Conclusions of the research: Feeding 25OHD3 in place of the majority of dietary D3 improved broiler chicken vitamin D status and resulted in a satellite cell-mediated muscle hypertrophy response in breast (PM), but not thigh (BF) muscles (Hutton et al., 2013) ƒ The differential response in functionally different muscles as well as the cell signaling mechanisms by which skeletal muscle satellite cells respond to improved vitamin D status resulting from dietary Hy·D supplementation will require further investigation.

1. Role of vitamin D in
broiler chicken skeletal muscle
development and growth
Dr. Jessica D. Starkey
Adjunct Professor of Meat and Muscle Biology
Texas Tech University
Lubbock, TX USA

2. Skeletal muscle anatomy
FasciclesIndividual Muscle fibers
Epimysium
Perimysium Endomysium

3. Skeletal muscle anatomy

4. Satellite cell location
Endomysium
Sarcolemma
Satellite Cell
Post-Mitotic
Muscle Fiber
Nuclei

5. Satellite cell location and identification
Satellite cells are a
heterogeneous population of
stem cells that express various
combinations of several well-
known markers at different
times throughout myogenesis
and post-hatch skeletal muscle
growth
• Pax7
• Myf-5
• CD34
• M-Cadherin
• Caveolin-1
• Integrin alpha-7 and beta-1
• Sca-1

6. Skeletal muscle growth
Skeletal muscle fiber number is
fixed at hatch
• No significant amount of muscle
fiber hyperplasia post-hatch
Skeletal muscle fibers are
multinucleated cells and those nuclei
are post-mitotic and cannot divide
Skeletal muscle growth occurs
postnatally by hypertrophy
• Increasing the diameter and length
of the muscle fibers developed in ovo
• Muscle fiber hypertrophy requires
extensive myofibrillar protein
synthesis

7. Skeletal muscle growth
Skeletal muscle fiber hypertrophy requires additional DNA (nuclei) as the
muscle fibers grow longer and larger in diameter
• Satellite cells (muscle stem cells) are the source of the additional DNA
• Satellite cells proliferate and fuse with existing muscle fibers
• More DNA = More capacity for myofibrillar protein synthesis
Hypertrophied
Myofiber
SC Proliferation
& Fusion
Resting Myofiber

8. Vitamin D metabolism and ROVIMIX Hy·D®
Liver
Kidney
Dietary Vitamin D3
Cholecalciferol (Vitamin D3)
25-hydroxycholecalciferol (25OHD3, Circulating form)
1,25-dihydroxycholecalciferol (Active form)
GITSkin
7-dehydrocholesterol
UVB irradiation
(and other tissues)
hydroxylation
hydroxylation by 1- -hydroxylase

9. Vitamin D metabolism and ROVIMIX Hy·D®
Liver
Dietary Vitamin D3
Cholecalciferol (Vitamin D3)
25-hydroxycholecalciferol (25OHD3, Circulating form)
1,25-dihydroxycholecalciferol (Active form)
GITSkin
7-dehydrocholesterol
UVB irradiation
hydroxylation
ROVIMIX Hy·D
Kidney
(and other tissues)
hydroxylation by 1- -hydroxylase

10. The vitamin D and skeletal muscle connection
Lack of vitamin D receptor (VDR) disrupts muscle growth and development
(Endo et al., 2003)
• VDR knockout mice have 20% smaller muscle fibers
• Aberrant myogenic regulatory factor expression
Vitamin D status is positively associated with human muscle performance
(Ceglia, 2009) and supplementation increases fast-twitch muscle fiber
diameter (Sato et al., 2005)
Muscle fibers and myoblasts express VDR and 1- -hydroxylase (Boland,
1985; Zanello et al., 1997; Ceglia, 2010)
Low maternal vitamin D status can negatively impact lean tissue
development in human fetuses (Morley et al., 2006; Pasco et al., 2008)
Maternal supplementation with Hy·D before and during gestation resulted in
fetal pigs with significantly more muscle fibers in their loin muscles at d 90
of gestation (Hines, et al., 2013)
Myoblasts from fetal pigs with improved vitamin D status remain
proliferative longer in culture (Hines et al., 2013)

