Medullary bone attributes in aged Lohmann LSL-lite …...For Review Only Medullary bone attributes in aged Lohmann LSL-lite layers fed different levels of calcium and top-dressed 25-hydroxy

For Review Only

Medullary bone attributes in aged Lohmann LSL-lite layers

fed different levels of calcium and top-dressed 25-hydroxy vitamin D3

Journal: Canadian Journal of Animal Science

Manuscript ID CJAS-2018-0062.R1

Manuscript Type: Article

Date Submitted by the Author: 20-Jun-2018

Complete List of Authors: Akbari Moghaddam Kakhki, Reza; University of Guelph, Animal Bioscience

Heuthorst , Thomas; University of Guelph, Department of Animal Biosciences Wornath - Vanhumbeck, Alisha; University of Guelph, Department of Animal Biosciences Neijat, mohamed; University of Guelph, Animal Biosciences Kiarie, Elijah; University of Guelph, Department of Animal Biosciences

Keywords: Aged hens, Calcium, 25-hydroxyvitamin-D3, Bone health

https://mc.manuscriptcentral.com/cjas-pubs

Canadian Journal of Animal Science

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Calcium and 25-OH vitamin D3 for aged hens - Akbari Moghaddam Kakhki et al.

Medullary bone attributes in aged Lohmann LSL-lite layers fed different levels of calcium

and top-dressed 25-hydroxy vitamin D3

R. Akbari Moghaddam Kakhki, T. Heuthorst, A. Wornath-Vanhumbeck, M. Neijat and E.

Kiarie*1

Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada.

*Corresponding author: [email protected]

1 Presented in part at the 2018 ASAS-CSAS Annual meeting & Trade show, July 8-12, 2018, Vancouver, BC.

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R. Akbari Moghaddam Kakhki, T. Heuthorst, A. Wornath-Vanhumbeck, M. Neijat and E.

Kiarie. Medullary bone attributes in aged Lohmann LSL-lite layers fed different levels of

calcium and top-dressed 25-hydroxy vitamin D3. Can. J. Anim. Sci. 98: XXX-XXX. Structural

bone depletion over the course of lay cycle predisposes hens to skeletal problems. We

investigated the effects of dietary Ca and top-dressed 25-hydroxy vitamin D3 (25OHD3) on

attributes (relative weight, ash content (AC) and concentration (ACN)) in whole ulna, femur,

tibia and sub-parts of femur and tibia (epiphysis, medullary and cortical) in 74-wk old Lohmann

LSL-lite layers. Four levels of Ca (3.0, 3.5, 4.0 and 4.5%) and three levels of 25OHD3 (0, 69 and

138 µg/kg) were tested. All diets had basal level of 3,300 IU of vitamin D3/kg. Eighty-four, 74-

wk old hens were placed in individual cages and 13 spare hens sacrificed for baseline samples.

Diets (n=7) were fed to 81-wks of age and hens sacrificed for bone samples. There was no (P >

0.05) diet effects on whole bone attributes. Interaction (P < 0.05) between Ca and 25OHD3 on

femur sub-parts was such that 25OHD3 linearly increased medullary ACN and concomitantly

decreased cortical ACN at all Ca levels. In tibia, 25OHD3 (P < 0.05) increased AC and ACN in

medullary and reduced these parameters in cortical. The results suggested that sub-parts and not

whole medullary bone attributes are more amenable to dietary interventions in aged hens.

Keywords: Aged hens, calcium, 25-hydroxyvitamin-D3, bone health

Abbreviations used: AC, ash content; ACN, ash concentration; 25OHD3, 25-hydroxy vitamin

D3; SRW, relative weight of sub-part to the whole bone; TBRW, total bone relative weight of

the whole bone to body weight; CI, change index.

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Introduction

Egg is one the most affordable source of animal proteins in Canada and contributes

significantly to the national economy. To meet demand, genetic selection and improvements in

nutrition and management have led to dramatic increases in productivity, sustainability and

profitability of Canadian egg industry (Egg Farmers of Canada 2018). However, the modern hen

experiences decrease in the amount of fully mineralized structural bone as the lay cycle

progresses leading to high incidences of osteoporosis, an important welfare, health, and

economic challenge for the layer industry (Kim et al. 2007) and associated with 20-35% of all

mortality and depopulation in cage housing systems (Whitehead and Fleming 2000).

The balance between intestinal Ca absorption, renal excretion and bone mineral

metabolism maintains Ca homeostasis to meet Ca requirements for eggshell (Elaroussi et al.

1994). Eggshell formation requires about 10% Ca from skeletal reserves (Gilbert 1983). In

modern layers, this daily bone remodeling for eggshell formation occurs more than 300 times

during the laying cycle which represent 900 grams of Ca from the hen skeletal system in her

lifespan (Anderson et al. 2013). Thus, mineral reservoirs in bones and Ca intake are critical for

optimal eggshell and maintenance of a healthy skeletal system. Shell calcification occurs mainly

during dark period when hens have little or no access to feed (de Matos 2008). Calcium

requirements are higher during eggshell formation than any other stage of hen life cycle (Leeson

and Summers, 2000), hence, at sexual maturity, lamellar cortical bone changes to non-structural

medullary bone in response to the rise in estrogen (de Matos 2008), synchronized with Ca intake

(Kim et al. 2007) and acts as a labile source of Ca (Whitehead 2004). Regulation of Ca

homeostasis in avian is mainly due to the interrelation of vitamin D3, parathyroid and sex

hormones on target organs including liver, kidney, gastrointestinal tract, and bone. Extensive

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research has been conducted on the impact of dietary Ca and vitamin D3 on bone health (Kaur et

al. 2013; Keshavarz 2003). The vital role of vitamin D as a calcitropic hormone is associated

with regulation of Ca metabolism (Sanders and Edwards Jr 1991) and largely supplied in poultry

diets in form of cholecalciferol (Rath et al. 2000). Following cholecalciferol absorption in the

intestine, conversion to 25-hydroxycholecalciferol (25OHD3) occurs in liver. The 25OHD3 is

the most active precursor of 1,25-dihydroxycholecalciferol, which is generated by hydroxylation

at carbon 1 of 25OHD3 in the kidney (Do Nascimento et al. 2014). Hence, supplementation of

vitamin D in form of 25OHD3 might be more efficient in bolstering Ca metabolism compared

with cholecalciferol since it can bypass the first stage of conversion in the liver (Keshavarz

2003).

Genetic progress in the last four decades has created “long life and productive” layers with

lower feed consumption, smaller body size, earlier sexual maturity and higher egg production

(Anderson et al. 2013). However, the daily Ca requirement recommended by NRC reduced from

3.85 g of Ca /hen/d (NRC, 1984) to 3.25 g of Ca /hen/d in the latest NRC edition (NRC, 1994).

Furthermore, the hen ability to absorb Ca from the intestine decreases with age (Keshavarz and

Nakajima, 1993). Subsequently, further studies should evaluate whether Ca and vitamin D

nutrition affect bone mineral content in medullary bones in late lay cycle. The objective of the

current study is to investigate effects of different dietary levels of Ca and top dressed 25OHD3

on weight, ash content and concentration in whole ulna, femur, tibia and sub-parts of tibia and

femur including epiphysis, medullary and cortical in 74-wk old Lohmann LSL-lite layers.

