.6

28.8

7.32

a10

.96

0.22

8c,d

6.25

c27

.53b

45.0

b18

.4c

33to

3680

.2a

−–

83.3

83.3

27.0

6.80

b,c

9.06

0.32

1a6.

64b

28.2

1a,b

46.1

b18

.7b

37to

4069

.8b

––

83.2

83.1

29.0

6.88

b,c

10.8

80.

269b

6.39

b,c

28.9

5a47

.7a

18.8

b

41to

4468

.8b

−–

80.8

82.1

28.9

6.66

c12

.12

0.26

1b,c

7.23

a28

.33a

,b48

.6a

19.0

a

SEM

0.37

361.

3489

0.53

620.

5601

0.54

460.

2354

0.06

360.

4626

0.00

610.

0367

0.13

120.

1400

0.02

48Pro

bLev

el<

.000

10.

0032

<.0

001

0.22

370.

4709

0.00

13<

.000

10.

2238

0.00

570.

3032

0.00

550.

0018

<.0

001

Per

iod

<.0

001

––

0.42

760.

7815

0.05

090.

0006

0.29

48<

.000

1<

.000

10.

0039

<.0

001

<.0

001

Lev

elx

Per

iod

0.74

79–

–0.

9999

0.99

90.

9629

0.47

580.

8871

0.63

930.

5161

0.47

50.

4075

0.45

33

a–dM

eans

wit

hin

aco

lum

nw

itho

uta

com

mon

supe

rscr

ipt

differ

sign

ifica

ntly

byTuk

ey’s

test

(P<

0.05

).1 V

alue

sar

ean

alyz

ed,an

dva

lues

betw

een

pare

nthe

ses

are

Cu

inta

ke(m

g/he

n/d)

.2 E

ggs

prod

uced

asa

perc

enta

geof

tota

lliv

ehe

nsat

the

mom

ent

ofm

easu

rem

ent.

3 Tot

alse

ttab

leeg

gsat

the

end

ofth

eex

peri

men

t.4 T

otal

eggs

atth

een

dof

the

expe

rim

ent.

5 Hem

atoc

rit.

6 Hem

oglo

bin.

7 Cer

ulop

lasm

in.



Tab

le4.

Bro

iler

bree

der

hen

egg

char

acte

rist

ics

asaf

fect

edby

incr

ease

ddi

etar

yC

u.

Egg

shel

l

Egg

wei

ght,

gY

olk,

%2

Alb

umen

,%

3E

ggsh

ell,

%Y

olk

Cu,

ppm

4

Spec

ific

grav

ity,

g/cm

3Pal

isad

ela

yer,

μm

Mam

mill

ary

laye

r,μm

Mem

bran

e,μm

Thi

ckne

ss,

μm

Cu,

ppm

1(m

g/d)

2.67

(0.3

8)62

.0c

29.4

b61

.7a

8.90

1.30

b1.

082

241.

892

.6b

58.4

b41

8.9

5.82

(0.8

3)62

.7b,

c29

.6b

61.4

a9.

011.

80a,

b1.

082

240.

810

6.5a

69.9

a,b

424.

09.

38(1

.34)

64.3

a30

.4a

60.6

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Figure 1. Scanning electron cross-sections of eggshells from broiler breeder hens fed a Cu-deficient diet (2.67 ppm) (A), and diets with5.82 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.

(P < 0.05) when they were fed diets with 9.38 ppm Cuor greater as compared to the non-supplemented diet.Breeder Ht, serum Cu, hatching chick Ht, and bodyweight and length of hatching chicks increased (P <0.05) as dietary Cu reached 12.92 ppm.

Egg weight and yolk percentage were significantly in-creased 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 yolkCu concentration was highest (P < 0.05) when dietaryCu was 12.92 ppm or above. Scanning electronic mi-croscopy photographs (Figure 1) showed that dietaryCu deficiency led to a less stable eggshell ultrastruc-ture compared with the level above 5.82 ppm dietaryCu (P < 0.05). The Cu deficient diet (2.67 ppm Cu)led to decreased eggshell membrane thickness, whereasthe eggshell mammillary layer was affected by dietary

Cu (P < 0.05) only in the period between 29 and 32weeks.

Requirements of Cu were determined using EA,BLQ, and QP regression models. These are shownin Tables 5 and 6 in ppm as well as in mg perhen day. A sharp increase in egg production was ob-served at the first Cu level supplemented (5.82 ppm),while the maximum responses were obtained with6.2 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 ofCu estimated for total settable eggs per hen were8.1 ppm (1.16 mg/hen/d), 9.0 ppm (1.29 mg/hen/d),and 13.4 ppm (1.91 mg/hen/d) Cu.

