in ovo feeding with minerals and vitamin d3 improves bone

In ovo feeding with minerals and vitamin D3 improves bone properties inhatchlings and mature broilers

R. Yair,∗,† R. Shahar,∗ and Z. Uni†,1

∗The Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment,The Hebrew University, Rehovot, Israel; and †Department of Animal Science, The Robert H. Smith Faculty of

Agriculture, Food and Environment, The Hebrew University, Rehovot, Israel

ABSTRACT The objective of this study was to ex-amine the effect of in ovo feeding (IOF) with inor-ganic minerals or organic minerals and vitamin D3on bone properties and mineral consumption. Eggswere incubated and divided into 4 groups: IOF withorganic minerals, phosphate, and vitamin D3 (IOF-OMD); IOF with inorganic minerals and phosphate(IOF-IM); sham; and non-treated controls (NTC). IOFwas performed on embryonic day (E) 17; tibiae and yolksamples were taken on E19 and E21. Post-hatch, onlychicks from the IOF-OMD, sham, and NTC were raised,and tibiae were taken on d 10 and 38. Yolk mineralcontent was examined by inductively coupled plasmaspectroscopy. Tibiae were tested for their whole-bonemechanical properties, and mid-diaphysis bone sectionswere indented in a micro-indenter to determine bonematerial stiffness (Young’s modulus). Micro-computedtomography (μCT) was used to examine cortical andtrabecular bone structure. Ash content analysis wasused to examine bone mineralization. A latency-to-lie

(LTL) test was used to measure standing ability of thed 38 broilers. The results showed that embryos fromboth IOF-OMD and IOF-IM treatments had elevatedCu, Mn, and Zn amounts in the yolk on E19 and E21and consumed more of these minerals (between E19and E21) in comparison to the sham and NTC. OnE21, these hatchlings had higher whole-bone stiffnessin comparison to the NTC. On d 38, the IOF-OMD hadhigher ash content, elevated whole-bone stiffness, andelevated Young’s modulus (in males) in comparison tothe sham and NTC; however, no differences in standingability were found. Very few structural differences wereseen during the whole experiment. This study demon-strates that mineral supplementation by in ovo feedingis sufficient to induce higher mineral consumption fromthe yolk, regardless of its chemical form or the presenceof vitamin D3. Additionally, IOF with organic mineralsand vitamin D3 can increase bone ash content, as wellas stiffness of the whole bone and bone material in themature broiler, but does not lead to longer LTL.

Key words: in ovo feeding, bone, broiler, mineral, vitamin D3.2015 Poultry Science 94:2695–2707

http://dx.doi.org/10.3382/ps/pev252

INTRODUCTION

Embryonic nutrition has a pronounced effect onprogeny performance. Nutritional insufficiencies duringthe embryonic period and early life can induce adap-tive responses with long-lasting adverse consequences(Tygesen et al., 2008; Matthiesen et al., 2010; Smithet al., 2010; Langley-Evans, 2015). This phenomenonwas previously termed “programming” (Lucas, 1991).

Unlike mammals, the broiler embryo develops inde-pendent from the mother, and therefore, the deposi-tion of the finite nutrient storages to the egg by thehen is crucial to enable optimal embryonic development(Richards, 1997; Moran, 2007; Uni et al., 2012).

C© 2015 Poultry Science Association Inc.Received January 20, 2015.Accepted July 15, 2015.1Corresponding author: [email protected]

The deposition of minerals to the different egg com-partments is crucial for optimal embryonic developmentbecause minerals are important for the development ofthe skeletal, muscular immune, and cardiovascular sys-tems of the broiler embryo (Caskey et al., 1944; Wilson,1997; Favero et al., 2013; Oviedo-Rondon et al., 2013).

The hen deposits minerals to the egg via two routes:through the ovary to the yolk or through the oviduct tothe albumen, shell, and shell membrane (Richards andPackard, 1996). Each of these compartments containsa different variety of minerals; the yolk is the majormineral source for the embryo during incubation, con-taining most of the P, Zn, Cu, Mn, and Fe, while thealbumen is the major source of Na and K in the eggand contains very low levels of P, Fe, Cu, Mn, and Zn(Richards and Packard, 1996; Richards, 1997; Yair andUni, 2011). The shell contains high amounts of Ca andlow amounts of Fe, Mg, Mn, P, and Zn; however, onlyhigh amounts of Ca, a much lower amount of Mg, andnegligible amounts of Fe, Mn, and P are released from

2695

Dow

nloaded from https://academ

ic.oup.com/ps/article-abstract/94/11/2695/2453312 by guest on 11 April 2019

mailto:[email protected]

2696 YAIR ET AL.

the shell and made available for the embryo (Packardand Packard, 1991; Richards and Packard, 1996; Yairand Uni, 2011).

Modern broilers underwent genetic selection for high-growth and metabolic rates, resulting in annual im-provements in BW gain (due to increased muscle mass),feed efficiency, and meat yields (Havenstein et al., 2003;Tona et al., 2004; Vieira and Angel, 2012); however,with these improvements it became evident that somesystems, such as the skeletal system, were not keep-ing up with the increase in muscle mass (Dibner et al.,2007). As a result, modern-day broilers suffer from nu-merous bone problems that were found to be closelyassociated with their fast growth rate (Thorp, 1994; Ju-lian, 1998; Angel, 2007; Dibner et al., 2007; Shim et al.,2012; Prisby et al., 2014).

Leg problems are among the major causes for eco-nomic losses in the chicken house (Sullivan, 1994).These problems were estimated to cause economic lossesof 80 to 120 million dollars annually in the United States(Sullivan, 1994). To our knowledge, no recent estima-tions were published; however, in a survey of commer-cial broiler flocks, Knowles et al. (2008) found that morethan 27.6% of the birds suffered from poor locomotion,and 3.3% were almost unable to walk. Such impairedwalking ability is expected to reduce production dueto reduced feed consumption, increased frequency ofdowngrades and condemnations, and elevated mortality(Sullivan, 1994). Accordingly, it is reasonable to assumethat leg problems are still a cause for major economiclosses.

In addition, leg problems can dramatically affectbroiler welfare by causing lameness and impaired walk-ing ability, inducing acute and chronic pain, reducingaccess to feed and water, and even causing mortality(Angel, 2007; Dibner et al., 2007; Naas et al., 2009;Ruiz-Feria et al., 2014). Due to these economic andwelfare issues, it is very important to minimize the in-cidence of leg problems in broilers.

In order to reduce the incidence of leg problems, inthe last 25 years attempts were made to select broilersfor improved skeletal integrity (Williams et al., 2000;Kapell et al., 2012). Some progress was reported byKapell et al. (2012), who showed that selection by ac-curately scoring selection candidates and using a strin-gent culling policy of discarding any selection candidatewith clinical leg defects has led to a reduction in theincidence of some leg defects, such as tibial dyschon-droplasia (TD) and crooked toes, but not of hock burn.Despite this selection effort, recent works still haveshown that fast-growing broilers have a high incidenceof leg problems: Dinev et al. (2012) found that 24.22 to27.70% of broilers from 3 commercial lines suffer fromsome degree of TD. Wideman et al. (2013) found thatfaster-growing commercial broilers are more susceptibleto lameness. Additionally, leg problems are affected bynutrition and management, not only by growth rate.For example, the content and chemical form of Ca, P,Cu, Mn, Zn, and Vitamin D3 in the diet can influence

bone development and leg problems (Richards, 1997;Angel, 2007; Dibner et al., 2007; Favero et al., 2013),while incubation conditions have an important effecton bone properties, leg problems, and standing abilityof modern broilers (Oviedo-Rondon et al., 2008; Grovesand Muir, 2014; Van der Pol et al., 2014). Accordingly,methods other than selection should also be used in or-der to reduce the incidence of leg problems in modernbroilers.