11. The vitamin D and skeletal muscle connection
Feeding Hy·D (25OHD3) to broiler chickens in place of at least a portion of
D3 in the diet has previously been shown to result in:
• improved vitamin D status as a result of more efficient absorption in the
upper intestine (Bar et al., 1980)
• increased body weight (Bar et al., 2003; Fritts and Waldroup, 2003)
• improved feed conversion efficiency (Gain:Feed) and meat yield
(Cantor and Bacon, 1978; Yarger, et al. 1995)
– Can improved vitamin D status cause changes in satellite cell
activity?
– Are satellite cells involved in the muscle yield response?
– Is the yield response a direct result of muscle fiber hypertrophy?

12. Effect of vitamin D status improvement with
25-hydroxycholecalciferol on broiler chicken
growth performance, satellite cell activity, and
skeletal muscle growth characteristics
Hutton et al., 2013. J. Anim. Sci. in review

13. Objective: to determine if improvement of broiler chicken vitamin D status
as a result of replacing a significant proportion of dietary vitamin D3 with
Hy·D (25OHD3) could influence satellite cell activity and skeletal muscle
growth characteristics in 2 functionally different muscles
Day old, male Ross 708 broiler chicks (n = 150)
Start-Grow Cages (Alternative Design)
• Random assignment to 1 of 12 cages (n = 12 or 13 per cage)
– On d 42, birds were redistributed within treatment to 9 cages (n = 4 or 5
per cage)
• 23 h of light per day in a temperature-
controlled facility
• Ad libitum access to feed and water
Hutton, et al., 2013
Materials and Methods

14. Diet Composition
Starter Phase (d 0 – 14) Grower Phase (d 15 – 49)
Treatment Treatment
Ingredient, % CTL Hy·D CTL Hy·D
Corn 56.4 56.4 63.8 63.8
Soybean meal 33.5 33.5 25.9 25.9
Porcine meat and bone meal 4.0 4.0 5.0 5.0
Soybean oil 2.9 2.9 2.8 2.8
Dicalcium phosphate 1.2 1.2 0.8 0.8
Limestone 0.6 0.6 0.4 0.4
Sodium chloride 0.5 0.5 0.4 0.4
DL-methionine 0.3 0.3 0.3 0.3
Choline chloride 0.1 0.1 0.1 0.1
Mineral premix 0.1 0.1 0.1 0.1
Vitamin premix 0.2 0.2 0.2 0.2
Lysine hydrochloride 0.1 0.1 0.1 0.1
Threonine 0.1 0.1 0.1 0.1
Calculated values
Vitamin D3, IU per kg of diet 5,000 2,240 5,000 2,240
Hy·D (25OHD3), IU per kg of diet 0 2,760 (69 µg) 0 2,760 (69 µg)
Ca, % 1.0 1.0 0.9 0.9
P, % 0.5 0.5 0.4 0.4
ME, Mcal/kg of diet 3,050 3,050 3,130 3,130
CP, % 22.3 22.3 19.9 19.9
Lysine, % 1.4 1.4 1.2 1.2
Methionine, % 0.7 0.7 0.6 0.6
Tryptophan, % 0.3 0.3 0.2 0.2

15. Materials and Methods
Serial harvest 10 birds/treatment per week (d 0, 7, 14, 21, 28, 35, 42, 49)
Bromodeoxyuridine (BrdU) injection to label mitotically active cells
Serum collection to determine circulating 25OHD3 concentration
Euthanasia by CO2 asphyxiation and cervical dislocation 2 h after BrdU
Pectoralis major (PM) and Biceps femoris (BF) muscles
• Individual muscle weights
• Cryohistology, Immunofluorescence, and Microscopy
– Satellite cell enumeration and mitotic activity
• Myf-5+ and Pax7+
• Myf-5+;BrdU+ and Pax7+;BrdU+
– Muscle fiber cross-sectional area, µm2
– Nuclear density
Hutton, et al., 2013

16. *
*
*
* *
* *
0
25
50
75
100
0 7 14 21 28 35 42 49
25OHD3,ng/mL
Days
CTL 25OHD3
Results:
Broiler vitamin D status over time
*P < 0.01
Hutton, et al., 2013

