ANNEX to Final Report on Project AW1127

ANNEX to Final Report on Project AW1127

Glossary of terms used in the report

Bending stress Breaking strength in relation to x-sectional area of bone

Bone stiffness Deformation of bone during 3-point test

Breaking strength Energy required to break bone under 3-point test

Contig Contiguous segment of DNA constructed from a number of sequences

Cortical area Histomorphic measure of the midshaft x-sectional area of the ring of cortical bone

(cortex)

Cortical porosity Cortical bone in the hen is not solid. It contains holes and gaps between areas of

mineralised bone. Porosity is a measure of the proportion of the overall cortex

EST Expressed sequence tag. Sequences from expressed genes

Gene library Collection of genes

Housekeeping gene Ubiquitous gene present in all tissues

MB medullary bone

MB score A visual assessment on a scale of 0 to 3 of the amount of infilling of the internal

cavity of the humerus with medullary bone

mm Al eq. mm of aluminium equivalent (radiographic calibration from aluminium stepwedge)

Osteoblast bone forming cell

Osteoclast bone resorbing cell

PCR Polymerase chain reaction

True cortical area Cortical area corrected for porosity, i.e. the x-sectional area of the cortex

composed of mineralised bone

QTL Quantitative trait locus

RD Radiographic density

Figure 1. Aviary with birds under experiment in Objective 3

Table 1. Candidate genes for (a) osteoporosis and (b) bone mass (Objective 1).

The lists are not exhaustive and the genes are not ranked in order of importance. Some are systemic regulators of bone mass and thus expression would need to be measured in the tissue of origin.

(a)

Vitamin D receptor

Interleukin-6

Transforming growth factor-b1

Oestrogen receptor-a

Calcitonin receptor

Apolipoprotein E

Osteocalcin

Collagen type Ia1

Interleukin-1 receptor antagonist

(b)

Parathyroid hormone receptor

Parathyroid hormone

Insulin-like growth factor-I

Fibroblast growth factor-1

Fibroblast growth factor-2

Osteoprotegerin

c-Abl kinase

Osteonectin

Thrombospondin 2

Biglycan gene

RANK

RANK Ligand

TRAF6

NFkB

Amylin

Bone sialoprotein

Cbfa1

Glucocorticoid receptor

b3-adrenergic receptor

b3-integrins

a2-HS-glycoprotein

Oestradiol receptor

Calcitonin

Calcitonin gene related peptide

Calmodulin-2

c-fos

Pu.1

c-Src

Thrombopoetin receptor

Matrix Gla protein

Nitric oxide synthase

Epidermal growth factor

Interleukin-1a

Interleukin-4

Table 2. List of up and down regulated genes in bone. Those genes shown in yellow are up regulated in the high line whilst those in pink are down regulated in the high line. The 1st column contains the EST code, the 2nd the delta log value, the 3rd column the gene name if known, the 4th the chromosome (Un = unknown, M = mitochondrial) colours in this column show locations coincident with QTL, the 5th column has position in bases along the chromosome and the 6th column the significance attached to the difference between the lines (Objective 1).