Materials and Methods

Birds and management

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The experimental protocol (#3634)was approved by the University of Guelph Animal

Care Committee and birds were cared for in accordance with the Canadian Council on Animal

Care guidelines (CCAC 2009).

Experimental diets

The diets were formulated to meet or exceed specifications of Lohmann LSL-lite

(Lohman 2016) with exception of Ca (Table 1). Four levels of Ca (3.0, 3.5, 4.0 and 4.5%) and

three levels of top-dressed 25OHD3 (0, 69 and 138 µg/kg) were tested. Sample of 25OHD3

(1.25% 25OHD3, Hy-D®) was provided by DSM Nutritional Products Ayr, ON, Canada.

Limestone was included at the ratio of 5.7:1 (wt/wt) coarse (≥2 mm) and fine (<2 mm) particle

size. All diets had a basal level of 3,300 IU of cholecalciferol/kg of feed.

Experimental procedures and sampling

A total of 97, 72-wk old Lohmann LSL-lite hens were randomly selected from the

University of Guelph Arkell Poultry Station flock. The hens were maintained in conventional

cages (5-6 hens per cage) prior to selection for the current study. Birds were moved to individual

cages (45.72 cm deep, 41.91 cm high in the back, 45.72 cm high at the front and 25.4 cm wide).

Initially, the birds were fed a regular layer mash for two wks period for adaptation to cages and

for recording egg production data as the basis for experimental diets allocation. The room was

environmentally controlled (20oC) with lighting program of 14-h (incandescent, 15 lux, 06:00 to

19:00 h) and 10-h of dark. Following a 2-wk adaptation period, hens were allocated to 12

treatments in a completely randomized design to give 7 replicates per treatment. The 13 spare

birds were sacrificed for baseline left tibia, femur and ulna samples. Birds had free access to feed

and water up to the end of 81-wk of age. All birds were palpated in early hours (04:00 to 06:00 h,

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pre-oviposition) prior to lighting at 06:00 h within the last 4 d of the experiment and hens found

with a hard shell in the shell gland were sacrificed. Left tibia, femur and ulna samples were

dissected, defleshed and stored at -20˚C for further analysis.

Sample processing and analyses

Feed samples were submitted to the commercial lab (SGS, Guelph, ON, Canada) for

determination of Ca (method 985.01) concentration (AOAC 1995). Bone sub-parts were

separated following according to Clunies et al. (1992) with some modifications. A standard

assessment was developed to specify epiphysis region with consideration of possible differences

between bone sizes (Figure 1). Briefly, distal epiphysis was from 3.2 mm at the endpoint of

curvature. For proximal epiphysis, since there was no curvature or observable markings (in tibia

or femur), noticeable broken angle from left or right side (in way that live bird stands) was

considered as starting point and 6.4 mm after this point was considered as edge of proximal

epiphysis. Diaphysis was cut longitudinally and moist medullary bone removed by scraping with

aid of scalpel and the remainder designated cortical. The epiphysis, cortical and medullary

sections were dried at 105˚C for 24 h, weighed to calculate relative weight of sub-part to the

whole bone (SRW) and total whole bone weight to live body weight (TBRW). Subsequently,

dried sub-parts were ashed at 600˚C for 12 h (Jin et al. 2001) and reweighed for measuring ash

content (AC) and ash concentration (ACN) in sub-part and total bone.

Calculations and statistical analyses

Calculations for TBRW, SRW, and ACN in sub-parts and the total bone were as follows:

TBRW =Totalbonedriedweight(g)

Bodyweight(g)× 100

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SRW =Subpartdriedweight(g)

Totalbonedriedweight(g)× 100

ACNoftotalbone =Totalboneashcontent(g)

Totalbonedriedweight(g)× 100

ACNofsubpart =Sub − partashcontent(g)

Subpartdriedweight(g)× 100

To demonstrate how dietary treatments modified bone attributes through the lay cycle,

the measured bones attributes (total bone and sub-parts) at 81-wk were divided by baseline

values and multiplied by 100 and reported as a change index (CI). Data were subjected to GLM

procedures of SAS with Ca, 25OHD3 and interaction being the fixed effects. Contrast

coefficients for linear and quadratic effects of Ca and 25OHD3 were generated using the

interactive matrix language of SAS (Snedecor and Cochran 1980). Significance was declared at

P < 0.05.

Results

Diets and feed intake

Average daily feed intake was not influenced by the interaction between Ca and 25OHD3 or

main effects (Data are not shown). Based on feed intake and assayed Ca, Ca intake was 3.4, 4.1,

4.2 and 4.8 g of Ca/bird (b)/d for birds fed diets calculated to contain 3.0, 3.5, 4.0 and 4.5 % Ca,

respectively.

Bone attributes at 74-wk of age

Attributes of ulna, femur and tibia measured at 74-wk of age are presented in Table 2. The

TBRW was 0.102, 0.228 and 0.262% for ulna, femur and tibia respectively. Ash content values

for ulna, femur and tibia was 0.754, 2.23 and 2.76 g, respectively. Corresponding values for

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ACN were 43.48, 55.83 and 61.57% for ulna, femur and tibia, respectively. The RW, AC and

ACN of femur medullary were 17.50%, 0.38 g, 45.98% and corresponding values for cortical

were 35.70%, 0.94 g and 67.22%, respectively (Table 2). The values of RW, AC and ACN of

tibia medullary were 15.83%, 0.33 g, 47.48% and corresponding values for cortical were

46.36%, 1.48 g and 71.05%, respectively.

Whole bone attributes and change index

There was no interaction (P>0.05) between Ca and 25OHD3 or their main effect on

TBRW, AC and ACN of ulna, femur and tibia at 81-wk of age (Table 3). Averaged TBRW, AC

and ACN of ulna were 0.114%, 0.78 g and 41.97%, respectively. The TBRW, AC and ACN

values of femur were 0.243 %, 2.08 g and 52.56 %, respectively and corresponding TBRW, AC

and ACN values for tibia were 0.274 %, 2.58 g and 58.28%, respectively.

There was no interaction (P>0.05) between Ca and 25OHD3 or main effect of Ca (P>0.05)

on CI of TBRW, AC and ACN of ulna, femur and tibia (Table 4). Supplementation of 25OHD3

up to 69 µg/kg linearly decreased (P=0.001) CI value of ulna TBRW. Further increase in the

supplemental level of 25OHD3 decreased (P<0.05) CI of AC and ACN of ulna. Feeding

25OHD3 had no effect on CI of femur and tibia (P>0.05). However, there was tendency for

quadratic and linear change in CI of femur AC in response to feeding Ca (P=0.063) and

25OHD3 (P=0.090), respectively.

Sub-parts attributes and change index

Ash concentration in femur medullary and cortical was affected by the interactive effect

of 25OHD3 and Ca (P<0.05; Table 5). Supplemental 25OHD3 at each level of Ca linearly

increased ACN of medullary part and concomitantly decreased cortical ACN. However, the

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interaction between Ca and 25OHD3 did not influence SRW and AC of femur sub-parts

(P>0.05). There was no Ca effect (P>0.05) on SRW, AC and ACN of femur sub-parts. Feeding

25OHD3 at highest level decreased (P<0.001) SRW of femur cortical in a linear manner.

