Effect of the Texel muscling QTL (TM-QTL) on spine characteristics in purebred Texel lambs.

Previous work showed that the Texel muscling QTL (TM-QTL) results in pronounced hypertrophy in the loin muscle, with the largest phenotypic effects observed in lambs inheriting a single copy of the allele from the sire. As the loin runs parallel to the spinal vertebrae, and the development of muscle and bone are closely linked, the primary aim of this study was to investigate if there were any subsequent associations between TM-QTL inheritance and underlying spine characteristics (vertebrae number, VN; spine region length, SPL; average length of individual vertebrae, VL) of the thoracic, lumbar, and thoracolumbar spine regions. Spine characteristics were measured from X-ray computed tomography (CT) scans for 142 purebred Texel lambs which had been previously genotyped. Least-squares means were significantly different between genotype groups for lumbar and thoracic VN and lumbar SPL. Similarly for these traits, contrasts were shown to be significant for particular modes of gene action but overall were inconclusive. In general, the results showed little evidence that spine trait phenotypes were associated with differences in loin muscling associated with the different TM-QTL genotypes.

Evaluation of Video Image Analysis (VIA) technology to predict meat yield of sheep carcasses on-line under UK abattoir conditions.

The Meat and Livestock Commission's (MLC) EUROP classification based scheme and Video Image Analysis (VIA) system were compared in their ability to predict weights of primal carcass joints. A total of 443 commercial lamb carcasses under 12 months of age and mixed gender were selected by their cold carcass weight (CCW), conformation and fat scores. Lamb carcasses were classified for conformation and fatness, scanned by the VIA system and dissected into primal joints of leg, chump, loin, breast and shoulder. After adjustment for CCW, the estimation of primal joints using MLC EUROP scores showed high coefficients of determination (R(2)) in the range of 0.82-0.99. The use of VIA always resulted in equal or higher R(2). The precision measured as root mean square error (RMSE) was 27% (leg), 13% (chump), 1% (loin), 11% (breast), 5% (shoulders) and 13% (total primals) higher using VIA than MLC carcass information. Adjustment for slaughter day and gender effects indicated that estimations of primal joints using MLC EUROP scores were more sensitive to these factors than using VIA. This was consistent with an increase in stability of the prediction model of 28%, 11%, 2%, 12%, 6% and 14% for leg, chump, loin, breast and shoulder and total primals, respectively, using VIA compared to MLC EUROP scores. Consequently, VIA was capable of improving the prediction of primal meat yields compared to the current MLC EUROP carcass classification scheme used in the UK abattoirs.

Muscle fibre characteristics of two contrasting sheep breeds: Scottish Blackface and Texel.

This study evaluates the effects of breed and sex, together with those of birth weight and litter size, on muscle fibre type characteristics in Texel (TEX) and Scottish Blackface sheep (SBF). The M.longissimus thoracis et lumborum (LTL) of TEX had a significantly higher total muscle cross-sectional area (16%), a higher total fibre number (20%) and a higher muscle CT density (5%) than the SBF but had a similar average muscle fibre size. The frequency of slow fibres in the LTL in TEX was lower than in SBF (7.5% vs. 9.6%). Muscle fibre histochemistry similarly demonstrated that the oxidative fibre frequency in TEX was 10% lower than in SBF. The inter-fibre lipid content in TEX was also significantly lower than in SBF. Correspondingly, TEX displayed higher frequency (91.7% vs. 90.9% in SBF) and higher relative total area (92.5% vs. 90.4% in SBF) of fast fibres. These breed differences in muscle fibre traits indicate underlying genetic variation, and future analyses will evaluate the link of these traits to meat quality and assess the usefulness of these traits in breeding programmes.

Quality pork genes and meat production.

Functional genomics, including analysis of the transcriptome and proteome, provides new opportunities for understanding the molecular processes in muscle and how these influence its conversion to meat. The Quality Pork Genes project was established to identify genes associated with variation in different aspects of raw material (muscle) quality and to then develop genetic tools that could be utilized to improve this quality. DNA polymorphisms identified in the porcine PRKAG3 and CAST genes illustrate the impact that such tools can have in improving meat quality. The resources developed in Quality Pork Genes provide the basis for identifying more of these tools.