17. Results:
Body weight, g
CTL 25OHD3 SEM P-value
d 7 0.23 0.23 0.002 0.81
d 14 0.58 0.58 0.007 0.69
d 21 1.13 1.15 0.015 0.52
d 28 1.77 1.77 0.030 0.97
d 35 2.46 2.46 0.045 0.92
d 42 2.94 2.89 0.086 0.71
d 49 3.42 3.31 0.18 0.63
Hutton, et al., 2013

18. Results:
Growth Performance
CTL 25OHD3 SEM P-value
Starter (d 0 to 14)
ADG, g 38.2 37.6 0.55 0.41
ADFI, g 49.0 48.0 0.26 0.01
Gain:Feed 0.78 0.78 0.01 0.77
Grower (d 15 to 49)
ADG, g 72.2 66.6 3.22 0.24
ADFI, g 117.8 114.3 3.18 0.44
Gain:Feed 0.61 0.58 0.03 0.40
Overall (d 0 to 49)
ADG, g 72.2 66.6 3.22 0.24
ADFI, g 116.0 113.3 1.82 0.31
Gain:Feed 0.62 0.59 0.03 0.37
Hutton, et al., 2013

19. Pectoralis major Biceps Femoris
CTL 25OHD3 SEM P-value CTL 25OHD3 SEM P-value
d 7 7.7 8.0 0.4 0.58 1.8 1.7 0.2 0.61
d 14 26.9 29.5 1.6 0.26 3.4 3.6 0.2 0.62
d 21 60.5 64.1 2.8 0.37 6.7 6.9 0.4 0.71
d 28 103.6 104.8 5.8 0.88 10.8 12.2 0.9 0.26
d 35 168.6 177.5 6.7 0.37 20.8 21.5 1.9 0.79
d 42 223.4 231.8 11.3 0.60 21.5 25.9 1.9 0.79
d 49 305.3 282.8 16.6 0.48 35.3 29.3 2.3 0.11
Results:
Muscle weights, g
Hutton, et al., 2013

20. Pectoralis major Biceps Femoris
CTL 25OHD3 SEM P-value CTL 25OHD3 SEM P-value
d 7 3.51 3.56 0.04 0.94 0.84 0.76 0.19 0.84
d 14 4.87 5.40 0.31 0.43 0.62 0.65 0.11 0.87
d 21 5.65 5.57 0.22 0.84 0.62 0.60 0.08 0.84
d 28 6.36 6.46 0.19 0.78 0.67 0.75 0.07 0.51
d 35 6.89 7.17 0.20 0.32 0.85 0.87 0.06 0.85
d 42 7.45 7.85 0.21 0.19 0.86 0.90 0.05 0.70
d 49 8.52 8.54 0.19 0.95 0.99 0.90 0.05 0.47
Results:
Muscle yield as a proportion of body weight
Hutton, et al., 2013

21. Results:
Myf-5+ satellite cell density per mm2
Pectoralis major Biceps Femoris
CTL 25OHD3 SEM P-value CTL 25OHD3 SEM P-value
d 7 1,209 1,270 224 0.85 1,015 1,151 178 0.59
d 14 66 184 47 0.09 130 183 40 0.36
d 21 219 164 50 0.45 116 53 47 0.35
d 28 142 185 47 0.52 106 117 32 0.81
d 35 120 113 30 0.87 134 100 29 0.42
d 42 28 29 5 0.94 110 84 23 0.43
d 49 192 137 51 0.40 118 97 49 0.76
Hutton, et al., 2013

22. Pectoralis major Biceps Femoris
CTL 25OHD3 SEM P-value CTL 25OHD3 SEM P-value
d 7 18 25 6 0.42 3 4 2 0.56
d 14 5 5 2 0.89 7 4 2 0.39
d 21 3 6 1 0.01 11 10 2 0.48
d 28 3 4 2 0.53 6 3 2 0.13
d 35 2 2 1 0.43 4 5 1 0.45
d 42 8 7 2 0.83 3 5 1 0.17
d 49 6 3 1 0.12 3 2 1 0.26
Results:
Mitotically active Pax7+ satellite cell density per mm2
Hutton, et al., 2013