Increasing 25OHD3 tended to increase RW of femur medullary (P=0.071). Supplementation of

25OHD3 linearly increased (P=0.013) AC of femur medullary in concomitant with a linear

reduction in AC of femur cortical (P=0.004).

There was an interaction between Ca and 25OHD3 (P<0.05) on CI of SRW, AC and ACN

values of femur medullary and cortical (Table 6). This interactive effect was such that femur

medullary CI values of SRW, AC and ACN increased in a linear fashion with 25OHD3 at each

level of Ca. Concomitantly, SRW, AC and ACN values of femur cortical decreased in response

to incremental level of 25OHD3 at each level of Ca. Change index of SRW, AC and ACN of

femur epiphysis were neither affected by the interaction between Ca and 25OHD3 nor their main

effects (P>0.05). However, feeding 25OHD3 tended to increase (P= 0.085) CI of AC in femur

epiphysis.

Neither interaction between Ca and 25OHD3 nor the Ca main effect changed SRW, AC

and ACN of tibia sub-parts (P>0.05, Table 7). Supplementation of 25OHD3 at the highest level

led to an increase in SRW, AC and ACN (P<0.05) of tibia medullary. Concomitantly, feeding

25OHD3 at the highest level decreased SRW, AC and ACN (P<0.05) of tibia cortical. However,

tibia epiphysis attributes were not affected (P>0.05) by top-dressing 25OHD3.

There was no interaction (P>0.05) between Ca and 25OHD3 on CI values of tibia sub-parts

(Table 8). However, interactive effect of Ca and 25OHD3 tended to influence CI of medullary

SRW (P=0.090) and ACN (P=0.076). Change index for tibia medullary SRW linearly increased

(P=0.002) in response to increasing Ca from 3.4 to 4.1 g/b/d. Feeding 250HD3 linearly increased

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CI of tibia medullary SRW with the highest 25OHD3 intake resulting in 14.7% (P<0.001) but

linearly reduced CI of tibia cortical SRW by 3.7% (P=0.001). The SRW of cortical and epiphysis

was not influenced by levels of Ca (P>0.05). Change index of medullary AC was linearly

increased by levels of Ca (P=0.005) and 25OHD3 (P<0.001; Table 8). In addition, corresponding

CI value of tibia epiphysis was decreased (P<0.001) by Ca intake over 4.2 g/b/d. Feeding

25OHD3 led to 8.7% reduction (P= 0.001) in CI of cortical AC value. The CI of medullary and

epiphysis ACN was reduced (P<0.05) by Ca intake over 4.2 g/b/d. Supplementation of 25OHD3

led to increase (P<0.001) in CI of AC by 12.6% in tibia medullary accompanied by reduction

(P<0.001) in corresponding value of cortical by 3.3 %. The CI value of ACN of tibia medullary

was not affected by levels of Ca (P>0.05) and corresponding value of tibia epiphysis was not

influenced by feeding 25OHD3 (P>0.05).

Discussion

Progressive deterioration of structural bone in the course of lay cycle increases

susceptibility to fractures and osteoporotic mortality (Whitehead 2004). Optimal Ca nutrition is

critical for maintaining bone strength (Cheng and Coon 1990). However, in the current study, Ca

intake of 3.4 to 4.8 g/b/d did not affect TBRW, AC and ACN of ulna, femur and tibia bones.

Similarly Ca intake of 3.5 to 4.3 g/b/d had no impact on tibia weight, AC and Ca and P content

in 42-wk old W36 Hy-line (Pastore et al. 2012). Likewise Ca intake of 4.1 to 4.6 g g/b/d with

constant Ca: P (12:1) did not influence whole bones parameters in 73-wk old Lohmann brown

hens (Safaa et al. 2008). In contrast, Ca intake of 2.1 to 4.2 g /b/d (deficient to surfeit) with a

constant dietary level of available P (0.40 %) linearly increased tibia mineral density in 21-wk

old W36-Hyline hens (An et al., 2016). One reason for the contrasting bone attributes responses

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to dietary Ca level in later study, is due to bone Ca mobilization which was stimulated to cover

for the Ca demand in birds fed Ca-deficient diet (Pastore et al. 2012). Other reasons for the

varied bone attributes responses to Ca levels includes the differences in sampling time relative to

oviposition, age, strain, Ca forms and environmental condition (Roland Sr et al. 1996).

Vitamin D plays a critical role in Ca and P absorption, bone mineralization and regulation

of PTH (Garcia et al. 2013). In agreement with our findings, Frost et al. (1990) did not observe

any change in AC of the tibia at 10 h post-oviposition in response to incremental levels of

cholecalciferol from 12.5 to 37.5 µg/kg in 65-wk old W36 Hy-line. Although, they observed that

AC was decreased by increasing level of cholecalciferol at oviposition. The ACN of tibia was

neither affected at oviposition nor at 10 h post-oviposition in response to supplementation of 1-

25OH2D3 up to 1.0 µg/kg. These findings demonstrated that source of vitamin D and sampling

time relative to oviposition can influence bone attributes responses.

Decrease in AC and ACN of femur and tibia during lay cycle was inconsistent with the

findings of Fleming et al. (1998) who observed 7.1% increase in tibia mineral density 50 wk old

ISA Brown layers. Feeding 25OHD3 linearly reduced ulna TBRW and AC, however, Ca did not

alleviate adverse impact of aging on ulna, femur and tibia attributes over the 8 wks experimental

period. Therefore, patterns of bone loss over the lifetime of laying hens might be varied

depending on the bone type and structure (Fleming et al. 1998). With the surge of estrogen in

bloodstream at the onset of sexual maturity, function of osteoblast alters from generating

lamellar cortical bone to produce spicules of medullary bone in the endosteal surface. This

process requires an adequate level of vitamin D for mineralization (de Matos 2008). As lay cycle

progresses, osteoclastic resorption of structural bone continues mainly in long bones of the

wings, legs and vertebrae leading to decrease in structural bone content (Whitehead 2004).

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Therefore, evaluation of bone attributes in terms of bone sub-parts may lead to better

understanding of bone dynamic.

There is little information on interaction between Ca and 25OHD3 or another form of

vitamin D on medullary bone sub-parts attributes in poultry. Vitamin D3, PTH, calcitonin and

sex hormones mainly regulate Ca hemostasis in birds (de Matos 2008). Absorption of dietary Ca

across duodenum and jejunum membrane is expedited by 1-25OH2D3 through increased

synthesis of Ca-binding proteins, mainly calbindin (de Matos 2008). In addition, 1-25OH2D3

stimulate synthesis of multiple bone proteins produced by osteoblasts (Chew et al. 1992). In the

current study, ACN of medullary part of femur increased by increasing 25OHD3 at each level of

Ca. de Matos (2008) reported that the medullary bone is replenished when hens received

adequate dietary Ca. However, ACN of cortical was reduced by 25OHD3 at each level of Ca

suggesting active resorption (Chew et al. 1992). The same pattern of the interactive effect of Ca

and 25OHD3 was observed on CI values of SRW, AC and ACN of medullary and cortical.