It was previously shown that a shortage in Cu, Mn,P, and Zn during the embryonic period and post-hatchleads to impaired bone development (Caskey et al.,1944; Dibner et al., 2007). We previously showed thatduring the last days of incubation, the amount of Cu,Mn, P, and Zn in the yolk is low, and therefore, the em-bryo consumes little if any of those minerals during thatperiod (Yair and Uni, 2011). Correspondingly, most me-chanical and geometric properties of the tibia and femurremain unchanged or even deteriorate during that pe-riod (Yair et al., 2012). Accordingly, it was previouslysuggested that the limited availability of minerals dur-ing the embryonic period and the first weeks post-hatchlimits skeletal development during its rapid-growth pe-riod, thus increasing the incidence of leg problems (Dib-ner et al., 2007; Yair et al., 2012).

Previous work has shown that embryonic enrichmentwith organic Cu, Fe, Mn, and Zn, phosphate, vitaminD3, and carbohydrates using the in ovo feeding (IOF)methodology (Uni and Ferket, 2003; Uni and Ferket,2004) increased the content of these minerals in theyolk and their consumption by the embryo pre-hatch(Yair and Uni, 2011).

However, it is unclear if this effect is due to the en-richment with minerals per se, or due to the organicform of the added minerals, vitamin D3, carbohydrates,or their combination. Additionally, similar IOF led tohigher tibial stiffness, but only until 14 d post-hatch(Yair et al., 2013).

Consequently, the objectives of this work were: 1) todetermine whether embryonic enrichment with minerals(in inorganic form) is sufficient to induce higher embry-onic mineral consumption, and 2) to examine the effectof IOF with a solution of organic trace minerals (OTM),phosphate, and vitamin D3, which was prepared basedon our conclusions from the previous trials (Yair andUni, 2011; Yair et al., 2013) on the content and uptakeof minerals from the yolk, and to evaluate if it couldhave a longer lasting effect on the mechanical proper-ties of broiler bones (i.e., in the mature broiler, and notonly until d 14).

MATERIALS AND METHODS

Overview

The experiment was carried out in 2 sequential andidentical studies (treated as blocks). In each study,150 fertile eggs were collected from Cobb 500 hens (35and 37 wk of age, respectively, in the first and second

Dow



IN OVO FEEDING IMPROVES BONE PROPERTIES 2697

Table 1. Formulation of the breeder and broiler diets.

Broiler diet

Breeder diet Pre-starter (1 to 10 d) Starter (11 to 21 d) Grower (21 to 31 d) Finisher (31 to 38 d)

Protein % 15.5 22.0 21.0 20.0 19.0Fat % 4.0 6.0 7.0 7.5 8.2Ash % 11.0 5.6 5.1 4.8 4.6

Sodium % 0.35 0.18 0.15 0.15 0.15Available P % 0.35 0.5 0.42 0.40 0.39

Total P % 0.52 0.71 0.61 0.60 0.57Ca % 3.30 0.95 0.85 0.8 0.77

Metabolic energy kcal/kg 2,750 3,225 3,150 3,100 3,050

Premix

Mn ppm 80 100 100 100 100Zn ppm 100 90 90 90 90Fe ppm 60 30 30 30 30Cu ppm 10 50 50 50 50

Vitamin D3 IU/kg 3,000 4,000 4,000 4,000 4,000

Table 2. Content of the IOF solutions.1

IOF-OMD IOF-IM

Concentration Amount per Concentration Amount perChemical form (mg/mL) embryo (mg) Chemical form (mg/mL) embryo (mg)

Zn 2Organic 1.80 1.08 2ZnSO4 1.45 0.87Mn 2Organic 0.057 0.034 2MnSO4 0.055 0.033Cu 2Organic 0.088 0.053 2CuSO4 0.08 0.048P 3KH2PO4&4NaH2PO4 3.2 1.92 3KH2PO4&4NaH2PO4 3.53 2.12K 3KH2PO4 2.03 1.22 3KH2PO4 2.08 1.25Na 4NaH2PO4 1.13 0.68 4NaH2PO4 1.25 0.75

5Vitamin D3 Liquid 400 (IU/mL) 240 IU – – 0

1Both solutions were injected to the amniotic fluid on E17.2Organic minerals were supplemented as Glycine chelates (MAAC R©, Novus International, Inc. (St. Charles, MO)).3JT Baker (Phillipsburg, NJ).4Merck (Darmstadt, Germany).5Dor.Ky (Nes Tziona, Israel).

studies), which were housed in the Faculty of Agricul-ture (Rehovot, Israel) and fed according to Cobb’s pro-tocols (Table 1). The eggs were divided into 4 groups,with each group consisting of similar egg weight distri-bution. The 4 groups were: (1) IOF with OTM, phos-phate, and vitamin D3 (IOF-OMD); (2) IOF with in-organic trace minerals and phosphate (IOF-IM); (3)sham (IOF with the diluent alone); and (4) non-treated controls (NTC). The eggs were incubated (Pe-tersime, Zulte, Belgium) according to routine proce-dures (37.8◦C and 56% relative humidity). On em-bryonic day 17 (E17) the amniotic fluids of the IOF-OMD, IOF-IM, and sham groups were enriched withsolutions (0.6 mL per egg) by the IOF technique, asdescribed by Uni and Ferket (2003; 2004). The con-tent of the IOF solutions given to each embryo wasplanned to be as follows: 2.0 mg of P (in NaH2PO4and KH2PO4 forms), 1.0, 0.05, and 0.03 mg of Zn, Cu,and Mn as Glycine chelates (MAAC R©, Novus Interna-tional, Inc. (St. Charles, MO)), and 240 IU of solubi-lize vitamin D3 for the IOF-OMD. The IOF-IM wereplanned to have similar amount of P (in similar forms)than the IOF-OMD, similar amount of Zn, Cu, andMn as sulfates, and no vitamin D3. The mineral con-

tent of the solutions as analyzed by an inductively cou-pled plasma atomic emission spectroscopy (ICP-AES;Spectro Arcos, Kleve, Germany) was 1.92, 1.08, 0.053,and 0.034 mg of P, Zn, Cu, and Mn, respectively, forthe IOF-OMD, and 2.12, 0.87, 0.048, and 0.033 mgof P, Zn, Cu, and Mn, respectively, for the IOF-IM(Table 2). The sham group solution contained only thediluent of the IOF solutions (DDW with Tween 20,which was used to solubilize the vitamin D3).