23. Pectoralis major Biceps Femoris
CTL 25OHD3 SEM P-value CTL 25OHD3 SEM P-value
d 7 280 321 23 0.21 282 138 51 0.05
d 14 114 113 23 0.97 107 79 21 0.35
d 21 40 52 12 0.47 33 22 4 0.19
d 28 53 41 11 0.45 56 52 13 0.82
d 35 49 62 5 0.07 70 61 12 0.62
d 42 81 86 22 0.87 56 53 9 0.82
d 49 77 82 13 0.77 105 93 8 0.25
Results:
Pax7+ satellite cell density per mm2
Hutton, et al., 2013

24. Results:
Nuclear density per mm2
Pectoralis major Biceps Femoris
CTL 25OHD3 SEM P-value CTL 25OHD3 SEM P-value
d 7 3,959 3,940 58 0.93 3,549 3,696 167 0.54
d 14 2,832 2,762 158 0.75 3,176 3,249 211 0.80
d 21 2,334 2,446 76 0.31 2,236 2,278 63 0.64
d 28 2,009 2,251 85 0.05 2,086 2,216 72 0.22
d 35 1,987 1,951 101 0.82 1,955 1,894 45 0.35
d 42 1,736 1,883 112 0.36 1,764 1,756 68 0.93
d 49 1,799 1,708 109 0.56 1,824 1,818 101 0.97
Hutton, et al., 2013

25. Pectoralis major Biceps Femoris
CTL 25OHD3 SEM P-value CTL 25OHD3 SEM P-value
d 7 854 859 375 0.99 527 736 154 0.33
d 14 1,175 1,476 96 0.99 1,417 1,172 120 0.17
d 21 2,368 2,568 151 0.35 1,944 2,202 112 0.11
d 28 3,668 3,695 216 0.93 2,123 2,232 98 0.43
d 35 4,680 4,723 287 0.92 3,241 3,251 178 0.97
d 42 5,635 5,734 440 0.88 3,736 3,928 259 0.61
d 49 6,173 7,077 363 0.09 4,020 3,849 214 0.58
Results:
Skeletal muscle fiber cross-sectional area, m2
Hutton, et al., 2013

26. Results:
Summary
Feeding 25OHD3 resulted in:
Improved vitamin D status from d 7 to 49
PM (breast) muscle
• More Myf-5+ satellite cells on d 14
• More mitotically active Pax7+ satellite cells on d 21
• Greater nuclear density on d 28
• More Pax7+ satellite cells on d 35
• Larger muscle fiber cross-sectional area on d 49
• Satellite cell-mediated skeletal muscle hypertrophy response
SC Proliferation
& Fusion
Hypertrophied
Myofiber
Hutton, et al., 2013

27. Conclusions
Feeding 25OHD3 in place of the
majority of dietary D3 improved broiler
chicken vitamin D status and resulted in
a satellite cell-mediated muscle
hypertrophy response in breast (PM), but
not thigh (BF) muscles (Hutton et al.,
2013)
The differential response in functionally
different muscles as well as the cell
signaling mechanisms by which skeletal
muscle satellite cells respond to
improved vitamin D status resulting from
dietary Hy·D supplementation will
require further investigation

28. Conclusions
A 0.4% (8.4 g/bird) increase in yield as a
proportion of BW observed on 6-wk-old
Hy·D-fed broilers translates into an additional
50,000 metric tons of poultry protein from the
approx. 6 billion broilers marketed in the EU
in 2012
The results of the Hutton et al. (2013) study
provide initial evidence toward unraveling the
mechanism by which birds supplemented
with Hy·D (25OHD3) have exhibited
increased BW, Gain:Feed, as well as meat
yield compared with those fed only D3
(Cantor and Bacon, 1978; Yarger, et al. 1995;
Bar et al., 2003; Fritts and Waldroup, 2003)

29. Acknowledgements
DSM Nutritional Products
• Drs. Turner and Litta
Kansas State University
• Grain Science Feed Mill Staff
Texas Tech University
• Graduate and undergraduate
research assistants, Faculty,
and Staff