Osteoclast resorption is not specific to medullary bone but also to exposed structural bone

surfaces, which explains the osteoporotic structural bone loss. However, mineral content of

medullary bone are replenished by adequate dietary Ca consumption (de Matos 2008). Cheng

and Coon (1990) observed increase in dried weight and AC of medullary, cortical and total

femur in response to increasing in Ca intake of 2 to 4.5 g/b/d in 42-wk old DeKalb. They

concluded that for maximizing mineralization in the medullary and whole femur, higher Ca

intake is required than recommended value of 3.75 g (NRC, 1984). However, recommended Ca

intake was decreased to 3.25 g Ca /b/d in the latest NRC edition (NRC, 1994). Supplemental

25OHD3 had similar effects on tibia medullary and cortical. However, interactive effect of Ca

and 25OHD3 was not observed on the tibia, which might be due to different structures. Femur

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had medullary part with higher ACN, AC and SRW compared to the tibia. On the other hand,

tibia had cortical part with higher ACN, AC and SRW. Similarly, Clunies et al. (1992) reported

that femur had largest reserve of medullary among medullary bones followed by the tibiotarsus

in 40-wk old leghorn layers. In addition, in agreement with our observation, (Cheng and Coon

1990) reported lower AC in medullary part compared to other parts of bones.

Ca levels had no effect on SRW, AC and ACN of sub-parts in tibia. Relative weight, AC

and ACN of medullary part of tibia increased with 25OHD3 supplementation in concomitant

with reduction in corresponding indices in cortical. Clunies et al. (1992) observed that dietary Ca

level of 3.5 % (Ca and feed intake were not reported) increased medullary bone Ca content in 43-

wk old white leghorn compared to 2.5 and 4.5 % of Ca, which had no impact on medullary bone.

Medullary bone develops rapidly during early stage of lay (Whitehead 2004), particularly,

shortly before the onset of egg production, because its formation occurs concomitantly with the

maturation of the ovarian follicles (Dacke et al., 1999), and undergoes little change compared to

structural parts when birds were fed Ca-deficient diet (Taylor and Moore 1954).

Vitamin D metabolites especially 1-25OH2D3 is required for formation of osteoclast

(Takeda et al. 1999). In addition, active metabolites of vitamin D are required for maturation of

osteoblasts to exert their inhibitory impact through increasing osteoprotegerin, which can inhibit

osteoclastogenesis (Baldock et al. 2006). However, cancellous bone do not undergo this

inhibition process (Baldock et al. 2006). Zallone and Mueller (1969) reported that based on

histological assessment, only cortical part had osteoclastic activity at bone resorption. It has been

reported that AC of medullary part was decreased while cortical bone remained unaffected in

quails fed vitamin D-deficient diet (Takahashi et al. 1983). Kim et al. (2007) reported medullary

bone developed at the expense of losing medullary mineral density in 80-wk old white leghorn.

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Reduction in CI values of AC and ACN of the sub-parts in tibia can be attributed by less

synthesis of 1-25OH2D3 and subsequently reduction in plasma concentration of 1-25OH2D3

along with the decreased number of intestinal Ca transport is resulted from the ageing process.

Laying hens showed age-dependent (22 vs. 120-wk old white leghorn) responses to being fed a

Ca-deficient diet in terms of the utilization of their cortical as Ca source (Elaroussi et al. 1994).

In addition, Fleming et al. (1998) reported that diaphysis region of long bones is mainly

subjected to bone mass losses while cancellous part undergoes the mass loss in the early lay

cycle. Whereas CI value of SRW of tibia medullary was increased by levels of Ca and CI value

of ACN was reduced by the highest level of dietary Ca (4.5%; 4.8 g Ca/b/d), the CI value of

cortical remained unaffected, which was in agreement with the response of cortical part of the

femur. This finding can demonstrate Ca might not affect cortical part of tibia and femur bone in

aged hens, while medullary part had higher propensity to be maintained. Thus, total bone mineral

content may remain constant or even increase throughout laying period (Whitehead 2004).

The long-term maintenance tissues and organs involved in egg production should be

considered as breeding companies aim to develop “long life and productive” commercial layer

flock for producing 500 eggs in 100-wk (Bain et al. 2016). To cope with osteoporosis challenge

in achieving “long life and productive” commercial layer flock, Ca and vitamin D feeding should

be critically reconsidered. Based on our finding, it might be important to assess bone sub-parts

attributes than whole bone. These findings suggested that hens at this age have higher propensity

to maintain medullary bone at expenses of reducing cortical bone. Dietary interventions such as

increased offerings of Ca over 4.0 % and 25OHD3 more than 69 µg/kg may have adverse effects

on the structure of epiphysis and cortical bones at this age, respectively. In addition, medullary

bone starts to be formed at sexual maturity and continues to develop over the lay cycle, but

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cortical continues to lose the mineral content and even increasing dietary Ca level cannot prevent

or slow down the erosion. Thus, nutritional strategies aiming at minimizing osteoporosis should

not be focused on late-phase of lay cycle or when there is high risk of osteoporosis but in early

stages of skeletal development. Moreover, the pattern of change in measured attributes

demonstrated that bone type and sub-parts responded distinctively suggesting onset of

osteoporosis in laying hens is not uniform in all bone types.

Acknowledgments

This work was supported by National Sciences and Engineering Research Council of Canada,

Egg Farmers of Ontario and Canada, Canadian Poultry Research Council, OMAFRA and

Wallenstein Feeds & Supply. Author would like to acknowledge DSM Nutritional Products Ayr,

ON, Canada for providing 25OHD3 (Hy-D®) and monogastric nutrition lab students for their

help for sampling in early hours of the day. R. Akbari Moghaddam Kakhki is a recipient of the

Ontario Trillium Doctoral Scholarship.

References

Anderson, K., Havenstein, G., Jenkins, P. and Osborne, J. 2013. Changes in commercial laying

stock performance, 1958-2011: thirty-seven flocks of the North Carolina random sample

and subsequent layer performance and management tests. World. Poult. Sci. J. 69:489-

514. doi: 10.1017/S0043933913000536.

AOAC. 1995. Official methods of analysis of AOAC International. Arlington, Va: AOAC Intl pv

(loose-leaf).

Page 15 of 32



For Review Only

16

Bain, M., Nys, Y. and Dunn, I. 2016. Increasing persistency in lay and stabilising egg quality in

longer laying cycles. What are the challenges? Br. Poult. Sci. 57:330-338. doi:

10.1080/00071668.2016.1161727

Baldock, P. A., Thomas, G. P., Hodge, J. M., Baker, S. U., Dressel, U., O'Loughlin, P. D.,

Nicholson, G. C., Briffa, K. H., Eisman, J. A. and Gardiner, E. M. 2006. Vitamin D

action and regulation of bone remodeling: suppression of osteoclastogenesis by the

mature osteoblast. J. Bone. Miner. Res. 21:1618-1626. doi: 10.1359/jbmr.060714

CCAC. 2009. Guidelines on the care and use of farm animals in research, teaching and testing.

Pages 1-168. Canadian Council on Animal Care, Ottawa, ON, Canada.

https://www.ccac.ca/Documents/Standards/Guidelines/Farm_Animals.pdf. Accessed

April 2018.