Yolk and Bone Samples On E0, day of set, andE17, 4 eggs (in each study) were randomly selected andtheir yolk sacs taken for mineral analysis. On E19 andE21 (day of hatch), 4 eggs from each group (in eachstudy) were randomly selected, and the yolk sacs andembryonic tibiae were removed. Upon hatching of theremaining eggs, hatchlings from the NTC, IOF-OMD,and sham groups were identified by a neck tag num-ber and moved to a pen, and the floor was coveredwith soft pinewood shavings. As the IOF-IM group wasused to see if embryonic enrichment with inorganic min-erals without other nutrients (not organic, since theymight influence consumption) is sufficient to increasemineral consumption pre-hatch, the IOF-IM hatchlingswere not raised post-hatch. The chicks were housed in

Dow



2698 YAIR ET AL.

one pen (stocking density of 8 to 10 chicks/m2) andgiven ad libitum access to feed (Table 1) and water.On d 10 post-hatch, 4 randomly selected broilers fromeach group (in each study) were selected, while on d38 post-hatch (marketing age), 10 broilers from eachgroup (in each study) were randomly selected. The se-lected broilers were sacrificed by cervical dislocation,their sex was determined by identifying the testis or theovaries, and tibiae from both legs were removed. Theselected bones at all time points were cleaned of allsoft tissues, externally measured (weight and length),wrapped in saline-soaked gauze, and stored at –20◦C.The selected yolk sacs were weighed, homogenized us-ing a T-25 Ultra-Turrax homogenizer (IKA, Staufen,Germany), and stored at –20◦C.

Mineral Analysis Samples (100 to 150 mg) fromeach yolk sac or albumen homogenate were digestedwith a mixture of 2 mL 30% H2O2 and 4 mL 70% HNO3inside a 50-mL plastic tube for 6 h in a 95◦C bath. Thedigested samples were analyzed for their mineral con-tent using ICP-AES (Spectro Arcos, Kleve, Germany).

Structural Analysis The left tibia of each embryoand chick was scanned using a high-resolution micro-computed tomography (μCT) scanner (SkyScan 1174,Belgium). The X-ray source was set at 50 kV and 800μA. A total of 225 projections were acquired over anangular range of 180◦. An aluminum filter of 0.25 mmthickness for samples from E19 and E21, and 0.5 mmfor samples from d 10 and d 38 was used to decreasebeam-hardening effects. The image slices were recon-structed using customized software (NRecon, SkyScan,Belgium). Scans were performed at the highest resolu-tion possible, depending on bone size (7.9, 10.2, 16.1,and 30.1 μm, for E19, E21, d 10, and d 38, respec-tively). In addition, for each set of scanning parameters,2 phantoms of known mineral density (0.25 g/cm3 and0.75 g/cm3) also were scanned in order to allow calibra-tion of the attenuation levels directly to bone mineraldensity (BMD) values.

Cortical bone analysis was performed at mid-diaphysis of the examined bone, selecting a 100-sliceregion of interest. The following cortical properties weremeasured: bone volume fraction as % of total tissue vol-ume (BV/TV, %), BMD (g/cm3), cortical area (mm2),medullary area (the area of the medullary cavity sur-rounded by the cortex) (mm2), polar moment of inertia(mm4), and mean cortical thickness (μm).

Cancellous bone analysis was performed at the distalepiphyseal area of each examined bone over a 100-sliceregion of interest. The first slice of the selected regionof interest was set at the most proximal area where thecross-section was filled with trabeculae, and the regionof interest consisted of the next 100 slices in the dis-tal direction. A customized software (CTan, SkyScan,Belgium) was used to manually specify the limits ofthe trabecular area on each slice in order to separateit from the cortical shell surrounding it; measurementswere taken only from within these limits. The follow-ing trabecular properties were measured: bone volume

fraction as % of total tissue volume (BV/TV, %), meantrabecular thickness (μm), and mean trabecular sepa-ration (μm). Cancellous bone analysis was performedonly on d 10 and d 38, due to insufficient amounts oftrabecular struts at younger ages.

Mechanical Testing After completion of the struc-tural analysis, tibiae were biomechanically tested. Test-ing of the smaller bones (E19 to d 10) was performedwith a custom-built micromechanical testing device; anaxial-motion DC motor (PI M-235.2 DG, Physik In-strumente, GmbH, Germany) moved a metal shaft intothe testing chamber in small sub-micron steps whilebeing able to apply substantial force (>100 N). Themetal shaft was connected in series to a 120 N load cell(AL311, Sensotec, Honeywell, United States, ± 0.4 N),to which was attached in turn a movable anvil that wasdesigned to contact the bone sample at its mid-point.The movement of the anvil was measured using an opticencoder with 50 nm resolution (Model RGH22H30D63,Renishaw, United Kingdom). In order to test the bonesin an environment that approximates their physiologi-cal state, they were tested while immersed in saline. Theload and displacement values were monitored throughan in-house written program (Labview, National Instru-ments, Austin, TX).

Biomechanical testing of the bones from d 38 wassimilarly performed using an Instron 3345 materialstesting machine (Instron, Norwood, MA). The boneswere tested in a plastic container that allowed themto be immersed in saline during testing. Force and dis-placement data were collected at 20 Hz by Instron soft-ware (BlueHill, Buckinghamshire, United Kingdom).

All bones were tested by the 3-point bending method.The bones were placed on 2 supports having roundedprofiles (0.5 mm radius) to limit stress concentrationat the point of load application, such that the supportswere equidistant from the ends of the bone, and bothcontacted the posterior aspect of the diaphysis. The dis-tance between the supports was adjusted to be the max-imal distance possible in the cylindrical part of the dia-physis. Each bone was loaded on its anterior aspect by amoving prong with rounded profile at the mid-point be-tween the bottom supports and at the mid-point alongits length. Loading was conducted at a constant rateof 2 mm/min up to fracture point, as identified by asudden decrease in load.

The resulting load-displacement curves were used tocalculate whole-bone stiffness (slope of the linear por-tion of the load-displacement curve), maximal load, andwork to fracture (WTF) (Lanyon et al., 1982).

In order to determine the stiffness of the material ofthe cortical bone of mature broilers, tibiae (n = 5) fromd 38 obtained from the second study were tested bya micro-indenter using a Vickers hardness tip (Evanset al., 1990; Bonser and Casinos, 2003); 5 mm-thicktransverse bone sections were cut from the mid diaph-ysis using a slow water-cooled diamond saw (BuehlerIsomet, Lake Bluff, IL). The sections were than hand-polished from both sides using emery paper (800, 1,200,

Dow




Figure 1. A sample of an indentation done on a mature broiler bone(d 38). The length of the indentation diagonals are noted as d1 andd2. 1Tibiae cross sections were indented using a Future-Tech FM-300micro hardness tester.

2,400, and 4,000 grit) and a polishing cloth with 3μm diamond suspension (Buehler Minimet Polisher,Lake Bluff, IL). Vickers hardness was measured using aFuture-Tech FM-300 micro hardness tester with a loadof 25 g and a dwell time of 10 sec. Each bone sectionwas indented in the center of its anterior part; 10 in-dentations per sample were performed in 2 parallel linesof 5 indentations. The distance between each two adja-cent indentations was 200 μm. Micro-hardness at eachindentation site was determined by using the followingformula:

VHN =1.8544P

d2

VHN is Vickers hardness number (kg/mm2), P is thetest load (kg), and d is the average indentation diago-nal length (mm) (Figure 1). The Young’s modulus (E)was calculated by using the conversion formula fromhardness to stiffness published by Evans et al. (1990):

E =

√(V HN − 17.5)

0.104

Ash Content After the tibiae were mechanicallytested, a small section from the mid diaphysis of eachbone (about 10 to 20% of the bone’s length) was re-moved and processed for ash quantification, following apreviously described protocol (Yair et al., 2013).

Standing Ability Test In order to assess differencesin standing ability and leg weakness of chicks from thevarious treatment groups, a latency-to-lie (LTL) test(Weeks, 2001; Berg and Sanotra, 2003) was performedin the second study. On d 35, 12 chicks per group weresubjected to a modified LTL test as described by Berg

and Sanotra (2003). Briefly, each chick was put in asmall plastic tub filled with 3 cm of water (∼32◦C),surrounded by disposable cardboard sheets to limit thechick’s ability to see and jump outside the test area.The time until the chick could not continue standingand attempted to lie was measured by a stopwatch. Ifthe bird was still standing after 600 sec, the test wasinterrupted.