Effect of growth promoters on pig muscle structural protein and proteolytic enzyme levels in vivo and in vitro.

To elucidate the biochemical mechanism by which the growth hormone porcine somatotrophin (PST) promotes skeletal muscle growth, we have determined the activity of a comprehensive range of protein catabolizing proteolytic enzymes and structural proteins (determined by analytical electrophoresis, SDS-PAGE) in longissimus dorsi muscle from control and PST treated pigs. There was no significant difference in the levels of muscle structural proteins, or in the activity of muscle proteolytic enzymes at point of slaughter in control or PST treated animals. Similarly, in post-mortem muscle proteolysis time course experiments at pH 5.5 or pH 7.5, there was no significant difference in the rate of structural protein degradation by endogenous muscle proteases (determined via SDS-PAGE) using muscle from control or PST treated animals. In addition, investigation of a range of beta-agonist related drugs (clenbuterol, salbutamol, pirbuterol, fenoterol) showed no effect (10(-4)-10(-8) M) in vitro on the activity of individual protease types in control muscle, or on the degradation rate of muscle structural proteins by endogenous proteases in time course experiments at pH 5.5 or pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line.

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.

Myotoxic activity of the crude venom and the principal neurotoxin, taipoxin, of the Australian taipan, Oxyuranus scutellatus.

1 The crude venom of the Australian taipan. Oxyuranus scutellatus and its principal neurotoxin, taipoxin, were injected into the anterolateral aspect of one hind limb of the rat. 2 The effects of the venom and toxin on the morphology and physiology on the underlying soleus muscles were examined. 3 Both the crude venom and the toxin caused necrosis and degeneration of the muscle. Damage to the peripheral muscle fibres could be seen at the light microscopic level as early as 3 h after injection of the toxic compounds. 4 The necrotic response was accompanied by an infiltration of phagocytic cells and an extensive oedema. The wet weight of the damaged muscles was almost doubled by 6 h. 5 In individual muscle fibres, necrosis was associated with the disruption of the plasma membrane and the disorganization of the myofibrils. The basal lamina of the muscle fibres was left intact. 6 Denervated mammalian muscles and innervated avian muscles were also destroyed by tiapoxin, but immature avian muscle growing in tissue culture was resistant. 7 Of the 3 subunits of taipoxin, only the basic alpha-taipoxin was itself myotoxic. However, its potency was enhanced by the presence of the acid gamma subunit. The role of the neutral beta-subunit is unclear. 8 The period of necrosis and degeneration lasted for approximately 48 h, after which the muscle fibres began to regenerate. Regeneration took place within the surviving basal lamina, with the formation of myotubes by three days, and small, immature muscle fibres by five days. Regeneration was virtually complete by 21 days.

The effects of the subcutaneous injection of the crude venom of the Australian common brown snake, Pseudonaja textilis on the skeletal neuromuscular system.

1 The effects of the crude venom of the Australian common brown snake on the mammalian neuromuscular system have been investigated. 2 The venom was injected subcutaneously into the dorso-lateral aspect of one hind limb of the rat. The limb was paralyzed within 90 min and remained paralysed for 2 to 3 days. 3 The exposed muscles failed to respond to indirect excitation, and individual fibres were not depolarized at the nerve-muscle junction by exposure to carbachol. 4 The wet weight, histological appearance, resting potential and input resistance of the muscle fibres and their ability to generate directly elicited action potentials were unaffected by exposure to the venom. 5 Administration of venom to isolated preparations caused a reduction in the amplitude of miniature endplate potentials, with no change in frequency. The quantal content of evoked endplate potentials was unchanged. 6 It was concluded that the crude venom was largely devoid of presynaptic activity and myotoxicity, and that its primary site of neurotoxicity was directed to the postsynaptic membrane.

Evidence that the hypertrophic action of clenbuterol on denervated rat muscle is not propranolol-sensitive.

1. The effect of propranolol on the clenbuterol-induced protein anabolism in innervated and denervated soleus and plantaris muscles of the rat was studied. 2. The response to the beta-agonist, clenbuterol, in both innervated and denervated muscles, was not significantly inhibited by the beta-antagonist, propranolol. 3. The results provide further evidence to suggest that the action of clenbuterol on skeletal muscle protein accretion may not be directly mediated by beta-adrenoceptors.