Cheng, T. K. and Coon, C. N. 1990. Sensitivity of various bone parameters of laying hens to

different daily calcium intakes. Poult. Sci. 69:2209-2213. doi: 10.3382/ps.0692209

Chew, D., Nagode, L. and Carothers, M. 1992. Disorders of calcium: hypercalcemia and

hypocalcemia. WB Saunders, Philadelphia. 116-117

Clunies, M., Emslie, J. and Leeson, S. 1992. Effect of dietary calcium level on medullary bone

calcium reserves and shell weight of leghorn hens. Poult. Sci. 71:1348-1356. doi:

10.3382/ps.0711348

de Matos, R. 2008. Calcium metabolism in birds. Veterinary Clinics: Exotic Animal Practice

11:59-82. doi: 10.1016/j.cvex.2007.09.005

Do Nascimento, G., Murakami, A., Guerra, A., Ospinas-Rojas, I., Ferreira, M. and Fanhani, J.

2014. Effect of different vitamin D sources and calcium levels in the diet of layers in the

Page 16 of 32



For Review Only

17

second laying cycle. Rev. Bras. Cienc. Avic. 16:37-42. doi: 10.1590/1516-635x160237-

42

Egg Farmers of Canada. 2009. Our sustaibaility story. http://www.eggfarmers.ca/wp-

content/uploads/2018/02/2018_Egg-Farmers-of-Canada_Sustainability-Story.pdf.

Accessed April 2018.

Elaroussi, M. A., Forte, L. R., Eber, S. L. and Biellier, H. V. 1994. Calcium homeostasis in the

laying hen. 1. Age and dietary calcium effects. Poult. Sci. 73:1581-1589. doi:

10.3382/ps.0731581

Fleming, R., McCormack, H. and Whitehead, C. 1998. Bone structure and strength at different

ages in laying hens and effects of dietary particulate limestone, vitamin K and ascorbic

acid. Br. Poult. Sci. 39:434-440. doi: 10.1080/00071669889024

Frost, T. J., D. A. Roland, S. and Untawale, G. 1990. Influence of Vitamin D3, la-

Hydroxyvitamin D3, and 1, 25-Dmydroxyvitarnin D3 on Eggshell Quality, Tibia

Strength, and Various Production Parameters in Commercial Laying Hens. Poult. Sci.

69:2008-2016. doi: 10.3382/ps.0692008

Garcia, A. F. Q. M., Murakami, A. E., do Amaral Duarte, C. R., Rojas, I. C. O., Picoli, K. P. and

Puzotti, M. M. 2013. Use of vitamin D3 and its metabolites in broiler chicken feed on

performance, bone parameters and meat quality. Asian-Australas J. Anim. Sci. 26:408.

doi: 10.5713/ajas.2012.12455

Gilbert, A. 1983. Calcium and reproductive function in the hen. Proc. Nutr. Soc. 42:195-212.

doi: 10.1079/PNS19830024

Page 17 of 32



For Review Only

18

Jin, S., Sell, J. and Haynes, J. 2001. Effect of dietary vitamin K1 on selected plasma

characteristics and bone ash in young turkeys fed diets adequate or deficient in vitamin

D3. Poult. Sci. 80:607-614. doi: 10.1093/ps/80.5.607

Kaur, R., Rathgeber, B. M., Thompson, K. L. and Macisaac, J. 2013. Uterine fluid proteins and

egg quality characteristics for 2 commercial and 2 heritage laying hen lines in response to

manipulation of dietary calcium and vitamin D3. Poult Sci 92:2419-32. doi:

10.3382/ps.2012-02983

Keshavarz, K. 2003. A comparison between cholecalciferol and 25-OH-cholecalciferol on

performance and eggshell quality of hens fed different levels of calcium and phosphorus.

Poult. Sci. 82:1415-22. doi: 10.1093/ps/82.9.1415

Kim, W., Donalson, L., Bloomfield, S., Hogan, H., Kubena, L., Nisbet, D. and Ricke, S. 2007.

Molt performance and bone density of cortical, medullary, and cancellous bone in laying

hens during feed restriction or alfalfa-based feed molt. Poult. Sci. 86:1821-1830. doi:

10.1093/ps/86.9.1821

Lohman, L. 2016. Classic international (2016) Variety LSL-Lite commercial management guide.

Lohman international. Tierzucht. www Itz de.

Pastore, S. M., Gomes, P. C., Rostagno, H. S., Albino, L. F. T., Calderano, A. A., Vellasco, C.

R., Viana, G. d. S. and Almeida, R. L. d. 2012. Calcium levels and calcium: available

phosphorus ratios in diets for white egg layers from 42 to 58 weeks of age. Rev. Bras.

Zootec. 41:2424-2432. doi: 10.1590/S1516-35982012001200007

Rath, N., Huff, G., Huff, W. and Balog, J. 2000. Factors regulating bone maturity and strength in

poultry. Poult. Sci. 79:1024-1032. doi: 10.1093/ps/79.7.1024

Page 18 of 32



For Review Only

19

Roland Sr, D., Bryant, M., Rabon, H. and Self, J. 1996. Influence of calcium and environmental

temperature on performance of first-cycle (Phase 1) commercial Leghorns. Poult. Sci.

75:62-68. doi: 10.3382/ps.0750062

Safaa, H. M., Serrano, M., Valencia, D., Frikha, M., Jiménez-Moreno, E. and Mateos, G. 2008.

Productive performance and egg quality of brown egg-laying hens in the late phase of

production as influenced by level and source of calcium in the diet. Poult. Sci. 87:2043-

2051. doi: 10.3382/ps.2008-00110.

Sanders, A. and Edwards Jr, H. 1991. The effects of 1, 25-dihydroxycholecalciferol on

performance and bone development in the turkey poult. Poult. Sci. 70:853-866. doi:

10.3382/ps.0700853

Snedecor, G. and Cochran, W. 1980. Statistical Methods, 503 Iowa. USA: Iowa State Univ.

Press.

Takahashi, N., Shinki, T., Abe, E., Horiuchi, N., Yamaguchi, A., Yoshiki, S. and Suda, T. 1983.

The role of vitamin D in the medullary bone formation in egg-laying Japanese quail and

in immature male chicks treated with sex hormones. Calcif. Tissue. Int. 35:465-471.

Takeda, S., Yoshizawa, T., Nagai, Y., Yamato, H., Fukumoto, S., Sekline, K., Kato, S.,

Matsumoto, T. and Fujita, T. 1999. Stimulation of osteoclast formation by 1, 25-

dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic

cells: studies using VDR knockout mice. Endocrinology 140:1005-1008. doi:

10.1210/endo.140.2.6673

Taylor, T. and Moore, J. 1954. Skeletal depletion in hens laying on a low-calcium diet. Brit. J.

Nutr. 8:112-124. doi:10.1079/BJN19540020.