Ethical Approval Embryos and chicks were sacri-ficed at predetermined time points as described. Theexperiment was approved by the Ethics Committee forAnimal Experimentation, Faculty of Agricultural, Foodand Environmental Sciences, the Hebrew University ofJerusalem.

Statistical Analysis Data from the sampling daywere subjected to 3-way full-factorial ANOVA with 4groups for E19 and E21 (NTC, IOF-OMD, IOF-IM,and sham) or 3 groups for d 10 and d 38 (NTC, IOF-OMD, and sham), 2 studies, 2 sexes, and all of theirinteractions. The group means from E19 and E21 werecompared by the Tukey Kramer HSD test, while resultsfrom bones of chicks aged 10 and 38 d were compared byStudent’s t-test. All statistical analyses were conductedusing the JMP software (SAS Institute Inc., Cary, NC),and differences were considered statistically significantat P < 0.05.

RESULTS

Yolk Mineral Levels

The amounts of Cu, Mn, P, and Zn in the yolk duringincubation are presented in Figure 2. On E19, the IOF-OMD and IOF-IM groups had 5.6 to 6.8 fold higher Cu,3.3 to 4.5 fold higher Mn, and 2.8 to 3.1 fold higher Znin comparison to the NTC and sham control groups.On E21, the IOF-OMD and IOF-IM groups had 2.4 to5.5 fold higher Cu, 2.3 to 3.2 fold higher Mn, and 3.3to 4.7 fold higher Zn in comparison to the NTC andsham control groups. No differences in P content wereobserved among the groups on E19 and E21.

The consumption of Cu, Mn, P, and Zn from the yolkbetween E19 and E21 (measured for each group by sub-tracting the average mineral level on E21 from its levelon E19) is presented in Figure 3. Since we cannot mea-sure the individual consumption value for each embryo,only the average consumption of each group was esti-mated. Therefore, no statistical analysis was performedand the groups were compared numerically. Generally,the IOF-OMD and IOF-IM had similar consumption ofCu, Mn, and Zn, which was higher than the consump-tion of the sham and NTC groups. For example, theIOF-OMD consumed 27.9 and 58.3% more Zn than thesham and NTC, respectively, while the IOF-IM con-sumed 50.5 and 86.4% more Zn than the sham andNTC, respectively.

Body and Tibia Morphometric Parameters Bodyweight, tibia weight, and tibia length are presented in

Dow



2700 YAIR ET AL.

Figure 2. Content of A) Cu, B) Mn, C) P, and D) Zn in the yolk of embryos from the NTC, sham, IOF-OMD, and IOF-IM groups duringincubation (n = 8). a-bSignificant differences among groups are marked with different superscripts (P < 0.05). 1Values are means ± SE of malesand females that did not differ. 2At E17 the amniotic fluids of the IOF-OMD, IOF-IM, and sham groups were injected with a solution of organicminerals, phosphate, and vitamin D3 (IOF-OMD); inorganic minerals and phosphate (IOF-IM); or the diluent of the IOF-OMD and IOF-IM(sham). The NTC were non-treated controls.

Tables 3 and 4. There were no statistical differencesamong the groups in those parameters.

Bone Mineralization Tibia ash weight percent andBMD are presented in Figure 4. Mineralization differ-ences between the groups were seen only on d 38; theIOF-OMD group had 1.2 and 1.6% higher ash contentcompared to the sham and NTC groups, respectively.

Mechanical Properties Tibial whole-bone stiffness,maximal load, and WTF, as well as cortical bone mate-rial Young’s modulus are presented in Figure 5. Gener-ally, the IOF-OMD group showed elevated whole-bonestiffness in comparison to the NTC and sham groupsduring the compared sampled days (other than d 10and E19 for the sham group). On E19, the IOF-OMDhad 40% higher stiffness than the NTC (but not thanthe IOF-IM and sham). Interestingly, between E19 andE21, the stiffness values of all the groups decreased by

20 to 45%. On E21, the IOF-OMD group (which hadthe smallest decrease between E19 and E21) still had90 and 52% higher stiffness than the NTC and sham,respectively. Additionally, the IOF-IM tibiae were 55%stiffer than the NTC. On d 38 the IOF-OMD tibiaewere 20% and 12% stiffer than the NTC and the sham,respectively. These observations were coupled with themeasurement of Young’s modulus (on d 38) that was 26to 27% higher than the NTC and sham (in the males).No differences between the groups were seen in the max-imal load and WTF.

Structural Properties The BV/TV, cortical area,medullary area, polar moment of inertia, and cross-sectional thickness of the tibial cortices are presentedin Figure 6. Generally there were only small differencesbetween the groups; however, between E19 and E21, themedullary area of the NTC, sham, and IOF-IM grew

Dow




Figure 3. Consumption of Cu, Mn, Zn (μg/day), and P (mg/day)from the yolk of the NTC, sham, IOF-OMD, and IOF-IM groups be-tween E19 and E21. 1These are numeric values calculated from themeans of each group on E19 and E21, so no statistical testing wasdone. 2At E17 the amniotic fluids of the IOF-OMD, IOF-IM, and shamgroups were injected with a solution of organic minerals, phosphate,and vitamin D3 (IOF-OMD); inorganic minerals and phosphate (IOF-IM); or the diluent of these solutions (sham). The NTC were non-treated controls.

by 60 to 70%, while in the IOF-OMD group it grew byonly 35%, leading to a smaller medullary area of theIOF-OMD on E21 in comparison to the NTC group.

The BV/TV, trabecular thickness and trabecularseparation of the trabecular bone are presented inTable 5. Differences were seen in BV/TV only on d38, but they were inconsistent; the sham showed re-duced BV/TV in females (in comparison to the NTCand IOF-OMD groups) and increased BV/TV in males(in comparison to the NTC group).

Standing Ability The average standing time of eachgroup in the LTL test is presented in Figure 7. No sta-tistical differences were seen among the groups.

DISCUSSION

This study demonstrates that embryonic enrichmentwith minerals and vitamin D3 by IOF has the poten-tial to improve bone quality as seen by increased ashcontent, whole-bone stiffness, and Young’s modulus.

Inserting minerals into the amniotic fluid elevatesyolk mineral content 36 h post IOF (E19). This shows atransfer of minerals from the amniotic fluid to the yolksac as was previously described (Yair and Uni, 2011),and can be explained by transfer of amniotic fluid to theyolk sac via the gastrointestinal tract and the vitellinediverticulum (Esteban et al., 1991).

Following this mineral enrichment by IOF methodol-ogy, the higher levels of Cu, Mn, and Zn in the yolksof the IOF-OMD and IOF-IM groups on E19 led toelevated consumption of those minerals from the yolkbetween E19 and E21. The negative consumption of Cuand Mn seen in the NTC and sham could be a result ofan entrance of Cu and Mn to the yolk from the otheregg compartments, such as the shell and amniotic fluid,which contain low amounts of Cu and Mn (Richards,1997; Yair and Uni, 2011).

On the other hand, no significant differences in thelevels and consumption of P were found. This might beexplained by the fact that the P amount in the in ovosolutions was only 2 to 2.2% of the initial yolk P amount(at E0), while the amounts of Cu, Mn, and Zn in theIOF solutions were more than 150% of their amountsin the yolk at E0.

