Craggs, Lucinda
(2010)
Manipulation of growth & meat quality by vitamin D and its analogues.
PhD thesis, University of Nottingham.
Abstract
Recent published work indicates a role for pre-slaughter dietary vitamin D supplementation to promote post mortem meat tenderization in cattle (Montgomery et al., 2000; Foote et al., 2004; Montogmery et al., 2004), pigs (Wilborn et al., 2004) and sheep (Wiegand et al., 2001; Boleman et al., 2004). The hypothesis being that vitamin D supplementation at supra-nutritional levels is able to cause increases in the calcium status of the animals, increasing the activity of the calcium-dependant proteolytic enzymes, the calpains, which are responsible for meat tenderization (Koohmaraie & Geesink, 2006). Muscle fibre type is a variable factor in muscle and is related to meat quality (Klont et al., 1998). Vitamin D has been suggested to play a role in regulating skeletal muscle function through the creation of vitamin D receptor knockout mice models (Endo et al., 2003) and observations that muscle weakness and falling risk in vitamin D deficient patients is linked to a loss of fast muscle fibres (Aniansson et al., 1986; Larsson et al., 1979; Sorenson et al., 1979; Sato et al., 2005).
This thesis investigated two vitamin D pre-slaughter diet regimes on their effects on meat quality of the most economically important cut of the carcass, the longissimus dorsi (LD) muscle (Molina et al., 2005) and the expression of the calpain system. Trial 1 fed sheep vitamin D at 2.0 X 10[superscript]6 IU/day for four days prior to slaughter and found that this had no effect on shear force of LD chops. Calcium levels were unchanged but mRNA levels of calpain I and II were increased 3.7 and 10% respectively (P=0.099 and P=0.014) but there was no effect on calpastatin mRNA nor changes in the calpain system at the protein level. Trial 2 fed sheep the same dose of vitamin D for 7 days with an additional calcium bolus, resulting in a 10% increase in calcium concentrations of both serum and LD. Toughness of LD chops was increased (P<0.01), there was no effect on mRNA of the calpain system but there was an increase in the protein levels of calpain II and calpastatin by 16 and 17% respectively (P=0.05 and P=0.087).
A microarray study of rat primary myoblasts treated with 1,25(OH)[subscript]2D[subscript]3 for 24 hours highlighted a number of responsive genes significantly up and down regulated 1.5 fold or more (P<0.05). Pathway analysis identified novel targets of 1,25(OH)[subscript]2D[subscript]3 with a possible relationship to muscle growth and function; these included C/EBPβ metallothionein 2A and the MAPK, ERK. Three muscle cell strains, the rat primary muscle cells, L6 Aston and C2C12, were assessed for myosin heavy chain (MHC) gene expression using semi-quantitative PCR and western blotting analysis. The muscle cell line demonstrating the broadest range of MHC genes relevant to mature muscle tissue was used for the final experiments; this was the C2C12 cell line demonstrating expression of the slow MHC 1/β, an isoform which was absent or showed much lower expression in the other cells.
C2C12 cells treated with 1α(OH)D[subscript]3 for 48 hours at varying stages of development responded in changes in myogenic regulatory factors (MRFs), MHCs and novel target gene expression. Real time PCR analysis of C2C12 cells treated with the active vitamin D metabolite 1α(OH)D[subscript]3 affirmed C/EBPβ mRNA expression to be upregulated (P<0.001) and MAPK ERK 1/2 phosphorylation to be down regulated (P<0.001) by 1α(OH)D[subscript]3 in muscle cells. The effect of 1α(OH)D[subscript]3 in myoblasts was reduce proliferation and promote differentiation, as myotubes formed the effect of 1α(OH)D[subscript]3 was to promote MHC gene expression of an intermediate oxidative fibre type, increasing expression of MHC 1/β and 2A, decreasing MHC 2B.
In conclusion, there is no apparent benefit of a pre-slaughter dietary vitamin D feeding regime on meat quality, but the active metabolites of vitamin D, 1α(OH)D[subscript]3 and 1,25(OH)[subscript]2D[subscript]3, exert changes in gene expression and MAPK signalling which are likely to affect muscle growth and fibre type, and is of relevance in terms of both meat quality and muscle function in the elderly.
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