mature femur
Clone identity / Delta log / gene / Chr. / position / p-value
C0005392M3 / 0.805 / collagen type i alpha 2 (col1a2) / chr2 / 22.9 Mb / 0.02
C0005362K5 / 0.823 / collagen type i alpha 1 (col1a1) / chr9 / 9.0 Mb / 0.04
C0005537B8 / 1.222 / adipocyte fatty acid binding protein (afabp) / chr2 / 120.8 Mb / 0.01
ChEST176A9 / 1.223 / alpha2,3-sialyltransferase (st3gal) / chr1 / 78.3 Mb / 0.01
ChEST50L20 / 1.249 / pleckstrin homology domain containing family c / chr5 / 54.9 Mb / 0.02
ChEST239J15 / 1.276 / nexin / chr9 / 5.3 Mb / 0.00
ChEST158P22 / 1.292 / ovotransferrin (gal d iii) / chr9 / 9.0 Mb / 0.00
ChEST609C21 / 1.293 / ovotransferrin (gal d iii) / chr9 / 9.0 Mb / 0.04
ChEST837O24 / 1.306 / carbonic anhydrase ii (ca2) / chr2 / 122.2 Mb / 0.00
ChEST277M22 / 1.357 / no homology (in ankyrin gene region) / chr12 / 8.7 Mb / 0.00
Juvenile Femur
ChEST608K3 / 0.68 / decorin (dcn) / chr1 / 40.8 Mb / 0.03
C0005052G16 / 0.731 / clone chest72k8 no homology to known gene / chr17 / 2.8 Mb / 0.02
C0005056K19 / 0.748 / osteonectin (sparc) / chr13 / 11.8 Mb / 0.01
C0005056E19 / 0.784 / no homology to known gene / chr6 / 1.6 Mb / 0.01
ChEST283H8 / 0.798 / no homology to known gene (in beta catenin gene region) / chr2 / 42.5 Mb / 0.03
C0005534C14 / 0.811 / collagen type i alpha 2 (col1a2) / chr2 / 22.9 Mb / 0.02
ChEST213F7 / 0.82 / no homology to known gene / chrZ / 26.5 Mb / 0.04
C0005387G2 / 0.827 / goose-type lysozyme (lyg2) / chr1 / 125.6 Mb / 0.04
ChEST498N13 / 1.207 / inositol polyphosphate-4-phosphatase isoform a (inpp4a) / chr1 / 125.2 Mb / 0.01
ChEST179E20 / 1.21 / eukaryotic translation elongation factor 1 alpha 1 (ef-1-alpha-1) / chr3 / 79.8 Mb / 0.04
C0005526I15 / 1.215 / s-100 calcium-binding protein a12 (s100a12) / chrUn / 145.3 Mb / 0.04
C0005526M24 / 1.215 / s-100 calcium-binding protein a12 / chrUn / 145.3 Mb / 0.01
ChEST931K18 / 1.303 / no homology to known gene / chr5 / 14.3 Mb / 0.03
C0005391M13 / 1.304 / part beta-defensin 1 (gal1) like part set domain protein / chr4 / 29.8 Mb / 0.01
C0005361P21 / 1.334 / thymosin beta-4 (t beta 4) / chr1 / 116.6 Mb / 0.05
C0005388A15 / 1.376 / thymosin beta-4 (t beta 4) / chr1 / 116.6 Mb / 0.03
C0005057J5 / 1.535 / gallinacin 1 prepropeptide (gal1) / chr3 / 105.4 Mb / 0.04

Table 3. List of up and down regulated genes in shell gland. Those genes shown in yellow are up regulated in the high line whilst those in pink are down regulated in the high line. The 1st column contains the EST code, the 2nd the delta log value, the 3rd column the gene name if known, the 4th the chromosome (Un = unknown, M = mitochondrial) colours in this column show locations coincident with QTL, the 5th column has position in bases along the chromosome and the 6th column the significance attached to the difference between the lines (Objective 1).