Table 3. Body weights of the NTC, sham, IOF-OMD, and IOF-IM groups.1,2

Males Females

Sample day NTC Sham IOF-OMD IOF-IM NTC Sham IOF-OMD IOF-IM

Body weight (g) ∗E17 Mean 25.08 23.99SE 0.87 0.87

∗E19 Mean 34.00 33.05 32.53 33.93 33.07 32.92 30.10 32.48SE 1.09 1.09 0.81 1.03 0.81 0.81 1.09 1.03

$E21 Mean 44.59 45.09 45.40 44.44 44.61 44.24 45.02 48.44SE 0.56 0.63 0.61 0.99 0.53 0.47 0.50 1.33

#d 7 Mean 167.7 161.8 165.0 160.3 161.1 161.1SE 2.1 2.4 2.4 2.1 1.8 1.9

#d 14 Mean 436.5 431.9 435.3 407.4 407.8 409.6SE 5.6 6.6 6.3 5.5 4.7 5.1

#d 21 Mean 905.4 892.1 894.9 809.4 805.8 804.2SE 11.6 13.8 13.2 11.2 9.8 10.7

#d 28 Mean 1544 1547 1547 1319 1321 1304SE 18 21 20 17 1529 17

#d 38 Mean 2418 2447 2416 1983 1976 1964SE 29 35 33 28 25 27

1No statistical differences between the groups were found (P > 0.05.)2At E17 the amniotic fluids of the IOF-OMD, IOF-IM, and sham groups were injected with a solution of organic

minerals, phosphate, and vitamin D3 (IOF-OMD); inorganic minerals and phosphate (IOF-IM); or the diluent of thesesolutions (sham). The NTC were non-treated controls. The IOF-IM group was not raised after hatch (E21).

∗n = 8; $55 < n < 65 for the NTC, sham, and IOF-OMD groups; n = 15 for the IOF-IM group; #55 < n < 65.

Dow



2702 YAIR ET AL.

Table 4. Tibia weight and length of the NTC, sham, IOF-OMD, and IOF-IM groups.1,2

Males Females

Sample day NTC Sham IOF-OMD IOF-IM NTC Sham IOF-OMD IOF-IM

Bone weight (g) ∗E17 Mean 0.17 0.13SE 0.01 0.01

∗E19 Mean 0.27 0.27 0.27 0.24 0.25 0.27 0.24 0.26SE 0.02 0.02 0.01 0.02 0.01 0.01 0.02 0.02

∗E21 Mean 0.45 0.43 0.39 0.39 0.40 0.46 0.43 0.43SE 0.03 0.04 0.03 0.03 0.03 0.02 0.03 0.03

∗d 10 Mean 2.49 2.26 2.42 2.26 2.30 2.04SE 0.14 0.11 0.14 0.14 0.15 0.14

$d 38 Mean 19.90 19.87 19.08 14.54 14.42 15.14SE 0.51 0.41 0.40 0.36 0.32 0.35

Bone length (cm) ∗E17 Mean 2.32 2.28SE 0.07 0.07

∗E19 Mean 2.83 2.92 2.86 2.82 2.88 2.88 2.80 2.88SE 0.07 0.07 0.06 0.07 0.06 0.06 0.07 0.07

∗E21 Mean 3.16 3.07 3.02 3.16 3.06 3.15 3.13 3.18SE 0.04 0.05 0.03 0.03 0.03 0.03 0.03 0.04

∗d 10 Mean 5.07 4.94 4.96 4.95 5.02 4.93SE 0.07 0.06 0.07 0.07 0.08 0.07

$d 38 Mean 10.71 10.73 10.55 10.05 10.21 10.21SE 0.09 0.07 0.07 0.06 0.06 0.06

1No statistical differences between the groups were found (P > 0.05.)2At E17 the amniotic fluids of the IOF-OMD, IOF-IM, and sham groups were injected with a solution of organic

minerals, phosphate, and vitamin D3 (IOF-OMD); inorganic minerals and phosphate (IOF-IM); or the diluent of thesesolutions (sham). The NTC were non-treated controls. The IOF-IM group was not raised after hatch (E21).

∗n = 8; $n = 20.

Figure 4. Bone mineralization of the NTC, sham, IOF-OMD, and IOF-IM groups2 (n = 8). A) Ash content and B) bone mineral density3

(BMD). a-bSignificant differences among groups are marked with different superscripts (P < 0.05). 1Values are means ± SE of males and femalesthat did not differ. 2At E17 the amniotic fluids of the IOF-OMD, IOF-IM, and sham groups were injected with a solution of organic minerals,phosphate, and vitamin D3 (IOF-OMD); inorganic minerals and phosphate (IOF-IM); or the diluent of these solutions (sham). The NTC werenon-treated controls. The IOF-IM group was not raised after hatch (E21). 3BMD was measured on a section from the mid diaphysis of the tibiaby micro-computed tomography.

The amounts of most minerals in the egg cannotbe increased by increasing their concentration (in inor-ganic forms) in the maternal diet (Naber, 1979). There-fore, in recent years, work has focused on changing thechemical form of the minerals to organic forms dueto their ease of mobilization and use by the embryo,rather than increasing their concentrations in the hen

diet (Kidd et al., 1992; Hudson et al., 2004; Dobrzanskiet al., 2008; Favero et al., 2013; Oviedo-Rondon et al.,2013). It was previously shown that IOF with organicminerals, vitamin D3, and carbohydrates elevated min-eral levels and consumption from the yolk (Yair andUni, 2011). However, it is still unclear whether supple-menting the embryo with inorganic minerals also will

Dow




Figure 5. Bone mechanical properties of the NTC, sham, IOF-OMD, and IOF-IM groups2. A) Stiffness, B) maximal load, and C) WTF ofthe whole bone between E17 and d 38 (n = 8 for E17 to d 10, and n = 20 for d 38). D) Young’s modulus of the males and females on d 38(n = 5). a-c Significant differences among groups are marked with different superscripts (P < 0.05). For the Young’s modulus, significant interactionwas found between group and sex factors (P = 0.012). 1For A, B, and C, values are means ± SE of males and females. 2At E17 the amnioticfluids of the IOF-OMD, IOF-IM, and sham groups were injected with a solution of organic minerals, phosphate, and vitamin D3 (IOF-OMD);inorganic minerals and phosphate (IOF-IM); or the diluent of these solutions (sham). The NTC were non-treated controls. The IOF-IM groupwas not raised after hatch (E21). 3Stiffness, maximal load, and WTF were measured using a custom-built micro-mechanical testing device onE17 to d 10, while for d 38 they were measured using an Instron 3345 materials testing machine (Instron, Norwood, MA). Young’s modulus wascalculated using data obtained from micro indentations of mid diaphysis tibiae cross sections (by Future-Tech FM-300 micro hardness tester).

result in higher mineral content and utilization. In thiswork we also examined an IOF solution with just in-organic minerals (no vitamin D3 and carbohydrates) inorder to determine if inorganic minerals also can ele-vate yolk mineral content and consumption (regardlessof the chemical form of the added minerals or the ad-dition of vitamin D3). Our results show that mineralcontent and consumption of the IOF-OMD and IOF-IMgroups at E19 and E21 did not differ, which suggeststhat both organic and inorganic minerals have a similareffect on elevation of yolk mineral content. As the goalof the IOF-IM group was to determine if IOF with in-organic minerals can elevate yolk mineral content andconsumption, there was no need to grow the IOF-IM

chicks beyond the incubation period and, therefore, thistreatment group was not raised post-hatch.

