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Milestone 4 of COST action 925

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.

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Milestone 4 of COST action 925

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  1. 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

  2. „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

  3. Muscle fibre type – meat quality *Henckel et al., 1997

  4. Muscle fibre type – meat quality * Henckel et al., 1997

  5. Genetic correlations 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 Lean meat, % Backfat, mm Drip loss, % Reflectance, % pH 45 min p.m. Pig M. longissimus, n = 2024 (Fiedler et al., 2004 ) Rehfeldt/24

  6. Conclusions • 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

  7. Heritability 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) Rehfeldt/29

  8. Selection response Loin muscle area (cm²) 0.79 * 0.50 * Drip loss (%) 0.26 * - 0.03 Live weight (kg) 0.91 * 0.27 * pH45 value - 0.01* 0.01 Selection criteria Loin musclearea (+) Loin musclearea (+) Total fibre number (+) % white fibers (-) % giant fibers (-) Simulated selection without and with muscle fibre traits Selection responses (SI = 0.5) direct correlated *P < 0.05; n = 2024 (Fiedler et al. Anim. Breed. Genet. 2004) Rehfeldt/30

  9. Selection response Loin area (cm²) - 0.05 * 0.16 Live weight (kg) -0.66 0.09 Drip loss (%) - 0.52 * - 0.65 * pH45 value 0.03 * 0.05 * Selection criteria Drip loss (-) Drip loss (-) Total fibre number (+) % white fibres (-) % giant fibres (-) Simulated selection without and with muscle fibre traits Selection responses (SI = 0.1) direct correlated *P < 0.05; n = 2024 (Fiedler et al. Anim. Breed. Genet. 2004) Rehfeldt/31

  10. 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

  11. Repeatability of muscle fibre measurements (Cerisuelo et al. 2007; COST925, Viborg) • Experimental design • 23 pigs, • 5 M. longissimus samples/animal, • intraclass correlation coeficients(ICC) • Fiber typing by ATPase reaction • Results # of samples • total number of muscle fibres 3 (ICC=0.8) • Mean fibre area • Fibre type composition >5 • relative area occupied by each fibre type • Type IIa with highest intra-animal variability }

  12. 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

  13. Selection for growth rate in chicken • (Duclos et al. 2004, COST925, Porto) • High growth rate vs. low growth rat • HG 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-PCR • d18 in ovo: embryonic 3 • d7 post-hatch: neonatal • d43 post-hatch: adult • HG with lower neonatal MHC mRNA at d7 and d43 post-hatch • Differentiation of muscle fibres altered with growth rate

  14. 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: PAX7 Proliferation: PCNA Contractile differentiation of muscle fibres: MyHC

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

  16. 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

  17. Sscr15 7.3 4.6 DTypI DTypIIa DTypIIb Dang DRiesen Dall %TypI %TypIIa %TypIIb %Riesen %ang #/cm² #total LF1Ko LF24Ko LF1Schi LF24Schi pH1Ko pH24Ko pH24Schi Opto Sscr2 7.8 4.5 QTL for meat and muscle traits

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

  19. Proteomics for identification of marker for muscle hypertrophy Picard et al. 2004, COST925 Porto monogenic model: double muscled Belgium Blue molygenic model: divergently selected Charolais regulated 17 proteins Transcriptomics for identification of myostatin-loss of function Cassar-Malek et al. 2005, COST925 Volos 260 dpc DM fetuses vs. normal fetuses down: extracellular matrix, slow contractile protein up: regulation of transcrption, cell cycle, translation... longer proliferation, later differentiation

  20. LEFAUCHEUR & ECOLAN, 2004 COST925, Porto)

  21. Standard curves Thresholdcycle(Ct) Log CO -3.77 -3.77 -3.76 -3.68 -3.75 Slope

  22. real time RT PCR vs. ATPase

  23. Relative expression of MyHC isoforms in 3 pure breeds MyHC I MyHC IIa MyHC IIx MyHC IIb

  24. Relative expression of MyHC isoforms in discordant sib-pairs DUPI P < 0.05 Small eye muscle area Large eye muscle area

