FBN Dummerstorf COST 925, Cork; May 13&14, 2008 Milestone 4 of COST action 925 Possible new technologies to estimate the muscle fibre number for use in

FBN DummerstorfCOST 925, Cork; May 13&14, 2008

Milestone 4 of COST action 925

Possible new technologies to estimate the muscle fibre number for use in animal

production

Based on contributions to workgroup meetings of the action


„The number of muscle fibres and the growth rate of the individual muscle fibre determine the growth rate of muscles postnatal. … Moreover, a larger number of smaller muscle fibres, in contrast to a low number of larger muscle fibres, will lead to meat with a better technological quality/fish quality, …“(technical annex of COST925)

Significance of muscle fibre number and size and muscle fibre type composition

evidence from phenotypes evidence from genotypes

(Perspectives for) new technologies to measure fibre number and fibre type distribution


IMF pH1 colour cookloss

shearforce

Type [%]

Type 1 0 -0,14 0,31** 0,1 -0,6

Type 2a -0,31** 0,2 0,12 0,8 0,13

Type 2b 0,21* 0,11 -0,35*** -0,14 -0,4

Ø [µm²]

Type 1 0,7 -0,5 0,1 -0,13 -0,6

Type 2a -0,15 0,1 0,7 -0,22* -0,7

Type 2b -0,8 -0,6 0,23* -0,15 0,1* p<0,05; **p<0,01; ***p<0,001

Muscle fibre type – meat quality

*Henckel et al., 1997


colour taste juiciness tenderness preference

Type [%]

Type 1 -0,6 -0,3 0,9 0,1 0,3

Type 2a -0,28** -0,32** -0,20 -0,22** -0,29**

Type 2b 0,24 0,23** 0,5 0,6 0,16

Ø [µm²]

Type 1 0,6 -0,1 0,20 0,1 0,3

Type 2a 0,9 0,1 0,20 0,1 0,3

Type 2b -0,6 -0,7 0,7 0,6 0,1*p<0,05;**p<0,01;***p<0,001

* Henckel et al., 1997

Muscle fibre type – meat quality


Genetic correlations

Pig M. longissimus, n = 2024 (Fiedler et al., 2004 )

Rehfeldt/24

Lean meat, %

Backfat, mm

Drip loss, %

Reflectance, %

pH 45 min p.m.

Fibre Number

0.38

- 0.05

- 0.05

- 0.05

0.13

Fibre Diameter

0.52

- 0.12

0.64

0.32

- 0.37

Giant Fibres

0.06

0.24

0.77

0.79

- 0.78

White -FTG (%)

0.01

0.34

0.19

0.28

- 0.29


•Muscle fibre number and fibre size

correlate positively with lean accretion.

•Large fibres, high glycolytic potential,

high percentages of white, glycolytic

(FTG, IIb) and of giant fibres are

associated with poor meat quality.

•Explanation for the antagonism of lean

accretion and meat quality

Rehfeldt/25

Conclusions


Heritability

Rehfeldt/29

Fibre number 0.22 – 0.88

Fibre size 0.12 – 0.34

Fibres white-FTG (%) 0.19 – 0.58

Giant fibres (%) 0.20

Staun (1968, 1972), Dietl et al. (1993), Fiedler et al. (1991, 2004),Lahucky and Uhrin (1995), Larzul et al. (1997)

FBN DummerstorfCOST 925, Cork; May 13&14, 2008 Rehfeldt/30

Selection responses (SI = 0.5)

direct correlated

Simulated selection without and with

muscle fibre traits

* P < 0.05; n = 2024 (Fiedler et al. Anim. Breed. Genet. 2004 )

Selection criteria

Loin muscle area (+)

Loin muscle area (+)Total fibre number (+)% white fibers (-)% giant fibers (-)

Loin musclearea (cm²)

0.79 *

0.50 *

Live weight (kg)

0.91 *

0.27 *

pH45

value

- 0.01*

0.01

Drip loss(%)

0.26 *

- 0.03

Selection response

FBN DummerstorfCOST 925, Cork; May 13&14, 2008 Rehfeldt/31

Selection responses (SI = 0.1)

direct correlated

Simulated selection without and with

muscle fibre traits

* P < 0.05; n = 2024 (Fiedler et al. Anim. Breed. Genet. 2004 )

Selection criteria

Drip loss (-)

Drip loss (-)Total fibre number (+)% white fibres (-)% giant fibres (-)

Drip loss(%)

- 0.52 *

- 0.65 *

Live weight (kg)

-0.66

0.09

pH45

value

0.03 *

0.05 *

Loin area (cm²)

