Role of vitamin D in broiler chicken skeletal muscle

development and growth

Dr. Jessica D. Starkey

Adjunct Professor of Meat and Muscle BiologyTexas Tech University

Lubbock, TX USA

Skeletal muscle anatomy

FasciclesIndividual Muscle fibers

Epimysium

Perimysium Endomysium

Skeletal muscle anatomy

Satellite cell location

Endomysium

Sarcolemma

Satellite Cell

Post-Mitotic Muscle Fiber

Nuclei

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

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 nucleiare post-mitotic and cannot divideSkeletal muscle growth occurs postnatally by hypertrophy

• Increasing the diameter and lengthof the muscle fibers developed in ovo

• Muscle fiber hypertrophy requires extensive myofibrillar protein synthesis

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

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-dehydrocholesterolUVB irradiation

(and other tissues)

hydroxylation

hydroxylation by 1- -hydroxylase

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-dehydrocholesterolUVB irradiation

hydroxylation

ROVIMIX Hy·D

Kidney(and other tissues)

hydroxylation by 1- -hydroxylase

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)

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?

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

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

Diet Composition

Starter Phase (d 0 – 14) Grower Phase (d 15 – 49)Treatment Treatment

Ingredient, % CTL Hy·D CTL Hy·DCorn 56.4 56.4 63.8 63.8Soybean meal 33.5 33.5 25.9 25.9Porcine meat and bone meal 4.0 4.0 5.0 5.0Soybean oil 2.9 2.9 2.8 2.8Dicalcium phosphate 1.2 1.2 0.8 0.8Limestone 0.6 0.6 0.4 0.4Sodium chloride 0.5 0.5 0.4 0.4DL-methionine 0.3 0.3 0.3 0.3Choline chloride 0.1 0.1 0.1 0.1Mineral premix 0.1 0.1 0.1 0.1Vitamin premix 0.2 0.2 0.2 0.2Lysine hydrochloride 0.1 0.1 0.1 0.1Threonine 0.1 0.1 0.1 0.1

Calculated valuesVitamin D3, IU per kg of diet 5,000 2,240 5,000 2,240Hy·D (25OHD3), IU per kg of diet 0 2,760 (69 µg) 0 2,760 (69 µg)Ca, % 1.0 1.0 0.9 0.9P, % 0.5 0.5 0.4 0.4ME, Mcal/kg of diet 3,050 3,050 3,130 3,130CP, % 22.3 22.3 19.9 19.9Lysine, % 1.4 1.4 1.2 1.2Methionine, % 0.7 0.7 0.6 0.6Tryptophan, % 0.3 0.3 0.2 0.2

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 BrdUPectoralis 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 densityHutton, et al., 2013

**

*

* *

* *

0

25

50

75

100

0 7 14 21 28 35 42 49

25O

HD

3, n

g/m

L

Days

CTL 25OHD3

Results:Broiler vitamin D status over time

*P < 0.01

Hutton, et al., 2013

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

Results:Growth Performance

CTL 25OHD3 SEM P-valueStarter (d 0 to 14)

ADG, g 38.2 37.6 0.55 0.41ADFI, g 49.0 48.0 0.26 0.01Gain:Feed 0.78 0.78 0.01 0.77

Grower (d 15 to 49)ADG, g 72.2 66.6 3.22 0.24ADFI, g 117.8 114.3 3.18 0.44Gain:Feed 0.61 0.58 0.03 0.40

Overall (d 0 to 49)ADG, g 72.2 66.6 3.22 0.24ADFI, g 116.0 113.3 1.82 0.31Gain:Feed 0.62 0.59 0.03 0.37

Hutton, et al., 2013

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

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

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 0.76Hutton, et al., 2013

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

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

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 0.97Hutton, et al., 2013

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

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

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

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)

Acknowledgements

DSM Nutritional Products• Drs. Turner and Litta

Kansas State University

• Grain Science Feed Mill StaffTexas Tech University

• Graduate and undergraduate research assistants, Faculty, and Staff