mature shell gland
ChEST547D21 / 0.352 / glycerol kinase isoform 1 / chr1 / 109.1 Mb / 0.01
ChEST921B2 / 0.366 / ring1 and yy1 binding protein / chr12 / 16.3 Mb / 0.00
ChEST532C23 / 0.567 / cd109 antigen / chr3 / 79.7 Mb / 0.02
ChEST595G3 / 0.663 / calnexin / chr13 / 12.4 Mb / 0.01
ChEST303F21 / 0.666 / atpase h+ transporting lysosomal accessory / chr1 / 105.9 Mb / 0.04
ChEST154D3 / 1.502 / alpha-2-hs-glycoprotein / chr9 / 15.4 Mb / 0.02
ChEST179M19 / 1.535 / collagen (x) alpha 1 (col10a1) / chr3 / 62.4 Mb / 0.00
ChEST930J13 / 1.823 / collagen (vi) alpha 3 (col6a3). / chr7 / 4.7 Mb / 0.04
ChEST824M15 / 2.103 / collagen (iia) alpha 1 (col2a1). / chrUn / 134.2 Mb / 0.05
ChEST226A8 / 2.289 / transcription factor rreb-1 / chr2 / 64.4 Mb / 0.05
ChEST1009A5 / 2.558 / possibly beta catenin / chr2 / 42.5 Mb / 0.01
ChEST665M1 / 2.616 / possibly gemin 5 / chr13 / 10.9 Mb / 0.02
ChEST803N5 / 2.76 / growth factor receptor precursor / chr4 / 11.7 Mb / 0.03
juvenile shell gland
ChEST927G15 / 0.08 / baculoviral iap repeat-containing 6 / chr3 / 27,9 Mb / 0.005
ChEST812E2 / 0.08 / adenosine a2a receptor / chr15 / 8,4 Mb / 0.007
ChEST363F9 / 0.09 / v-ets avian erythroblastosis virus e26 oncogene / chr1 / 101,5 Mb / 0.04
ChEST21B4 / 0.10 / calcitonin-related polypeptide beta / chr5 / 8,0 Mb / 0.01
ChEST155G1 / 0.11 / serine (or cysteine) proteinase inhibitor clade / chr8 / 7,3 Mb / 0.01
ChEST664O20 / 0.11 / protein tyrosine phosphatase non-receptor type / chr3 / 17,4 Mb / 0.02
ChEST824J5 / 0.12 / flj31062 / chr1 / 20,3 Mb / 0.05
ChEST240H18 / 0.12 / nephrocystin 3 / chr2 / 41,4 Mb / 0.02
ChEST174B6 / 0.17 / carbohydrate sulfotransferase 11 / chr1 / 51,0 Mb / 0.03
ChEST938P24 / 0.17 / leucine rich repeat containing 16 / chr2 / 89,8 Mb / 0.04
ChEST390M10 / 0.21 / transcription elongation factor b polypeptide 3 / chr28 / 1,3 Mb / 0.013
ChEST110I13 / 0.22 / syndecan 3 / chr23 / 0.4 Mb / 0.02
ChEST185C12 / 0.28 / calcium-sensing receptor / chr1 / 69,2 Mb / 0.05
ChEST1032B14 / 0.33 / vacuolar h(+)-transporting atpase 116 kda / chr27 / 0.2 Mb / 0.0272
ChEST167N11 / 0.38 / hypothetical protein loc216613 / chr3 / 2,0 Mb / 0.05
ChEST88E24 / 2.08 / igf-ii mrna-binding protein 3 / chr2 / 30,3 Mb / 0.0277
ChEST1026P6 / 2.92 / mad mothers against decapentaplegic homolog 3 / chr10 / 19,5 Mb / 0.0356
ChEST179F1 / 3.15 / cartilage link protein / chrZ / 29,9 Mb / 0.0284
ChEST757L21 / 3.35 / fibroblast growth factor receptor 2 homolog / chr6 / 29,9 Mb / 0.0325
ChEST646A9 / 3.58 / collagen xiv (col14a1) / chr2 / 146,8 Mb / 0.0231
ChEST829H14 / 5.45 / hypothetical protein loc199953 / chr26 / 2,6 Mb / 0.032
ChEST673O11 / 16.66 / distal-less homeo box 5 / chrUn / 79,2 Mb / 0.0347

Table 4: Formulations of the starter, grower and layer diets (Objective 2).

Ingredient (g/kg) /

STARTER

/ GROWER / LAYER
Wheat / 321.5 / 801.39 / 702.2
Barley / 314.0
Soya meal / 310 / 152.11 / 164.7
Fish meal / 10
Soya oil / 10 / 10 / 28.5
Limestone / 9 / 12.79 / 82
Dicalcium Phosphate / 16.6 / 13.84 / 12.46
Salt / 3 / 2.74 / 2.88
Methionine / 0.9 / 0.78 / 1.45
Threonine / 1.35 / 1.35
Lysine HCl / 0.46
Yolk pigment / 1
Vitamin/mineral mix / 5* / 5* / 3+
Analysed:
Crude protein / 160.0
Calcium / 36.7
Total Phosphorus / 4.93

*To supply per kg diet:

Vitamin A 12,000IU; Vitamin D3 5,000IU; Vitamin E 30 mg; Vitamin K 3mg; VitaminB1 2mg; Vitamin B2 7mg; Vitamin B6 5mg; Vitamin B12 15mg; Nicotinic acid 50mg; Pantothenic acid 15mg; Folic acid 1mg; Biotin 200mg; Iron 80mg; Copper 10mg; Manganese 100mg; Cobalt 500mg; Zinc 80mg; Iodine 1mg; Selenium 200mg; Molybdenum 500mg.

+To supply per kg diet:

Vitamin A 7,200IU; Vitamin D3 3,000IU; Vitamin K 1.8mg; VitaminB1 1.2mg; Vitamin B2 4.2mg; Vitamin B6 3mg; Vitamin B12 9mg; ; Nicotinic acid 30mg; Pantothenic acid 9mg; Folic acid 0.6mg; Biotin 120mg; Iron 48mg; Copper 16mg; Manganese 60mg; Cobalt 300mg; Zinc 48mg; Iodine 600mg; Selenium 120mg; Molybdenum 300mg. Vitamin E supplements for the experimental diets provided 5, 30 and 295 mg tocopherol acetate/kg respectively.