The IOF-OMD treatment led to increased ash con-tent of mature broiler bones at d 38. As the mechani-cal performance of bones is determined by their struc-ture and composition (Weiner and Wagner, 1998; Shariret al., 2008), this increase in mineral density can explainthe increased stiffness and Young’s modulus (only inthe males) of the mature broilers from the IOF-OMDgroup.

On the other hand, with the exception of the changesin the medullary area at E21, there were very few differ-ences in geometry between the groups in this study andin a similar previous experiment (Yair et al., 2013); this

Dow



2704 YAIR ET AL.

Figure 6. Structural properties of the cortical bone of the NTC, sham, IOF-OMD, and IOF-IM groups1 (n = 8). A) Bone volume fraction(BV/TV), B) cortical area, C) medullary area, D) polar moment of inertia, and E) cross-sectional thickness2. a-bSignificant differences amonggroups are marked with different superscripts (P < 0.05). 1At E17 the amniotic fluids of the IOF-OMD, IOF-IM, and sham groups were injectedwith a solution of organic minerals, phosphate, and vitamin D3 (IOF-OMD); inorganic minerals and phosphate (IOF-IM); or the diluent of thesesolutions (sham). The NTC were non-treated controls. The IOF-IM group was not raised after hatch (E21). 2Structural properties of the corticalbone were measured by micro-computed tomography of the mid diaphysis.

observation suggests that bone geometry is conservedand almost unaffected by nutritional manipulations.

Interestingly, differences in Young’s modulus betweenthe IOF-OMD and the controls on d 38 were foundin the males, but not in the females. As broiler males

are much heavier (about 23% higher BW on d 38 ac-cording to Table 3) and, therefore, their bones have towithstand higher loads, this elevated Young’s modu-lus is of high importance. Similar results were foundby Bello et al. (2014), which showed that IOF of

Dow




Table 5. Structural properties of the trabecular bone of the NTC, sham, IOF-OMD, and IOF-IMgroups1 (n = 8). Bone volume fraction (BV/TV), trabecular thickness, and trabecular separation.2

Males Females

Sample day NTC Sham IOF-OMD NTC Sham IOF-OMD

BV/TV (%) D 10 Mean 37.48 37.70 37.91 41.03 40.47 38.91SE 1.89 1.49 1.89 1.89 2.00 1.89

∗d 38 Mean 41.45b 50.61a 45.11a,b 51.59x 45.98y 50.75x

SE 1.79 1.46 2.06 1.33 1.46 1.19Trabecular thickness (μm) d 10 Mean 0.10 0.11 0.11 0.11 0.10 0.11

SE 0.003 0.003 0.003 0.003 0.003 0.003d 38 Mean 0.21 0.22 0.21 0.22 0.21 0.20

SE 0.01 0.01 0.01 0.01 0.01 0.01Trabecular separation (μm) d 10 Mean 0.15 0.17 0.16 0.16 0.14 0.16

SE 0.01 0.01 0.01 0.01 0.01 0.01d 38 Mean 0.32 0.26 0.29 0.25 0.30 0.24

SE 0.03 0.02 0.03 0.02 0.02 0.02

a–bSignificant differences between groups within the males are marked with different letters (P < 0.05).x–ySignificant differences between groups within the females are marked with different letters (P < 0.05).∗Significant interaction between group and sex factors (P = 0.001).1At E17 the amniotic fluids of the IOF-OMD and sham groups were injected with a solution of organic

minerals, phosphate, and vitamin D3 (IOF-OMD) or the diluent of this solution (sham). The NTC were non-treated controls. The IOF-IM were not raised after hatch (E21).

2Structural properties of the trabecular bone were measured by micro-computed tomography of the distalepiphyseal area.

Figure 7. Latency to lie test1,2 (LTL) results (n = 12): Averagetime to sitting on d 38. 1Values are means ± SE of males and females.No statistical differences were found (P > 0.05). 2At E17 the amnioticfluids of the IOF-OMD and sham groups were injected with a solutionof organic minerals, phosphate, and vitamin D3 (IOF-OMD) or thediluent of this solution (sham). The NTC were non-treated controls.3LTL was measured by placing each broiler in a bath with shallowwater and measuring the time until sitting.

25-hydroxylcholecalceiferol (25-OH vitamin D3) led toelevated bone-breaking strength on d 28 post-hatch inmales but not in females. Yalcin et al. (2001) showedthat between d 16 and 48 post-hatch (but not ear-lier) males exhibit higher bone weight, width, and vol-ume than females, hinting at accelerated bone growth.Accordingly, it can be hypothesized that the benefits ofthe IOF procedure are more substantial for males onlyduring that increased bone growth rate period.

On E19, E21, and d 10, no differences in ash contentwere observed among the groups, and therefore, the

elevated bone stiffness of both IOF groups at hatchcannot be explained by differences in mineral content.Similar results showing increased bone mechanicalproperties at hatch were reported previously in embryosthat had undergone IOF with minerals, vitamins, andcarbohydrates and explained by a reduced medullaryarea (Yair et al., 2013). Such reduction in the medullaryarea at hatch might explain the elevated stiffness of theIOF-OMD group, but not of the IOF-IM group whosemedullary area did not differ from the control groups.

Changes in the medullary area are probably due todifferences in endosteal bone resorption rate; betweenE19 and E21, the medullary area of the NTC groupincreased at a much higher rate than the IOF-OMD(but not the IOF-IM), hinting at a higher resorptionrate of the NTC. A similar phenomenon of a higherendosteal resorption rate towards birth was found inmice, and it led to reduced bone mineral density (Shariret al., 2013); however, such reduction was not seen inthe current work. The difference in the medullary area(and bone resorption rate) seen in the chicks in thisstudy probably should be attributed to one of the in-gredients of the IOF-OMD solution, e.g., vitamin D3,phosphate, or the mineral-amino acid complexes of theorganic minerals. The authors are not aware of anyevidence to an effect of phosphate and organic min-erals on bone resorption rate. Vitamin D3 was foundto affect bone properties of broiler embryos: IOF of25-hydroxylcholecalceiferol led to higher bone-breakingstrength in male birds on d 28 post-hatch (Bello et al.,2014), while maternal supplementation with vitaminD3 or 25-hydroxylcholecalceiferol increased tibia ashpercent of progenies at hatch (Atencio et al., 2005).Although vitamin D also was found to have the abilityto suppress bone resorption (Ikeda and Ogata, 1999),

Dow



2706 YAIR ET AL.

there are contradictory reports, e.g., the addition of theactive form of vitamin D, calcitriol, to vitamin D defi-cient embryos on E14 elevated bone resorption betweenE14 and E17 (Narbaitz and Tsang, 1989). Thus, al-though it is possible that vitamin D3 is responsible forthe changes in the medullary area (and bone resorptionrate), more research is needed to conclude which com-pound is responsible for the changes in the medullaryarea on E21.

Similar to what was previously reported (Yair et al.,2013), in this work the effect of IOF with minerals andvitamin D3 can be seen in 2 periods: A rapid response(elevated stiffness and reduced medullary area) can beseen as early as E19 (36 h after the enrichment proce-dure) and E21, and a longer lasting effect (exhibited bythe increased ash content, stiffness, and Young’s modu-lus) in the mature broiler (d 38). Between these 2 peri-ods, on d 10 the control groups (NTC and sham) caughtup with all parameters, possibly due to compensatoryresponse (catch-up growth) as was previously seen (Yairet al., 2013).