Effects of the cyclo-oxygenase inhibitor, fenbufen, on clenbuterol-induced hypertrophy of cardiac and skeletal muscle of rats.

1. When rats were fed with clenbuterol for 7 days skeletal muscle mass increased by 21% in the tonic soleus and phasic plantaris muscles and a 16% hypertrophy of the heart was also induced. Fenbufen, fed to rats for the same period, blocked the hypertrophy of the heart but not that of the skeletal muscles. 2. When feeding of fenbufen commenced 3 days before the administration of clenbuterol, plasma prosta-glandin F2 alpha (PGF2 alpha) was reduced by 79%; there was again no effect of fenbufen on clenbuterol-induced increases in the RNA or protein content of plantaris, nor in the increased area of fast or slow twitch fibres in the soleus. In the heart the clenbuterol-induced increases in the RNA (+21%) and protein content (+20%) were totally inhibited. 3. The effects of clenbuterol on heart muscle appear to be mediated by a cyclo-oxygenase metabolite of arachidonic acid whilst the effects on skeletal muscle are not.

Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo.

The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.

Effect of prednisone on protease activities and structural protein levels in rat muscles in vivo.

To further elucidate the biochemical mechanism by which the corticosteroid prednisone induces differential changes in muscle mass (via altered protein synthesis/degradation rates) in normal or degenerating muscle tissues, we have determined the activity of a range of proteolytic enzyme types, together with levels of muscle structural proteins, in five innervated and denervated muscle types from control and drug treated rats. In both normal and wasting muscles, the activity of many protease types was substantially down-regulated following treatment with prednisone; however, accompanying net decreases in muscle mass were observed (although the structural protein composition of muscles was unaltered following drug treatment). We conclude that whilst overall rates of protein degradation in both normal and degenerating muscle may be reduced (via protease down-regulation) following prednisone treatment, the effect of the latter in reducing protein synthesis rates must be proportionately greater (even in actively degenerating tissue). Thus, the data do not support the hypothesis that the beneficial effect of prednisone in maintaining muscle mass in pathological tissues (e.g., Duchenne muscular dystrophy (DMD)) operates principally via down-regulation of protease action/protein catabolism.

Effect of protease inhibitors and clenbuterol on the in vitro degradation of dystrophin by endogenous proteases in human skeletal muscle.

The in vitro degradation of dystrophin protein by endogenous proteases in human skeletal muscle has been investigated using a tissue homogenate assay system with subsequent protein analysis via SDS polyacrylamide electrophoresis and immunoblotting (using a monoclonal antibody to the central rod region of dystrophin). The rate of dystrophin degradation and nature of the proteolytic fragments formed at pH 5.5 and pH 7.5 (corresponding to the two major protease groups of relevance to intracellular protein catabolism) were broadly similar; incorporation of protease inhibitors in the above system suggested that Ca2+ activated proteinase and cathepsin D are principally responsible for the degradation of dystrophin at pH 7.5 and pH 5.5 respectively. The rate of dystrophin degradation at pH 7.5 was reduced by approximately 20% in the presence of 10(-5) M clenbuterol, a beta-adrenoceptor agonist with therapeutic potential in the treatment of human muscle wasting diseases.

The effect of a growth promoting drug, clenbuterol, on fibre frequency and area in hind limb muscles from young male rats.

The effect of dietary administration of clenbuterol on soleus and extensor digitorum longus muscles was studied after 4 and 21 days. Both muscles showed an increase in wet weight with no significant change in total fibre number. After 4 days fibre cross-sectional areas were increased in soleus, but not in extensor digitorum longus, and after 21 days there was a change in fibre frequencies in extensor digitorum longus but not soleus muscles.

Propranolol apparently separates the physical and compositional characteristics of muscle growth induced by clenbuterol.

The effect of propranolol on clenbuterol-induced changes in muscle fibre size and protein content were studied. Propranolol did not inhibit the ability of clenbuterol to stimulate protein accretion but reduced the increase in muscle fibre size. The compositional and physical characteristics of clenbuterol-induced muscle growth thus appeared to be separated by propranolol.