Table 5: Body weight of hens and egg production and shell characteristics at different ages up to 60 weeks of age in laying hens fed different levels of vitamin E (Objective 2).

Dietary Vitamin E (mg/kg)

Age (weeks) / 10 / 35 / 300 /

SED

/

Significance

Body weight / 15 / 1223 / 9.66(SEM)
(g) / 32 / 1644 / 1657 / 1617 / 44.3 / NS
60 / 1854 / 1729 / 1765 / 61.8 / NS
60# / 1830a / 1660b / 1764 / 61.6 / * (covar NS)
Egg / 60 / 70.4 / 68.8 / 76.1 / 4.59 / NS
production (%) / 60# / 78.0 / 76.8 / 79.9 / 1.69 / NS (covar ***)
Egg weight / 25 / 53.71 / 54.27 / 53.17 / 0.869 / NS
(g) / 40 / 60.08 / 57.53 / 58.75 / 1.246 / NS
60 / 60.08 / 59.83 / 60.39 / 0.599 / NS
60# / 60.53 / 59.47 / 60.46 / 0.630 / NS (covar NS)
Shell / 25 / 411.2a / 413.7a / 395.4b / 6.80 / *
thickness / 40 /

Not

/ measured
(mm) / 60 / 378.1 / 380.9 / 371.5 / 4.60 / NS
60# / 380.4 / 375.5 / 373.1 / 4.67 / NS (covar NS)
Breaking / 25 / 33.15 / 34.65a / 31.88b / 1.012 / *
strength (N) / 40 / 30.58 / 30.44 / 30.00 / 1.41 / NS
60 / 28.02 / 28.17 / 27.77 / 0.701 / NS
60# / 28.43 / 27.58 / 27.82 / 0.719 / NS (covar NS)

# with egg production during final 6 weeks as covariate and excluding those hens whose egg production during the last six weeks was < 15. N excluded =5, 7, 2

NS: not significant (P>0.05); * : P<0.05; **: P<0.01; ***P<0.001.

Table 6: Biomechanics and dimensions of the tibia of laying hens at different ages fed different levels of vitamin E (Objective 2).

Dietary Vitamin E (mg/kg)

Age (weeks) / 10 / 35 / 300 /

SED

/

Significance

Tibia / 15 / 234.0 / 5.51 (SEM)
breaking / 32 / 181 / 178 / 178 / 19.4 / NS
strength / 60 / 263 / 296 / 222 / 43.1 / NS
(N) / 60# / 211 / 197 / 194 / 18.2 / NS (covar*)
Tibia / 15 / 299143 / 8615 (SEM)
stiffness / 32 / 248428 / 225687 / 234099 / 23984 / NS
(N/m) / 60 / 430240 / 446606 / 359048 / 55467 / NS
60# / 346827 / 331028 / 331543 / 30391 / NS (covar*)
Tibia / 15 / 9.71 / 0.125 (SEM)
weight (g) / 32 / 9.83 / 9.80 / 9.93 / 0.261 / NS
60 / 10.63 / 11.07 / 10.33 / 0.135 / NS
60# / 10.40 / 10.45 / 10.31 / 0.304 / NS (covar NS)
Tibia length / 15 / 111.6 / 0.631 (SEM)
(mm) / 32 / 114.2 / 114.0 / 113.7 / 1.043 / NS
60 / 116.2 / 114.9 / 114.3 / 1.075 / NS
60# / 116.4a / 113.1b / 114.7 / 1.069 / * (covar *)
Tibia width / 15 / 6.11 / 0.062 (SEM)
(mid-shaft, / 32 / 6.35 / 6.41 / 6.34 / 0.88 / NS
mm) / 60 / 6.65 / 6.63 / 6.41 / 0.11 / P=0.06
60# / 6.67 / 6.58 / 6.42 / 0.11 / NS (covar NS)

# with egg production during final 6 weeks as covariate and excluding those hens whose egg production during the last six weeks was < 15. N excluded =5, 7, 2

NS: not significant (P>0.05); * : P<0.05; **: P<0.01; ***P<0.001.

Table 7: Radiographic density (RD) of the keel and radiographic densities and dimensions of the tibiae of laying hens at different ages fed different levels of vitamin E (Objective 2).

Dietary Vitamin E (mg/kg)