Breeder hen requirements of Cu for Hb were es-timated 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)



Table 5. Requirements of Cu estimated for diverse broiler breeder responses adjusted with exponential asymptotic (EA), broken linewith 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 6.23BLQ Y = 74.7817 −0.3121 × (7.2833 −x)2 0.43 <.0001 7.28QP 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 8.15BLQ Y = 83.8056 −0.3454 × (9.0286 −x)2 0.13 0.0005 9.03QP 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 13.15EA 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 11.32QP 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 14.58BLQ Y = 29.0243 −0.0236 × (13.0201 −x)2 0.19 0.0002 13.02QP 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 16.22BLQ Y = 0.2721 −0.0004 × (14.5797 −x)2 0.11 0.0101 14.58QP 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 10.20BLQ Y = 28.4538 −0.0133 × (12.3415 −x)2 0.03 0.0027 12.34QP 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 6.80BLQ Y = 46.1032 −0.0549 × (7.089 −x)2 0.01 0.0368 7.09QP 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 12.94BLQ Y = 18.6696 −0.00181 × (13.8886 −x)2 0.02 0.0036 13.89QP 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.

using EA, BLQ, and QP models, respectively,whereas the requirement for Ht was estimated at15.6 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, serumCu concentration was maximized at 16.2 ppm(2.31 mg/hen/d), 14.6 ppm (2.08 mg/hen/d), and14.2 ppm (2.03 mg/hen/d), using EA, BLQ, and QPmodels, respectively.

The requirements of Cu for hatching chick Ht andbody weight and length were estimated at 10.2, 12.3,and 13.3 ppm (1.45, 1.76, and 1.90 mg/hen/d) usingEA, BLQ, and QP models; and 6.8 and 7.1 ppm (0.97and 1.01 mg/hen/d) and 12.9 and 13.9 ppm (1.84 and1.98 mg/hen/d) Cu using EA and BLQ models, re-spectively. The R squared obtained using the differenttested models for hatching chicks Ht, weight, and lengthwere very low. It was probably due to the high variationof the data among the different evaluation periods.

Estimations of Cu requirements for egg componentmeasures are shown in Tables 7 and 8 (ppm and mgper hen d, respectively). All models were well fitted inestimating Cu requirements for analysis done with eggs,except for egg yolk and albumen percentage, whichdid not fit for EA and BLQ models. The Cu require-ment for percent yolk and albumen were 11.0 ppm

(1.57 mg/hen/d) and 11.3 ppm (1.61 mg/hen/d) forpercent yolk and albumen, respectively, using theQP model. The requirement of Cu estimated for eggweight 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. Di-etary Cu requirements to maximize the Cu content inthe 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) us-ing EA, BLQ, and QP models, respectively. Eggshellmembrane was thickest when dietary Cu was fed at7.3 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.

DISCUSSION

In the present study, broiler breeder hens fed a non-supplemented Cu diet demonstrated signals of defi-ciency throughout most evaluated responses. Since Cuis necessary for adequate activities of a great number ofmetalloenzymes, biochemical reactions are not carriedout properly during Cu deficiency (Kaya et al., 2006;Scheiber et al., 2014; Cao et al., 2016).

The lack of interaction between dietary Cu and pe-riod is an indication that Cu requirements do not



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 0.89BLQ 0.88 1.10 1.10 1.07 1.04 1.04QP 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 1.16BLQ 1.09 1.37 1.36 1.33 1.29 1.29QP 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 1.98BLQ 1.37 1.71 1.71 1.66 1.62 1.61QP 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 2.08BLQ 1.58 1.97 1.97 1.91 1.86 1.86QP 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 2.31BLQ 1.76 2.21 2.20 2.14 2.08 2.08QP 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 1.45BLQ 1.49 1.87 1.86 1.81 1.76 1.76QP 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 0.97BLQ 0.86 1.07 1.07 1.04 1.01 1.01QP 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 1.84BLQ 1.68 2.10 2.10 2.04 1.98 1.98QP 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.