Although the IOF treatments led to changes in min-eral metabolism and bone mechanical, structural, andcompositional (ash content) properties, there were nosignificant differences in BW, as well as bone lengthand weight. Previous work with the IOF method hasshown that IOF can lead to elevated BW of the youngand mature broilers (Tako et al., 2004; Kornasio et al.,2011); however, these IOF solutions contained carbo-hydrates and β-hydroxy-β-methylbutyrate (a leucinemetabolite), which were directed to support intestinaldevelopment and breast muscle growth, while in thiswork the IOF solutions were aimed at supporting theskeleton and, therefore, contained minerals and vitaminD3, but no carbohydrates.

Although there was an increase in some bone mechan-ical properties as a result of IOF, it was insufficient toinduce changes in broiler standing ability, suggestingthat the selected IOF-OMD solution does not have asignificant positive effect on leg problems in broilers.It should be noted that, although walking ability andlameness of broilers are influenced by their bone proper-ties, they also are influenced by the muscles, cartilages,joints, and tendons (Berg and Sanotra, 2003; Ruiz-Feriaet al., 2014). Therefore, an IOF solution that aims toimprove the cartilage, joints, and tendons of broilersmight more effectively improve their walking ability.

In summary, here we show that IOF leads to in-creased mineral content of the yolk and embryonic con-sumption of minerals, as well as changes in the composi-tion and mechanical properties of the skeleton, but doesnot have positive functional effects, such as improvedstanding ability.

REFERENCES

Angel, R. 2007. Metabolic disorders: limitations to growth of andmineral deposition into the broiler skeleton after hatch and po-tential implications for leg problems. J. Appl. Poult. Res. 16:138–149.

Atencio, A., G. M. Pesti, and H. M. Edwards Jr. 2005. Twenty-five hydroxycholecalciferol as a cholecalciferol substitute in broilerbreeder hen diets and its effect on the performance and generalhealth of the progeny. Poult. Sci. 84:1277–1285.

Bello, A., P. Y. Hester, P. D. Gerard, W. Zhai, and E. D.Peebles. 2014. Effects of commercial in ovo injection of 25-hydroxycholecalciferol on bone development and mineralizationin male and female broilers. Poult. Sci. 93:2734–2739.

Berg, C., and G. S. Sanotra. 2003. Can a modified latency-to-lietest be used to validate gait-scoring results in commercial broilerflocks? Anim. Welfare. 12:655-659.

Bonser, R. H. C., and A. Casinos. 2003. Regional variation in corticalbone properties from broiler fowl–A first look. Br. Poult. Sci.44:350–354.

Caskey, C. D., L. C. Norris, and G. F. Heuser. 1944. A chronic con-genital ataxia in chicks due to manganese deficiency in the ma-ternal diet. Poult. Sci. 23:516–520.

Dibner, J. J., J. D. Richards, M. L. Kitchell, and M. A. Quiroz. 2007.Metabolic challenges and early bone development. J. Appl. Poult.Res. 16:126–137.

Dinev, I., S. A. Denev, and F. W. Edens. 2012. Comparative clini-cal and morphological studies on the incidence of tibial dyschon-droplasia as a cause of lameness in three commercial lines ofbroiler chickens. J. Appl. Poult. Res. 21:637–644.

Dobrzanski, Z., K. Mariusz, C. Katarzyna, G. Henryk, and O. Se-bastian. 2008. Influence of organic forms of copper, manganeseand iron on bioaccumulation of these metals and zinc in layinghens. J. Elementol. 13:309–319.

Esteban, S., J. Rayo, M. Moreno, M. Sastre, R. V. Rial, and J. A.Tur. 1991. A role played by the vitelline diverticulum in the yolksac resorption in young post-hatched chickens. J. Comp. Physiol.B. 160:645–648.

Evans, G. P., J. C. Behiri, J. D. Currey, and W. Bonfield. 1990.Microhardness and Young’s modulus in cortical bone exhibitinga wide range of mineral volume fractions, and in a bone analogue.J. Mater. Sci: Mater. Med. 1:38–43.

Favero, A., S. L. Vieira, C. R. Angel, A. Bos-Mikich, N. Lothhammer,D. Taschetto, R. F. A. Cruz, and T. L. Ward. 2013. Developmentof bone in chick embryos from Cobb 500 breeder hens fed dietssupplemented with zinc, manganese, and copper from inorganicand amino acid-complexed sources. Poult. Sci. 92:402–411.

Groves, P. J., and W. I. Muir. 2014. A meta-analysis of experimentslinking incubation conditions with subsequent leg weakness inbroiler chickens. PLoS ONE 9:e102682.

Havenstein, G. B., P. R. Ferket, and M. A. Qureshi. 2003. Growth,livability, and feed conversion of 1957 versus 2001 broilers whenfed representative 1957 and 2001 broiler diets. Poult. Sci. 82:1500–1508.

Hudson, B. P., W. A. Dozier, III, J. L. Wilson, J. E. Sander, andT. L. Ward. 2004. Reproductive performance and immune statusof caged broiler breeder hens provided diets supplemented witheither inorganic or organic source of zinc from hatching to 65 wkof age. J. Appl. Poult. Res. 13:349–359.

Ikeda, K., and E. Ogata. 1999. The effect of vitamin D on osteoblastsand osteoclasts. Curr. Opin. Orthop. 10:339–343.

Julian, R. J. 1998. Rapid growth problems: ascites and skeletal de-formities in broilers. Poult. Sci. 77:1773–1780.

Kapell, D. N. R. G., W. G. Hill, A.-M. Neeteson, J. McAdam, A.N. M. Koerhuis, and S. Avendano. 2012. Twenty-five years ofselection for improved leg health in purebred broiler lines andunderlying genetic parameters. Poult. Sci. 91:3032–3043.

Kidd, M. T., N. B. Anthony, and S. R. Lee. 1992. Progeny perfor-mance when dams and chicks are fed supplemental zinc. Poult.Sci. 71:1201–1206.

Knowles, T. G., S. C. Kestin, S. M. Haslam, S. N. Brown, L. E.Green, A. Butterworth, S. J. Pope, D. Pfeiffer, and C. J. Nicol.2008. Leg disorders in broiler chickens: Prevalence, risk factorsand prevention. PLoS ONE 3:e1545.

Kornasio, R., O. Halevy, O. Kedar, and Z. Uni. 2011. Effect of in ovofeeding and its interaction with timing of first feed on glycogenreserves, muscle growth, and body weight. Poult. Sci. 90:1467–1477.

Langley-Evans, S. C. 2015. Nutrition in early life and the program-ming of adult disease: A review. J. Hum. Nutr. Diet. 28:1–14.

Dow




Lanyon, L. E., A. E. Goodship, C. J. Pye, and J. H. MacFie. 1982.Mechanically adaptive bone remodelling. J. Biomech. 15:141–154.

Lucas, A. 1991. Programming by early nutrition in man. Pages 38–55in Ciba foundation symposium 156 - The childhood environmentand adult disease. John Wiley & Sons, Ltd.

Matthiesen, C. F., D. Blache, P. D. Thomsen, N. E. Hansen, andA-H. Tauson. 2010. Effect of late gestation low protein supplyto mink (Mustela vison) dams on reproductive performance andmetabolism of dam and offspring. Arch. Anim. Nutr. 64:56–76.

Moran, E. T. 2007. Nutrition of the developing embryo and hatch-ling. Poult. Sci. 86:1043–1049.