- 0.05 *

0.16

Selection response


Divergent selection for residual feed intake(Lefaucheur et al. 2007, COST925, Viborg)

Large White: RFI- (efficient animals) vs. RFI+ (luxurious animals)

at 108 kg body weight: RFI- leaner, hypertrophy of all muscle fibre types and increase type IIBW percentage


Repeatability of muscle fibre measurements(Cerisuelo et al. 2007; COST925, Viborg)

Experimental design23 pigs, 5 M. longissimus samples/animal, intraclass correlation coeficients(ICC)Fiber typing by ATPase reaction

Results # of samplestotal number of muscle fibres 3 (ICC=0.8)Mean fibre areaFibre type composition >5relative area occupied by each fibre type

Type IIa with highest intra-animal variability

}


Muscle fibers and flesh mass(Bugeon et al. 2007; COST925, Viborg)

rainbow trout: high fillet yield (65%) vs. low fillet yield (56%), Carcass traits, flesh quality, fibre measures Mean fibre diameter similar, muscle fibre number higher

muscle fibre hyperplasia


Selection for growth rate in chicken(Duclos et al. 2004, COST925, Porto)

High growth rate vs. low growth ratHG with higher number of fibres at hatch, similar fibre size at hatch but fibres differ in size within a few days

Rapid myosin heavy chain isoforms: embryonic 3, neonatal, adult; measured by real time RT-PCRd18 in ovo: embryonic 3d7 post-hatch: neonatald43 post-hatch: adultHG with lower neonatal MHC mRNA at d7 and d43 post-hatch

Differentiation of muscle fibres altered with growth rate


Broiler breast meat in relation to muscle hypertrophy(Berri et al. 2004, COST925, Porto)

selection for rapid growth led to muscle hypertrophy

Muscle fibre cross-sectional area (CSA) correlated with(+) body weight, breast muscle weight and yield(-) glycogen reserves, glycolytical potential, lactate, pH fall(+) ultimate pH, colour, (-) drip

(Berri et al. 2005, COST925, Volos)

Variations in growth rates alter expression of markers of Satellite cell number: PAX7Proliferation: PCNAContractile differentiation of muscle fibres: MyHC


Cellular aspects of breast muscle development in chickens with high or low growth rate(Duclos et al. 2005, COST925, Volos)


Divergent selection for body weight at 63 days of age in rabbits(Gondret et al. 2005, COST925, Volos)

at 63 days of age (n=20; L: 2.32 kg < C: 2.62 kg < H: 2.87 kg) decrease in the weight and total cross-sectional area of the semitendinosus (ST) Mean cross-sectional area of ST myofibers lowest in the L (-15%,

P < 0.001), but it did not differ among H and C lines. total number and type frequency of the myofibers were similar in the three groups

at same weight (n = 20; 51 d, 57 d, and 63 d):not influence ST myofiber histological characteristics


QTL for meat and muscle traits LF1Ko LF24Ko LF1Schi LF24Schi pH1Ko pH24Ko pH24Schi Opto

DTypI

DTypIIa

DTypIIb

Dang

DRiesen

Dall

%TypI

%TypIIa

%TypIIb

%Riesen

%ang

#/cm²

#total

0

1

2

3

4

5

6

7

8

9

10

D_FTG

D_mean

cond24ham

Sscr27.8

4.5

0

2

4

6

8

10

12

14

pH24ML

#giF

Sscr15

4.6

7.3


Effects of the Compact mutant myostatin allele in a mouse line with extreme growth(Bünger et al. 2004, COST925, Porto)


Proteomics for identification of marker for muscle hypertrophyPicard et al. 2004, COST925 Porto

monogenic model: double muscled Belgium Bluemolygenic model: divergently selected Charolaisregulated 17 proteins

Transcriptomics for identification of myostatin-loss of functionCassar-Malek et al. 2005, COST925 Volos

260 dpc DM fetuses vs. normal fetusesdown: extracellular matrix, slow contractile protein up: regulation of transcrption, cell cycle, translation...longer proliferation, later differentiation


LEFAUCHEUR & ECOLAN, 2004 COST925, Porto)


05

1015202530354045

1 2 3 4 5 6 7 8

18S MyHC I MyHC IIa MyHC IIb MyHC IIx

Th

resh

old

cyc

le

(Ct)

Log CO

-3.68 -3.75 -3.77 -3.77 -3.76Slope

Standard curves


real time RT PCR vs. ATPase


Relative expression of MyHC isoforms in 3 pure breeds

MyHC I MyHC IIa MyHC IIx MyHC IIb


Relative expression of MyHC isoforms in discordant sib-pairs

DUPI

P < 0.05Small eye muscle area

Large eye muscle area


Relative expression of MyHC isoforms in discordant sib-pairs

DUMI

P < 0.05

P < 0.05


Microarray analysis

DuPi: F2Duroc x Pietrain

F0

F1

Trait-associated Expression:

Selection of 6 discordant sib-pairs from 572 F2 DUPI resource population for drip and pH

QTL-Genotyp-associated Expression:

Selection of animals according to their genotype at the QTL of SSC5 and SSC18:a total of 18 animals per genotype per QTL


Microarray analysis

Signal intensity

Probe level analysis

Quality check

Variance Stabilization

Logarithmic scaling

PLIER, MAS 5 (Filter on present/absent)

Significance analysis

Mixed Model (SAS): fixed effects: `family´, `sex´, `QTL-genotype´random effect: `day of slaugther´co-variable: `weight at slaugther´

Pearson correlation


Correlation of MyHC isoform expression and DRIP and pH

DRIP pH1

MyHC I 0.16 (0.16) 0.33 (0.004)

MyHC IIa -0.12 (0.29) 0.33 (0.004)

MyHC IIx -0.08 (0.51) 0.41 (0.0002)

MyHC IIb 0.41 (0.0003) 0.12 (0.30)


Real-time PCR primers and probesHemmings et al. 2007, COST925, Viborg

• MHC mRNA isoforms are similar but do vary at the 5’ end• Specific primers and probes were designed within this region • Porcine IIB and IIX mRNA are identical in the region to which

real-time PCR primers and probe are designed– IIX primers and probe very likely to amplify IIB if present

5’ UTR5’ UTR CODING CODING SEQUENCESEQUENCEMHC I

MHC IIA

MHC IIX

MHC IIB?


15

20

25

30

35

40

-5.5 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5

log10 DNA concentration

cp

Effect of type IIA and IIX cDNA on type I standard curve

Hemmings et al. 2007, COST925, Viborg


Effect of type IIX and I cDNA on type IIA standard curve


15

20

25

30

35

40

-5.5 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5


cp


Effect of type I and IIA cDNA on type IIX standard curve


15

20

25

30

35

40

-5.5 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5


cp


0

5

10

15

20

25

30

35

40

45

50

d1 d60 d120 d180

AGE

% A

DU

LT M

HC

MHCIMHCIIAMHCIIX

MHC isoform expression at different ages in sheep



Real-time PCR primers and probes Hemmings et al. 2007, COST925, Viborg

• No interference was observed between isoforms

– Primers and probes are specific

• Primers and probes should detect all sheep adult MHC mRNA

• Therefore we propose that each isoform can be expressed relative to total MHC expression (I + IIA +IIX/IIB)


Myosin heavy chain expression in different muscles


• MHC Real-time PCR of samples of supraspinatus (SS), semitendinosus (ST) and longissimus dorsi (LD) muscles from lambs 65 ± 2 days of age (n=10)

SS ST LD SED p

%MHCI 34.7 13.4 7.3 5.00 <0.001

%MHCIIA 21.2 21.5 27.5 2.22 0.018

%MHCIIX/IIB 44.1 65.1 65.2 6.02 0.003


Application of Functional near infrared spectroscopy (fNIRS) to estimate numbers and types of muscle fibres in sheep (E. Sirin et al. 2007)

•Functional near infrared spectroscopy (fNIRS) – local concentration changes of oxygenated and deoxygenated hemoglobin

– oxygen consumption of tissues


Proportion of oxidative fibres, %

Decr

ease

in o

xygen

consu

mpti

on (

µm

ol)

Positive correlation between the proportion of oxidative muscle fibres (SDH-staining) and decrease in oxygen consumption

-400

-200

0

200

400

40 45 50 55 60 65 70

at 120 d of age:P<0.05; r=0,6

at 164 d of age: p<0.06 r=0.7 (E. Sirin et al. 2007)


0

5

10

15

20

25

30

0 2 4 6 8 10 12 14 16

Negative correlation between between the proportion of type I muscle fibres (m-ATPase activity) and the decrease in oxygen consumption

Decrease in oxygen consumption (µmol/sec.)

r=-0.68; P<0.05

Pro

port

ion o

f ty

pe I m

usc

le

fibre

s (%

)

(E. Sirin et al. 2007)


Negative correlation between the proportion of type IIB muscle fibres and the ratio of the decrease in oxygen consumption

30

40

50

60

70

0,30 0,40 0,50 0,60 0,70

Ratio of the decrease in oxygen consumption (µmol/sec.)

r=-0.79; P<0.05

Pro

port

ion o

f ty

pe IIB

musc

le

fibre

s (%

)



0

100

200

300

400

500

0 2 4 6 8 10

r=-0.69; P<0.05

Change in oxygen consumption (µmol/sec)

Num

ber

of

fibers

/unit

are

a

Negative correlation between the number of muscle fibres and change in oxygen consumption per sec.