change as hens age from 25 to 44 weeks. However, val-ues vary within the same database depending on themodel used to estimate its requirements (Robbins etal., 1979). Prediction of requirements do not have oneparticular true model; therefore, results of experimentsshould be the ones to dictate the model choice (Vede-nov and Pesti, 2008). Non-linear models, such as BLQand EA, have been thought to be advantageous com-pared to linear models, due to the fact that biologicalsystems rarely work in perfect linearity (Pesti et al.,2009). In the present study, adequate fit was sometimesnot found for one or more models and, in those cases,they were not included in this section. The EA andBLQ models fitted better and estimated lower require-ment values for most responses, whereas the QP modelprovided the best fit only for total egg production, yolk,and albumen percentage and estimated higher require-ments values. Requirements of Cu for hen d egg pro-duction varied between 6.2 and 12.9 ppm total Cu inthe diet, which represents a dietary intake of Cu from0.89 mg/hen/d to 1.84 mg/hen/day. Requirement fortotal egg production at the end of wk 44 was higher

(13.1 ppm or 1.87 mg/hen/d) than for egg productionper hen day.

Traditionally the tables of requirements for poultrydo not provide Cu requirements for broiler breeders. In-stead, supplemental levels are suggested, which rangefrom 10 to 15 ppm, intending to provide safety marginsin feed formulation (Mondal et al., 2010). Data from thepresent study indicate that the usually suggested sup-plemental Cu levels (NRC, 1994; Cobb-Vantress, 2013;Aviagen, 2017; Rostagno et al., 2017) are excessive. Asafety margin is generally used when it comes to mi-cronutrients due to numerous factors that can influ-ence the nutritional requirements (Applegate and An-gel, 2014), including an overall scarcity of research. Themaximum concentration of 25 ppm Cu in the diet as es-tablished by the EC (EFSA, 2016), therefore, seems tobe very safe in attending breeder hen needs, since itrepresents about 2 times what has been estimated asan average requirement in the present research. A non-supplemented broiler breeder hen diet formulated withcorn, soy, and wheat bran has an average of 8 ppmtotal Cu and, therefore, is expected to provide Cu in



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 14.94BLQ Y = 64.0796 −0.021 × (12.7022 −x)2 0.13 <.0001 12.70QP 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 14.98BLQ Y = 2.0983 −0.00409 × (16.3348 −x)2 0.11 <.0001 16.33QP 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 7.29BLQ Y = 71.9917 −0.4945 × (7.8596 −x)2 0.16 0.0006 7.86QP 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 10.56Layer4, 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.

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 2.13BLQ 1.54 1.92 1.92 1.86 1.81 1.81QP 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 2.14BLQ 1.98 2.47 2.47 2.40 2.33 2.33QP 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 1.04BLQ 0.95 1.19 1.19 1.15 1.12 1.12QP 1.69 2.11 2.11 2.05 1.99 1.99

Eggshell mammillary layer4, μm EA – 1.60 – – – 1.60BLQ – 1.54 – – – 1.54QP – 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.

amounts close to the requirements determined in thepresent study.

Dietary Cu fed to hens impacted their blood Hb andHt. A decrease in the Ht can be related to depressionin the phospholipid synthesis (Kaya et al., 2006) andin the platelet-mediated hemostasis in dietary Cu defi-ciency (Schuschke et al., 1994). A reduction in Ht wasreported by Baumgartner et al. (1978) when hens werefed a Cu deficient diet. Effects of dietary Cu also wereobserved in Hb, a response that also was previouslyreported 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 ppmCu (average of the EA and BLQ models, which pre-sented high R-squares). Kubena et al. (1972) also ob-served the same tendency when diets were tested with-out Cu supplementation (3 ppm) or were supplementedat 2.2 and 9.9 ppm. It has been earlier reported that Cufacilitates Hb formation in anemic chicks (Elvehjem andHart, 1929), and Cp-stimulated iron uptake requiresCp ferroxidase activity and utilizes a novel, trivalentcation-specific transport pathway (Attieh et al., 1999;Lenartowicz et al., 2015; Linder, 2016). Hephaestin andCp are Cu-dependent enzymes that can modify blood



Hb values (Reeves et al., 2005; Ha et al., 2016). In thepresent study, however, hen Cp activity when a deficientCu diet was fed (2.67 ppm Cu) was not affected. Othershave found the opposite, with 3.44 ppm Cu leading todecreases in Cp (Kaya et al., 2006). Concentration ofCu in the serum has been improved as dietary Cu in-creases (Zanetti et al., 1991; Schmidt et al. 2005), andmaximum serum Cu level obtained in the present studywas obtained at 15.0 ppm dietary Cu (2.14 mg/hen/d).The lack of significant differences in the Cp activity inresponse to dietary Cu increase found in the presentstudy and changes in the Cu contents in Hb, as well asin the serum observed, indicate that blood parametersare more sensitive to dietary Cu than Cp and that Cpis highly preserved. Average Cu requirements using the3 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 thoseneeded to maximize egg production. Blood Cu has beenshown to change in relation to growth rate and age(Abdel-Mageed and Oehme, 1990), which may explainthe differences in the amount of Cu in the serum amongperiods in this study.