Page 19 of 32



For Review Only

20

Whitehead, C. 2004. Overview of bone biology in the egg-laying hen. Poult. Sci. 83:193-199.

doi: 10.1093/ps/83.2.193

Whitehead, C. and Fleming, R. 2000. Osteoporosis in cage layers. Poult. Sci. 79:1033-1041. doi:

10.1093/ps/79.7.1033

Zallone, A. Z. and Mueller, W. J. 1969. Medullary bone of laying hens during calcium depletion

and repletion. Calcif. Tissue. Int. 4:136-146. doi: 10.1007/BF02279115

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Table 1. Composition of experimental diets, as fed basis a

Ingredients, %

Calcium level (%)

3.0 3.5 4.0 4.5

Corn 48.43 48.43 48.43 48.65

Soy bean meal 46% 19.97 19.97 19.97 20.01

Limestone coarse 5.82 6.94 8.06 9.17

Corn DDGSb 9.37 9.37 9.37 8.87

Wheat 5.00 5.00 5.00 5.00

Soy oil 3.50 3.50 3.50 3.57

Limestone fine 1.03 1.22 1.42 1.62

Mono calcium phosphate 1.64 1.64 1.64 1.65

Poultry VT Premixc 1.00 1.00 1.00 1.00

Salt 0.21 0.21 0.21 0.21

Sodium bicarbonate 0.16 0.16 0.15 0.15

DL-Methionine 0.09 0.09 0.09 0.09

Sand filler 3.78 2.47 1.16 0.00

Calculate provisions

Crude protein (%) 16.3 16.3 16.3 16.2

Ca (%) 3.00 3.50 4.00 4.50

Available P (%) 0.41 0.41 0.41 0.41

AME (kcal/kg) 2,820 2,820 2,820 2,820

SID Lys (%) 0.62 0.62 0.62 0.62

SID Met and Cys (%) 0.56 0.56 0.56 0.56 a

Each of Ca diet was split into three portions and top dressed with Hy-D® (1.25%), DSM

Nutritional Products, Ayr, ON, Canada to create 0, 69 and 138 µg of 25OHD3/kg of diet.

b Distiller's dried grains with solubles.

c Provided per kg of premix: vitamin A (retinol), 880 KIU; vitamin D3 (cholecalciferol),

330 KIU; vitamin E, 4,000 IU; vitamin K3 (menadione), 330 mg; vitamin B1 (thiamin), 400

mg; vitamin B2 (riboflavin), 800 mg; vitamin B3 (niacin), 5,000 mg; vitamin B5

(pantothenic acid), 1,500 mg; vitamin B6 (pyridoxine), 300 mg; vitamin B9 (folic acid),

100 mg; vitamin B12 (cyanocobalamin), 1200 mcg; biotin, 200 mcg; choline, 60,000 mg;

Fe, 6000 mg; Cu, 1000 mg; I, 1 mg, Se, 30 mg.

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Table 2. Measured attributes of medullary bones of baseline sampling at 74-wk of age.

Items Ulna Femur Tibia

Whole bone

Relative weight (%) a AVG 0.102±0.005 0.228±0.010 0.265±0.013

Max 0.112 0.208 0.241

Min 0.094 0.245 0.288

Ash content (g) AVG 0.754±0.035 2.23±0.108 2.76±134

Max 0.810 2.42 3.03

Min 0.670 2.03 2.54

Ash concentration (%) AVG 43.48±1.840 55.83±2.280 61.57±1.719

Max 47.60 59.65 66.21

Min 40.50 51.00 59.52

Medullary

Relative weight (%) b AVG - 17.50±0.852 15.83±0.746

Max 19.25 17.05

Min 16.13 14.42

Ash content (g) AVG - 0.38±0.018 0.33±0.016

Max 0.42 0.358

Min 0.35 0.301

Ash concentration (%) AVG - 45.98±2.081 47.48±1.995

Max 49.50 44.07

Min 49.50 50.98

Cortical


Max 38.74 50.05

Min 32.51 42.72

Ash content (g) AVG - 0.94±0.046 1.48±0.066

Max 1.03 1.60

Min 0.87 1.38


Max 72.20 76.28

Min 62.30 66.15

Epiphysis


Max 48.41 41.52

Min 43.52 34.90

Ash content (g) AVG - 0.92±0.038 0.97±0.047

Max 1.01 0.88

Min 0.86 1.05


Max 54.57 60.50

Min 46.30 53.22 a relative weight to the whole body weight.

b relative weight to the whole bone dried weight.

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Table 3. Effects of different levels of dietary calcium and top-dressed 25-hydroxy vitamin D3 (25OHD3) on medullary

bones attributes at week 81 of age.

Main Effects Ulna Femur Tibia

TBRWa AC

b ACN

c TBRW

a AC

b ACN

c TBRW

a AC

b ACN

c

Ca (%)

3.0 0.110 0.79 42.92 0.237 2.00 52.21 0.273 2.60 58.69

3.5 0.113 0.77 41.82 0.245 2.08 52.73 0.276 2.59 58.93

4.0 0.114 0.78 41.16 0.245 2.13 52.28 0.268 2.55 58.76

4.5 0.117 0.79 41.98 0.246 2.06 51.81 0.273 2.57 56.69

SEM 0.108 0.024 1.677 0.094 0.682 1.660 0.098 0.466 1.413

25OHD3 (µg/kg)

0 0.122 0.80 41.94 0.242 2.05 52.95 0.275 2.59 58.77

69 0.110 0.78 43.31 0.243 2.06 52.22 0.274 2.60 58.49

138 0.113 0.76 40.67 0.244 2.09 51.60 0.279 2.55 57.66

SEM 0.095 0.022 1.150 0.043 0.325 1.263 0.096 0.248 1.295

Probabilities

Ca 0.544 0.841 0.724 0.219 0.235 0.451 0.876 0.584 0.192

25OHD3 0.117 0.105 0.090 0.623 0.184 0.194 0.235 0.275 0.367

Ca × 25OHD3 0.644 0.780 0.889 0.952 0.221 0.873 0.105 0.142 0.244

Dose response

Ca level

Linear 0.266 0.995 0.404 0.199 0.754 0.225 0.813 0.440 0.105

Quadratic 0.334 0.326 0.402 0.847 0.465 0.275 0.994 0.825 0.502

25OHD3 level

Linear 0.109 0.087 0.312 0.332 0.072 0.131 0.196 0.145 0.219

Quadratic 0.442 0.789 0.078 0.803 0.858 0.568 0.683 0.322 0.353 a Total bone dried weight divided by body weight multiplied by 100, expressed as percentage.

b Total bone ash content; expressed as g of ash per each bone.

c Total bone ash concentration divided by total bone dried weight multiplied by 100; expressed as percentage.

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Table 4. Effects of different levels of dietary calcium and top-dressed 25-hydroxy vitamin D3 (25OHD3) on change

index values of medullary bone attributes at week 81 compared to 74 week of age a.