• fNIRS can be used to study the oxygenation status of muscle tissue in sheep

• fNIRS may be a new method to determine the metabolic types of muscle fibres in sheep non-invasively.

• further studies are required to optimise how fNIRS can reflect the muscle fibre numbers and metabolic types in various muscle samples at different age of animals

• It’s ability to determine meat quality based on a muscle fibre metabolism would be a valuable tool



Effects of muscle fiber type and size on EMG median frequency and conduction velocity

Kupa et al. 1995, J Appl Physiol

in vitro method for comparing surface-detected electromyographic median frequency (MF) and conduction velocity (CV) parameters with histochemical measurements of muscle fiber type composition and cross-sectional area (CSA)


Non-invasive measurement of muscle properties by surface electromyography (EMG)(Tygesen et al. 2004, Andersen et al. 2007)

Reference electrode

Stimulating electrodes

Recording electrodes

m. Biceps femoris

m. Longissimus dorsimeasurement of repeated action potentials in response to stimulation: peaks of variable amplitude and duration

compound muscle action potentials (CMAP)

Start of stimulation

mV

Time (s)

40

0

End of stimulation


EMG reflects postnatal growth in lambTygesen et al. 2004

5 10 15 20 25 30 35-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

BicepsLongissimus

Are

a (

mV

S-1

)

5 10 15 20 25 30 35

-25000

0

25000

50000

75000

100000

Slo

peL

(m

V S

-1)

P=0.006

5 10 15 20 25 30 35

-90000

-80000

-70000

-60000

-50000

-40000

-30000

-20000

-10000

0

10000

P<0.0001

Slo

peT

(m

V S

-1)

P=0.014


EMG for estimation of shear forceAnderson et al. 2007

correlations of CAMP parameters with shear force observed in pigs: shear force (N) = 62 – 183 x CAMP signal area (mV/s) (r = - 0.8, p=0.01)CMAP predictive for shear force

tenderness positively correlates with type I fibrestenderness negatively correlates with strength of perimysium

strong EMG signals with large area arise from a few large-cross-sectional area fibers, i.e. relatively little perimysium per unit volume of muscle tissue

perspective to link variation in fibre type frequency with meat quality


Determining the muscle fibre lengthPoulanne and Räsänen, 2007

M longissiumus dorsi caudal of 5th thoracic vertebraslice shown in Acubes of 1x1x1 cm shown in Bpieces of 300 mgseparation of fibres: Hooper method: HNO3 treatment, vigorous shakingDetermination of length of fractions (cumulative)Counting of tapered end 20000 fractions! (3 muscle x 3 slides x 5 cubes x >400 factions)

Length of fibre = ∑total length of fraction / (number of ends/2)

A B

Poulanne and Räsänen, 2007

FBN DummerstorfCOST 925, Cork; May 13&14, 2008Poulanne and Räsänen, 2007


number of fibres per muscle cross section is on average 1.2 million the total number of fibres in the whole muscle (length 60–70 cm, weight ca 4.0 kg) is about 4.2 million 1 fibre (173 mm long and diameter 40 μm) has a weight of about 1 mg.

For the first time the length of porcine muscle fibres determinedvery laborious, hard to automatize tapered ends are rare (4-14 within 2000 fractions per slide)

Poulanne and Räsänen, 2007

Determining the muscle fibre length


Serial sections of longissimus muscle from a Large White pig at 62 kg BW (131 d of age). mATPase after at pH 4.35 (A) and succino-dehydrogenase (SDH) activity (B)immunostaining with type I (C), IIa (E), IIx (G), and IIb (I) monoclonal antibodiesin situ hybridization for myosin heavy chain (MyHC) I (D), IIa (F), IIx (H), and IIb (J)

Lefaucheur et al.2004, J Anim Sci





Possible new technologies to estimate the muscle fibre number for use in animal production

Evidence for the importance of muscle fibre number and muscle fibre type distribution for growth and meat quality traits arise from various experiments on the level of

- phenotypic correlations, - selection experiments- positional and functional genomics information

within the COST 925-real time PCR for MyHC isoforms-In situ hybridisation, immunohistochemestry-functional near infrared spectroscopy (fNIRS), -electromyographymainly for the differentiation of muscle fibre type providing

-new phenotypes for selection and -new insight into the physiology of muscle fibre formation

Documents

FBN Dummerstorf COST 925, Cork; May 13&14, 2008 Milestone 4 of COST action 925 Possible new technologies to estimate the muscle fibre number for use in