Naas, I. A., I. C. L. A. Paz, M. S. Baracho, A. G. Menezes, L. G.F. Bueno, I. C. L. Almeida, and D. J. Moura. 2009. Impact oflameness on broiler well-being. J. Appl. Poult. Res. 18:432–439.

Naber, E. C. 1979. The effect of nutrition on the composition of theegg. Poult. Sci. 58:518–528.

Narbaitz, R., and C. P. W. Tsang. 1989. Vitamin D deficiency in thechick embryo: Effects on prehatching motility and on the growthand differentiation of bones, muscles, and parathyroid glands.Calcif. Tissue Int. 44:348–355.

Oviedo-Rondon, E. O., J. Small, M. J. Wineland, V. L. Christensen,P. S. Mozdziak, M. D. Koci, S. V. L. Funderburk, D. T. Ort,and K. M. Mann. 2008. Broiler embryo bone development isinfluenced by incubator temperature, oxygen concentration andeggshell conductance at the plateau stage in oxygen consumption.Br. Poult. Sci. 49:666–676.

Oviedo-Rondon, E. O., N. M. Leandro, R. Ali, M. Koci, V. Moraes,and J. Brake. 2013. Broiler breeder feeding programs and traceminerals on maternal antibody transfer and broiler humoral im-mune response. J. Appl. Poult. Res. 22:499–510.

Packard, M. J., and G. C. Packard. 1991. Patterns of mobilizationof calcium, magnesium, and phosphorus by embryonic yellow-headed blackbirds (Xanthocephalus Xanthocephalus). J. Comp.Physiol. B. 160:649–654.

Prisby, R., T. Menezes, J. Campbell, T. Benson, E. Samraj, I.Pevzner, and R. F. Wideman. 2014. Kinetic examination offemoral bone modeling in broilers. Poult. Sci. 93:1122–1129.

Richards, M. P., and M. J. Packard. 1996. Mineral metabolism inavian embryos. Poult. Avian Biol. Rev. 7:143–161.

Richards, M. P. 1997. Trace mineral metabolism in the avian embryo.Poult. Sci. 76:152–164.

Ruiz-Feria, C. A., J. J. Arroyo-Villegas, A. Pro-Martinez, J.Bautista-Ortega, A. Cortes-Cuevas, C. Narciso-Gaytan, A.Hernandez-Cazares, and J. Gallegos-Sanchez. 2014. Effects ofdistance and barriers between resources on bone and tendonstrength and productive performance of broiler chickens. Poult.Sci. 93:1608–1617.

Sharir, A., J. Milgram, G. Dubnov-Raz, E. Zelzer, and R. Shahar.2013. A temporary decrease in mineral density in perinatal mouselong bones. Bone. 52:197–205.

Sharir, A., M. M. Barak, and R. Shahar. 2008. Whole bone mechan-ics and mechanical testing. Vet. J. 177:8–17.

Shim, M. Y., A. B. Karnuah, A. D. Mitchell, N. B. Anthony, G. M.Pesti, and S. E. Aggrey. 2012. The effects of growth rate on legmorphology and tibia breaking strength, mineral density, mineralcontent, and bone ash in broilers. Poult. Sci. 91:1790–1795.

Smith, N. A., F. M. McAuliffe, K. Quinn, P. Lonergan, and A. C.O. Evans. 2010. The negative effects of a short period of mater-nal undernutrition at conception on the glucose-insulin system ofoffspring in sheep. Anim. Reprod. Sci. 121:94–100.

Sullivan, T. W. 1994. Skeletal problems in poultry: Estimated annualcost and descriptions. Poult. Sci. 73:879–882.

Tako, E., P. R. Ferket, and Z. Uni. 2004.Effects of in ovofeeding of carbohydrates and beta-hydroxy-beta-methylbutyrateon the development of chicken intestine. Poult. Sci. 83:2023–2028.

Thorp, B. H. 1994. Skeletal disorders in the fowl: A review. AvianPathol. 23:203–236.

Tona, K., O. M. Onagbesan, Y. Jego, B. Kamers, E. Decuypere, andV. Bruggeman. 2004. Comparison of embryo physiological param-eters during incubation, chick quality, and growth performance ofthree lines of broiler breeders differing in genetic composition andgrowth rate. Poult. Sci. 83:507–513.

Tygesen, M. P., A. H. Tauson, D. Blache, S. M. Husted, andM. O. Nielsen. 2008. Late foetal life nutrient restriction andsire genotype affect postnatal performance of lambs. Animal.4:574–581.

Uni, Z., and P. R. Ferket. 2003. Enhancement of development ofoviparous species by in ovo feeding. North Carolina State Uni-versity; Yissum research development company of the HebrewUniversity of Jerusalem, Jerusalem, Israel , assignee. Pat. No. US6592878.

Uni, Z., and R. P. Ferket. 2004. Methods for early nutrition and theirpotential. World’s Poult. Sci. J. 60:101–111.

Uni, Z., L. Yadgary, and R. Yair. 2012. Nutritional limitations duringpoultry embryonic development. J. Appl. Poult. Res. 21:175–184.

Van der Pol, C. W., I. A. M. Van Roovert-Reijrink, C. M. Maatjens,I. Van den Anker, B. Kemp, and H. Van den Brand. 2014. Effect ofeggshell temperature throughout incubation on broiler hatchlingleg bone development. Poult. Sci. 93:2878–2883.

Vieira, S. L., and C. R. Angel. 2012. Optimizing broiler performanceusing different amino acid density diets: What are the limits? J.Appl. Poult. Res. 21:149–155.

Weeks, C. A. 2001. Development of a new method for quantitativelyassessing lameness in broilers. Br. Poult. Sci. 42:S79–S80.

Wideman, R. F., Jr., A. Al-Rubaye, A. Gilley, D. Reynolds, H.Lester, D. Yoho, J. M. Hughes, and I. Pevzner. 2013. Suscepti-bility of 4 commercial broiler crosses to lameness attributable tobacterial chondronecrosis with osteomyelitis. Poult. Sci. 92:2311–2325.

Weiner, S., and H. D. Wagner. 1998. The material bone: Structuremechanical function relations. Annu. Rev. Mater. Sci. 28:271–298.

Williams, B., S. Solomon, D. Waddington, B. Thorp, and C. Far-quharson. 2000. Skeletal development in the meat-type chicken.Br. Poult. Sci. 41:141–149.

Wilson, H. R. 1997. Effects of maternal nutrition on hatchability.Poult. Sci. 76:134–143.

Yair, R., R. Shahar, and Z. Uni. 2013. Prenatal nutritional manipula-tion by in ovo enrichment influences bone structure, composition,and mechanical properties. J. Anim. Sci. 91:2784–2793.

Yair, R., and Z. Uni. 2011. Content and uptake of minerals in theyolk of broiler embryos during incubation and effect of nutrientenrichment. Poult. Sci. 90:1523–1531.

Yair, R., Z. Uni, and R. Shahar. 2012. Bone characteristics of late-term embryonic and hatchling broilers: Bone development underextreme growth rate. Poult. Sci. 91:2614–2620.

Yalcin, S., S. Ozkan, E. Coskuner, G. Bilgen, Y. Delen, Y. Kur-tulmus, and T. Tanyalcin. 2001. Effects of strain, maternal ageand sex on morphological characteristics and composition of tibialbone in broilers. Br. Poult. Sci. 42:184–190.

Dow



in ovo feeding with minerals and vitamin d3 improves bone

Documents