Deficiencies observed with the lowest Cu contents inthe diets fed in the present study resulted in decreasesin yolk Cu concentration, as well as in Ht of hatch-ing chicks. Contents of Cu in eggs are dependent onthe dietary supply to breeder hens (Kim et al., 2016)and, therefore, deficiencies or excesses affect egg qualityand subsequent performance of the progeny (Whiteheadet al., 1985). In the present study, it seemed that thedeposition of Cu in the yolk had a non-linear behav-ior, reaching maximum Cu depositions at 15.7 ppm di-etary Cu (or 2.24 mg/hen/d). Egg mineral constituentsare transferred from the hen, which originate fromthe feed.

Copper deprivation impairs eggshell quality, as itwas observed by scanning electronic microscopy in thepresent study. Eggshells are composed of ultrastruc-tural layers divided into shell membranes, mammil-lary knobs, palisade, and a cuticle (Arias et al., 1993;Hunton, 2005). Activation of lysyl oxidase by Cu is nec-essary for the collagen synthesis in the eggshell mem-brane (Leach et al., 1981). This enzyme is responsi-ble for oxidative deamination of the lysine side chains,which form crosslinks and thus confer characteristics ofinsolubility, flexibility, and structure for the depositionof other egg components (Linder and Hazegh-Azam,1996; Akagawa et al., 1999). In the present study, thethickening of the eggshell membrane provided by Cu in-take did not increase the total thickness of the shell orpalisade layer, or of the mammillary layer in most peri-ods. There was an increase in thickness of the eggshellmammillary layer only at 29 to 32 wk using 11.7 ppmCu (or 1.77 mg/hen/d), but there was no change in thethickness of the palisade layer at any time. Thus, theamount of Cu that is deposited in the eggshell mustbe influenced by other factors. Duan et al. (2016) ob-served that the thickness of the mammillary layer was

affected by mammillary density and might be regulatedby some protein interactions, which can be responsiblefor nucleation sites formation.

Although the membrane thickness does not regulatethe amount of mineral deposition in the other eggshelllayers, it appears to be important for structural orga-nization. Therefore, a well-structured outer shell mem-brane is necessary for correct mineralization and con-sequently to produce resistant eggshells (Nys et al.,1999; Nys et al., 2004). Any modification of the eggshellmembranes by Cu deficiency occurs due to inhibitionof fiber formation or crosslinking, characterized by anabnormal distribution of the eggshell membrane fibers(Mabe et al., 2003), and consequently altering its me-chanical properties (Hincke et al., 2012). The lowestthickness of the eggshell membrane observed using lowCu levels in the present study may have resulted in anincreased occurrence of unsettable eggs. The require-ment of 7.6 ppm Cu (1.08 mg/hen/d) for the maximumeggshell membrane thickness obtained in this experi-ment was similar to the requirement of the maximumtotal settable eggs of 8.6 ppm Cu (1.22 mg/hen/d) us-ing the mean EA and BLQ models. Results obtainedindicate that Cu most likely plays an important rolein thickening of the eggshell membrane, and, there-fore, it is expected to affect eggshell breaking resistance.Some studies suggest that the dry eggshell membraneweight decreases in eggs with eggshell deformation, aswell as the membrane strength having a positive corre-lation with eggshell breaking strength (Britton, 1977;Essary et al., 1977). In addition, adequately structuredeggshell membranes provide the proper formation of theair chamber immediately after oviposition, which is de-pendent on separation between the inner and the outershell membranes (Vieira, 2007).

In conclusion, the data from this study indicate a Curequirement range between 6.2 and 16.3 ppm (0.89 to2.33 mg/hen/d), depending on production objectives.The average of all Cu requirement estimates obtainedin the present trial was 12.5 ppm Cu (1.79 mg/hen/d),whereas averaged values for EA, BLQ, and QP mod-els were 11.4, 11.3, and 14.4 ppm Cu (1.63, 1.62, and2.06 mg/hen/d), respectively.

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