  25. Relative expression of MyHC isoforms in discordant sib-pairs DUMI P < 0.05 P < 0.05

  26. Microarray analysis Trait-associated Expression: Selection of 6 discordant sib-pairs from 572 F2 DUPI resource population for drip and pH F0 F1 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 DuPi: F2 Duroc x Pietrain

  27. Microarray analysis Signal intensity Probe level analysis Quality check PLIER, MAS 5 (Filter on present/absent) Variance Stabilization Logarithmic scaling Significance analysis Mixed Model (SAS): fixed effects: `family´, `sex´, `QTL- genotype´ random effect: `day of slaugther´ co-variable: `weight at slaugther´ Pearson correlation

  28. Correlation of MyHC isoform expression and DRIP and pH

  29. 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’ UTR CODING SEQUENCE MHC I MHC IIA MHC IIX MHC IIB? Real-time PCR primers and probesHemmings et al. 2007, COST925, Viborg

  30. Effect of type IIA and IIX cDNA on type I standard curveHemmings et al. 2007, COST925, Viborg

  31. Effect of type IIX and I cDNA on type IIA standard curveHemmings et al. 2007, COST925, Viborg

  32. Effect of type I and IIA cDNA on type IIX standard curve Hemmings et al. 2007, COST925, Viborg

  33. MHC isoform expression at different ages in sheepHemmings et al. 2007, COST925, Viborg

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

  35. Myosin heavy chain expression in different musclesHemmings et al. 2007, COST925, Viborg 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)

  36. 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

  37. Positive correlation between the proportion of oxidative muscle fibres(SDH-staining) and decrease in oxygen consumption Decrease in oxygen consumption (µmol) Proportion of oxidative fibres, % 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)

  38. Negative correlation betweenbetween the proportion of type I muscle fibres (m-ATPase activity) and the decrease in oxygen consumption Proportion of type I muscle fibres (%) Decrease in oxygen consumption (µmol/sec.) r=-0.68; P<0.05 (E. Sirin et al. 2007)

  39. Negative correlation betweentheproportion of type IIB muscle fibres and the ratio of the decrease in oxygen consumption Proportion of type IIB muscle fibres (%) Ratio of the decrease in oxygen consumption (µmol/sec.) r=-0.79; P<0.05 (E. Sirin et al. 2007)

  40. Negative correlation betweenthe number of muscle fibres and change in oxygen consumption per sec. Number of fibers/unit area Change in oxygen consumption (µmol/sec) r=-0.69; P<0.05 (E. Sirin et al. 2007)

  41. 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 (E. Sirin et al. 2007)

  42. 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)

  43. m. Longissimus dorsi m. Biceps femoris Reference electrode Stimulating electrodes Recording electrodes mV 40 0 Time (s) Start of stimulation End of stimulation Non-invasive measurement of muscle properties by surface electromyography (EMG) (Tygesen et al. 2004, Andersen et al. 2007) measurement of repeated action potentials in response to stimulation:  peaks of variable amplitude and duration • compound muscle action potentials (CMAP)

  44. EMG reflects postnatal growth in lamb Tygesen et al. 2004

  45. EMG for estimation of shear force Anderson 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 fibres tenderness 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

  46. Determining the muscle fibre length Poulanne and Räsänen, 2007 A B • M longissiumus dorsi caudal of 5th thoracic vertebra • slice shown in A • cubes of 1x1x1 cm shown in B • pieces of 300 mg • separation of fibres: Hooper method: HNO3 treatment, vigorous shaking • Determination 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) Poulanne and Räsänen, 2007

  47. Poulanne and Räsänen, 2007

  48. Determining the muscle fibre length • 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 determined • very laborious, hard to automatize • tapered ends are rare (4-14 within 2000 fractions per slide) Poulanne and Räsänen, 2007

  49. 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 antibodies • in situ hybridization for myosin heavy chain (MyHC) I (D), IIa (F), IIx (H), and IIb (J) Lefaucheur et al.2004, J Anim Sci

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