Main Effects Ulna Femur Tibia

TBRW AC ACN TBRW AC ACN TBRW AC ACN

Ca (%)

3.0 107.8 104.8 98.7 104.1 89.5 93.6 103.0 94.3 95.3

3.5 110.8 102.1 96.2 107.3 93.2 94.5 104.2 94.0 95.7

4.0 111.8 103.4 94.7 107.5 95.5 93.7 101.1 92.5 95.4

4.5 114.7 104.8 96.6 107.4 92.3 92.9 103.0 93.1 92.1

SEM 2.032 1.65 2.03 1.393 2.052 1.129 1.540 1.650 1.587

25OHD3 (µg/kg)

0 119.6a 106.1a 96.5ab 106.3 91.8 94.9 103.8 93.9 95.4

69 107.8b 103.4b 99.6a 106.7 92.3 93.6 103.4 94.1 95.0

138 110.8b 100.8c 93.5b 106.9 93.7 92.5 105.3 92.5 93.6

SEM 1.561 1.003 1.93 0.986 1.253 1.085 0.994 1.125 1.322

Probabilities

Ca 0.098 0.326 0.542 0.135 0.073 0.765 0.176 0.125 0.140

25OHD3 0.002 0.001 0.034 0.559 0.110 0.840 0.165 0.121 0.182

Ca × 25OHD3 0.854 0.798 0.619 0.986 0.152 0.181 0.274 0.239 0.154

Dose response, P-value

Ca level

Linear 0.068 0.684 0.653 0.133 0.157 0.321 0.543 0.110 0.115

Quadratic 0.952 0.124 0.254 0.365 0.063 0.215 0.458 0.457 0.252

25OHD3 level

Linear 0.001 <0.001 0.075 0.234 0.090 0.842 0.149 0.109 0.127

Quadratic 0.095 0.872 0.021 0.741 0.974 0.380 0.325 0.150 0.843

Note: Means within a row not sharing a lowercase letter differ significantly at the P < 0.05 level. SEM, standard error

of the mean. a calculated by dividing wk 81 values by baseline values and multiplied by 100.

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Table 5. Effects of different levels of dietary calcium and top-dressed 25-hydroxy vitamin D3 (25OHD3) on sub-parts

attributes of femur at week 81 of age.

Ca, %

25OHD

3,

µg/kg

SRWa (%) AC

b (g) ACN

c (%)

Medullary Cortical Ends Medullary Cortical Ends Medullary Cortical Ends

3.0 0 17.93 34.27 47.81 0.29 0.84 0.86 41.84b 65.11a 47.48

3.0 69 18.55 33.95 47.51 0.31 0.82 0.87 42.58ab 63.51ab 47.72

3.0 138 19.71 31.74 48.55 0.35 0.74 0.93 44.49a 62.16b 48.20

3.5 0 18.47 34.09 47.44 0.31 0.87 0.89 42.25ab 65.63a 47.89

3.5 69 19.70 34.25 46.04 0.33 0.85 0.89 42.90ab 64.00ab 48.88

3.5 138 20.91 31.91 47.18 0.37 0.78 0.96 44.83a 62.00b 48.80

4.0 0 19.10 34.61 46.28 0.33 0.89 0.89 42.21ab 65.67a 48.58

4.0 69 19.76 34.15 46.08 0.35 0.87 0.91 42.96ab 63.42ab 48.25

4.0 138 21.89 29.14 48.96 0.37 0.82 0.97 45.17a 59.48b 48.37

4.5 0 19.18 34.54 46.28 0.32 0.86 0.87 42.30ab 65.15a 47.90

4.5 69 19.60 33.62 46.78 0.34 0.84 0.88 41.42ab 62.32ab 47.29

4.5 138 21.94 29.20 48.87 0.38 0.76 0.94 45.26a 59.60b 48.27

SEM 1.567 1.143 1.057 0.115 0.157 0.191 0.982 0.660 1.173

Ca (%)

3.0 18.73 33.32 47.96 0.32 0.80 0.88 42.97 63.59 47.80

3.5 19.96 33.42 47.89 0.34 0.83 0.91 43.33 63.88 48.52

4.0 20.25 32.63 47.11 0.35 0.86 0.92 43.45 62.86 48.40

4.5 20.24 33.45 47.31 0.35 0.82 0.89 42.99 62.36 47.82

SEM 1.352 1.012 0.952 0.033 0.062 0.132 0.745 0.595 0.852

25OHD3

(µg/kg)

0 18.67 34.38a 46.95 0.31b 0.86a 0.88 42.15b 65.39a 47.96

69 19.40 33.99ab 46.60 0.33b 0.84ab 0.89 42.46b 63.31b 48.03

138 21.11 30.50b 48.39 0.37a 0.77b 0.95 44.94a 60.81c 48.41

SEM 1.25 0.828 0.902 0.011 0.025 0.125 0.721 0.439 0.705

Probabilities

Ca 0.181 0.451 0.193 0.079 0.198 0.515 0.140 0.158 0.813

25OHD3 0.071 0.023 0.289 0.021 0.032 0.186 0.024 0.001 0.785

Ca × 25OHD3 0.215 0.191 0.173 0.081 0.108 0.310 0.004 0.041 0.809

Dose response,

P-value

Ca level

Linear 0.106 0.368 0.097 0.067 0.145 0.388 0.282 0.206 0.950

Quadratic 0.273 0.545 0.319 0.816 0.790 0.213 0.416 0.647 0.769

25OHD3 level

Linear 0.059 <0.001 0.831 0.013 0.004 0.164 - - 0.180

×3.0 - - - - - - 0.007 0.002 -

×3.5 - - - - - - 0.009 0.001 -

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×4.0 - - - - - - 0.001 <0.001 -

×4.5 - - - - - - 0.001 0.005 -

Quadratic 0.703 0.882 0.761 0.160 0.121 0.911 - - 0.692

×3.0 - - - - - - 0.985 0.329 -

×3.5 - - - - - - 0.685 0.540 -

×4.0 - - - - - - 0.752 0.678 -

×4.5 - - - - - - 0.352 0.857 -

Note: Means within a row not sharing a lowercase letter differ significantly at the P < 0.05 level. SEM, standard error of

the mean. a Sub-part dried weight divided by total bone dried weight multiplied by 100, expressed as percentage.

b Sub-part ash content; expressed as g of ash per each part.

c Sub-part ash concentration divided by sub-part dried weight multiplied by 100; expressed as percentage.

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Table 6. Effects of different levels of dietary calcium and top-dressed 25-hydroxy vitamin D3 (25OHD3) on

change index values of sub-parts attributes of femur at week 81 compared to 74 week of agea.

Ca,

%

25OHD3,

µg/kg

SRW AC ACN


3.0 0 102.4d 96.0a 102.2 79.3d 88.8ab 92.9 91.0b 96.9a 93.5

3.0 69 106.0cd 95.1a 101.5 81.6d 86.7b 94.6 92.6b 94.5ab 93.9

3.0 138 112.6b 88.9b 103.7 92.4b 78.3c 100.5 96.7a 92.5b 94.9

3.5 0 105.5cd 95.5a 101.4 81.6d 92.0a 96.7 91.8b 97.7a 94.3

3.5 69 112.6b 96.0a 98.4 86.8c 89.9ab 96.4 93.3b 95.2ab 96.2

3.5 138 119.5a 89.4b 100.8 96.2a 83.0cb 104.3 97.5a 92.3a 96.1

4.0 0 109.2bc 96.9a 98.9 85.5c 94.7a 96.8 91.8b 97.7a 95.6

4.0 69 112.9b 95.7a 98.5 90.8b 92.6a 98.9 93.4b 94.4b 95.0

4.0 138 125.1a 81.6b 104.6 98.5a 87.3ab 105.0 98.2a 91.9b 95.2

4.5 0 109.6bc 96.7a 98.9 84.2d 91.0a 94.0 92.0b 97.0a 94.3

4.5 69 112.0b 94.2a 100.0 89.5bc 88.8a 95.7 90.0b 92.7b 93.1

4.5 138 125.4a 81.8b 104.4 100.0a 81.1c 102.1 98.4a 88.7c 95.0

SEM 2.170 1.520 1.645 1.972 1.830 2.950 1.033 1.102 1.619

Ca (%)