Clenbuterol, a beta agonist, induces growth in innervated and denervated rat soleus muscle via apparently different mechanisms.

Dietary administration of the anabolic agent, clenbuterol, has already been shown to inhibit or reverse denervation-induced atrophy in rat soleus muscles. We now show that the ameliorative effects of clenbuterol in denervated rat muscles are due principally to a large increase in protein synthesis. This results from both an increase in protein synthetic capacity and a normalised translational efficiency. The responses of innervated and denervated muscles are therefore fundamentally different, the changes in denervated muscles being reminiscent of the classical pleiotypic response of cells to growth factors.

Inhibition and reversal of denervation-induced atrophy by the beta-agonist growth promoter, clenbuterol.

Dietary administration of the growth promoter, clenbuterol, ameliorated denervation-induced atrophy in rat soleus muscles. In acutely denervated muscles the drug inhibited the appearance of atrophy, and in chronically denervated muscles the atrophy was almost fully reversed. Responses in slow twitch oxidative fibres were particularly marked.

The effect of the anabolic agent, clenbuterol, on overloaded rat skeletal muscle.

The dietary administration of clenbuterol to young male rats has been shown to produce a muscle specific hypertrophic growth response. This paper demonstrates that the combined effect of drug treatment and hypertrophic stimulus induced by tenotomy produced an additive effect on muscle growth. This effect was demonstrated in terms of both muscle composition (protein and RNA) and fibre size.

Fetal growth and development following temporary exposure of day 3 ovine embryos to an advanced uterine environment.

The effect of exposing Day 3 ovine embryos to an advanced uterine environment for a period of 3 days on subsequent fetal growth and development between Day 35 and Day 135 of gestation was studied. Day 3 embryos were recovered from superovulated donor ewes and transferred to synchronous final or asynchronous temporary recipients for 3 days. Embryos were recovered from these temporary recipients and transferred to Day 6 final recipients. Gravid uteri were recovered, weighed and dissected on Days 35, 45, 60, 90, 110, 125 and 135 of gestation. Fetal weight and length data were analysed by fitting non-linear Gompertz models of the form log(e) y = a - be(-ct), where y is fetal size and t is time from conception. Various terms including treatment, gestational age, embryo stage at transfer and fetal sex were fitted to this model. Fetal development was assessed by relating organ weight to fetal bodyweight using the linear allometric equation log(e) y = log(e) a + b log(e) x, where y is organ weight and x is fetal weight. Temporary exposure of Day 3 embryos to an advanced uterine environment did not increase the rate of embryo development and had no effect on fetal growth and development between Days 35 and 135 of gestation in this study. A single Gompertz model (log(e) y = 10.134 - 17.047e(-0.1733t)) explained 99.8% of the variation in fetal weight. Of terms fitted to this model only gestational age and fetal sex influenced fetal weight, with male fetuses being 5% heavier (P<0.05) than female fetuses. Fetal development was also unaffected by experimental treatment in this study. Allometric coefficients established for various fetal components agreed well with those from previously published studies.

Comparison of structural protein and proteolytic enzyme levels in degenerating and regenerating rat muscle induced by Notechis scutatus venom.

To develop a clear understanding of the biochemical mechanism of muscle degeneration and regeneration induced by a single dose of Notechis scutatus scutatus venom, we have correlated changes in the levels of a series of muscle structural proteins and proteolytic enzymes. The degradation of structural proteins post-injection fell into two broad groups; those completely degraded within 3-6 hr (e.g. C- and M-proteins, skelemin), and within 1-2 days (e.g. myosin, actin, troponin), respectively. Similarly, activation of proteases followed two general patterns; those enzymes showing substantially increased activity after 12-24 hr (lysosomal cathepsins, leucyl aminopeptidase) and those enzymes showing decreased activity after 12-24 hr, with substantially increased activity after 3-4 days (mainly cytoplasmic proteases). The data suggest that activation of cathepsins B, L and D and in particular leucyl aminopeptidase, may be responsible for the early stages of structural protein catabolism, and are thus potential therapeutic targets to prevent myonecrosis following envenomation.