3.0 107.0c 93.3 102.5 83.4c 84.6c 96.0 93.4 94.6 94.1

3.5 112.5b 93.6 100.2 88.2b 88.3b 99.2 94.2 95.1 95.5

4.0 115.8a 91.4 100.7 91.6a 91.5a 100.2 94.4 94.5 95.3

4.5 115.7a 90.9 101.1 91.2a 87.0bc 97.3 93.5 93.8 94.1

SEM 1.060 1.201 1.434 1.005 1.250 1.950 0.980 0.990 1.050

25OHD3

(µg/kg)

0 106.7b 96.3a 100.3 81.9c 91.6a 95.1 91.6b 97.3a 94.4

69 110.9b 95.2a 99.6 87.2b 89.5a 96.4 92.3b 94.2b 94.6

138 120.6a 85.4b 103.4 96.8a 82.4b 103.0 97.7a 90.5c 95.3

SEM 1.010 0.985 1.295 0.995 1.035 1.729 0.971 0.965 0.990

Probabilities

Ca 0.035 0.066 0.578 <0.001 0.005 0.557 0.548 0.099 0.362

25OHD3 0.001 0.005 0.232 0.001 0.001 0.085 0.001 <0.001 0.254

Ca ×

25OHD3 0.001 0.034 0.875 <0.001 <0.001 0.122 0.001 0.032 0.741

Dose response,

P-value

Ca level

Linear 0.001 0.053 0.685 <0.001 0.091 0.986 0.432 0.185 0.852

Quadratic 0.982 0.463 0.437 0.675 0.020 0.389 0.896 0.130 0.225

25OHD3

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level

Linear - - 0.423 - - 0.069 - - 0.189

×3.0 0.005 0.008 - <0.001 <0.001 - 0.001 0.012 -

×3.5 0.001 0.001 - <0.001 0.001 - 0.003 0.001 -

×4.0 <0.001 <0.001 - 0.011 0.005 - <0.001 <0.001 -

×4.5 <0.001 <0.001 - 0.001 0.005 - <0.001 <0.001 -

Quadratic - - 0.316 - - 0.857 - - 0.852

×3.0 0.852 0.951 - 0.895 0.678 - 0.852 0.654 -

×3.5 0.754 0.842 - 0.875 0.596 - 0.753 0.751 -

×4.0 0.720 0.751 - 0.678 0.385 - 0.651 0.699 -

×4.5 0.630 0.632 - 0.578 0.454 - 0.531 0.549 -

Note: Means within a row not sharing a lowercase letter differ significantly at the P < 0.05 level. SEM,

standard error of the mean. a calculated by dividing wk 81 values by baseline values and multiplied by 100.

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Table 7. Effects of different levels of dietary calcium and top-dressed 25-hydroxy vitamin D3 (25OHD3) on sub-parts

attributes of tibia at week 81 of age.

Main Effects SRW

a (%) AC

b (g) ACN

c (%)


Ca (%)

3.0 15.65 42.39 41.95 0.29 1.31 1.00 42.09 69.87 53.59

3.5 16.48 41.18 42.34 0.30 1.27 1.02 41.76 70.09 54.77

4.0 16.70 40.94 42.35 0.31 1.25 0.98 42.12 70.50 53.32

4.5 16.82 42.14 41.04 0.31 1.32 0.94 40.72 69.02 50.58

SEM 0.985 1.005 1.025 0.095 0.045 0.084 1.928 0.953 0.980

25OHD3 (µg/kg)

0 15.93b 44.23a 39.84 0.27b 1.38a 0.94 37.84b 70.91a 53.65

69 16.20ab 43.04ab 41.76 0.30ab 1.34a 0.99 41.42ab 69.93ab 53.19

138 18.52a 41.32b 40.16 0.36a 1.25b 0.94 43.59a 68.54b 52.95

SEM 0.752 0.852 1.254 0.021 0.032 0.102 1.865 0.785 0.799

Probabilities

Ca 0.112 0.085 0.647 0.126 0.452 0.088 0.091 0.734 0.108

25OHD3 0.003 0.002 0.916 0.001 0.045 0.866 0.044 0.033 0.872

Ca × 25OHD3 0.198 0.103 0.486 0.142 0.110 0.433 0.146 0.127 0.518


Ca level

Linear 0.095 0.233 0.236 0.072 0.188 0.062 0.070 0.905 0.137

Quadratic 0.489 0.064 0.294 0.745 0.130 0.895 0.584 0.151 0.080

25OHD3 level

Linear 0.014 0.001 0.917 <0.001 0.001 0.999 0.006 0.021 0.277

Quadratic 0.299 0.868 0.618 0.875 0.985 0.246 0.370 0.908 0.996


the mean.

a Sub-part dried weight divided by total bone dried weight multiplied by 100, expressed as percentage.

b Sub-part ash content; expressed as g of ash per each part.

c Sub-part ash concentration divided by sub-part dried weight multiplied by 100; expressed as percentage.

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Table 8. Effects of different levels of dietary calcium and top-dressed 25-hydroxy vitamin D3 (25OHD3) on change

index values of sub-parts attributes of tibia at week 81 compared to 74 week of agea.

Main Effects SRW AC ACN


Ca (%)

3.0 98.9b 91.4 110.9 87.8c 88.9 102.9ab 92.5a 98.3 93.8ab

3.5 104.1a 88.8 112.0 91.1b 86.0 105.4a 91.8ab 98.6 95.9a

4.0 105.5a 88.3 112.0 92.0b 85.0 101.3b 92.6a 99.2 93.3ab

4.5 106.3a 90.9 108.5 93.4a 89.3 97.2c 89.5b 97.1 88.5b

SEM 1.056 1.319 2.141 1.201 1.562 1.367 1.005 0.951 1.170

25OHD3 (µg/kg)

0 100.6b 95.4a 105.4 79.9c 93.6a 97.3 83.2c 99.8a 93.9

69 102.3b 92.8a 107.8 89.6b 90.5a 101.8 91.1b 98.4b 93.1

138 117.0a 89.1b 106.2 107.5a 84.9b 97.2 95.8a 96.5c 92.7

SEM 1.042 1.254 1.958 1.150 1.353 1.250 0.985 0.930 1.029

Probabilities

Ca 0.044 0.085 0.147 0.010 0.089 <0.001 0.011 0.134 0.005

25OHD3 <0.001 0.002 0.271 <0.001 0.001 0.101 <0.001 <0.001 0.872

Ca × 25OHD3 0.090 0.152 0.240 0.164 0.295 0.759 0.076 0.127 0.518


Ca level

Linear 0.002 0.233 0.541 0.005 0.125 <0.001 0.005 0.102 0.037

Quadratic 0.115 0.074 0.469 0.784 0.234 0.215 0.175 0.486 0.118

25OHD3 level

Linear <0.001 0.001 0.874 <0.001 <0.001 0.354 0.001 <0.001 0.277

Quadratic 0.745 0.868 0.114 0.653 0.865 0.090 0.857 0.979 0.996


the mean. a comparison between 74 and 81 wk of age was expressed based on percentage of change

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Figure Caption

Figure 1. Edges of approximated distal and proximal epiphysis and diaphysis regions.

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