P. J. Buttery

Tenderness - an enzymatic view.

One of the most common causes of unacceptability in meat quality is toughness. Toughness is attributed to a range of factors including the amount of intramuscular connective tissue, intramuscular fat, and the length of the sarcomere. However, it is apparent that the extent of proteolysis of key proteins within muscle fibres is significant determinant of ultimate tenderness. The objective of this manuscript is to describe the main endogenous proteolytic enzyme systems that have the potential to be involved in muscle post-mortem proteolysis and whether the experimental evidence available supports this involvement.

The simultaneous use of ribonucleic acid, 35 S, 2,6-diaminopimelic acid and 2-aminoethylphosphonic acid as markers of microbial nitrogen entering the duodenum of sheep

I. Three sheep, each fitted with a ruminal cannula and duodenal reentrant cannulas were given three isonitrogenous, isoenergetic diets in a Latin-Square design. Each diet contained 0 kg) approximately 400 g N as white fish meal, soya-bean meal or urea and approximately 600 g dry matter (DM) was barley grain. The diets were fed continuously and supplied about 28 g N/d. 2. Total duodenal digesta was collected manually for 72 h and the proportions of microbial N in that digesta were simultaneously estimated for all sheep using RNA, radioactive sulphur (%3), diaminopimelic acid (DAPA) and aminoethylphosphonic acid (AEPA) as markers. 3. Three of the estimation methods showed that the variable source of dietary N had the greatest (RNA P < 0.05, 85S P c 0.005, DAPA P c 0.1) effect on the proportions of microbial N in duodenal digesta, though differences between sheep accounted for some variation. 4. These methods also ranked the diets in the order: urea > soya-bean meal > fish meal with respect to the proportions of digesta N that were microbial in origin; the respective mean values for these diets with the different markers were: RNA 0.98,0.70,036; 36S 0.92,0.64,0.54; DAPA 0.80,0*47,0~42. 5. AEPA was found to be present in substantial quantities not only in isolated rumen protozoa, but also in dietary and bacterial material; an observation that invalidated its further use as a protozoal marker. 6. Calculations using values obtained from the 8% procedure showed that the proportions of dietary N degraded within the rumen were 0.38, 0-43 and 0.89 for the white fish meal, soya-bean meal and barley respectively. 7. The marker methods are compared and their advantages and disadvantages (real and apparent) are discussed. It is concluded that where microbial N estimates of a more general and comparative nature are required, the use of RNA as a marker is probably adequate. Where information for more exacting purposes is required, the use of appears to be more appropriate.

The effect of the β-2-adrenergic agonist clenbuterol or implantation with oestradiol plus trenbolone acetate on protein metabolism in wether lambs

The effects of Revalor (trenbolone acetate plus oestradiol) implantation or the inclusion of clenbuterol (a beta-2-adrenergic agonist) in the diet of wether lambs was studied. Using continuous intravenous infusion of [3H]tyrosine the fractional synthetic rate of mixed protein from three separate muscles was measured. Clenbuterol slightly increased growth rate but had a significant (P less than 0.02) effect on food conversion efficiency. The weight and protein content of the longissimus dorsi and vastus lateralis muscles were increased but no such changes were observed for the vastus intermedius. For the longissimus dorsi at least the increase was probably achieved by a reduction in fractional degradation rate of the muscle protein. Revalor significantly increased the growth rate and food conversion efficiency of the animals. This increase was not specific for muscle. Estimated degradation rates of muscle protein were lower in the treated animals. The possible mode of action of these materials was discussed. The results obtained again highlight the importance of protein degradation in controlling growth.

Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome

Clones corresponding to ovine stearoyl-CoA desaturase (SCD) cDNA were isolated from an adipose tissue cDNA library. All of these clones represented a single mRNA species as judged by restriction fragment and DNA sequence analysis. RNase protection analysis demonstrated that this SCD transcript is highly expressed in adipose tissue and liver, and in the mammary gland of lactating animals. A lower level of expression was detectable in a variety of other tissues including brain. Levels of the SCD transcript were decreased in adipose tissue during lactation, and this appears to be related to a marked decline in serum insulin and insulin-responsiveness of the tissue. Southern analysis of ovine and mouse genomic DNA demonstrated that the ovine SCD cDNA hybridised in a manner consistent with a single gene for SCD in ovine DNA; mouse genomic DNA produced a pattern of hybridisation consistent with the previously characterised mouse SCD-1 and SCD-2 genes. Three ovine cosmids were isolated that comprised the restriction fragments predicted by the genomic Southern analysis. The ovine SCD gene was predicted to be encompassed within a 23 kbp region that was present in all three cosmids. These results demonstrate that SCD is transcribed from a single gene in the ovine genome and this gene is insulin-responsive in ovine adipose tissue.

Effects of dietary quebracho tannin on nutrient utilisation and tissue metabolism in sheep and rats

The effect of feeding quebracho tannin, a mixture of condensed tannins, on dietary nutrient utilisation and nitrogen (N) retention and its effects on the gastrointestinal (GI) tract was investigated in sheep and rats. Sheep (n = 24) were fed on a pelleted diet of dried grass alone (controls) or containing quebracho tannin at 50 g kg−1 diet dry matter (DM) (tannin-fed animals) at a level sufficient to achieve a daily liveweight gain (DLWG) of 100 g day−1. Complete collections of faeces and urine were made for two seven-day periods after two and six weeks of feeding these diets (n = 6 per group). Apparent digestibilities of dry matter, N and neutral detergent fibre (NDF) were significantly (P < 0.001) reduced in tannin-fed animals at both measurement periods. No evidence was obtained to suggest that rumen micro-organisms can adapt to the presence of dietary tannins with prolonged feeding. Tannin-fed animals excreted significantly (P < 0.01) more N in faeces and less in urine than controls suggesting an alteration in N metabolism. Histological examination of samples of the GI tract obtained from pairs of sheep slaughtered after two, five and seven weeks of feeding the diets indicated ulceration and an increase in mucosal histiocytes, particularly in the jejunum and ileum of most tannin-fed animals. In a subsequent experiment, rats were fed ad libitum a ground chow containing either cellulose or quebracho tannin at 40 g kg−1 DM. Tannin-fed rats had significantly (P < 0.05) reduced feed intakes, DLWG, N retention and body fat deposition compared to controls. Protein synthesis rates in the duodenal mucosa were not increased in tannin-fed rats suggesting that enterocyte proliferation was not stimulated in this region of the GI tract. These studies indicate that feeding quebracho tannin to ruminants has both ruminal and post-ruminal effects that, together, result in reduced nutrient utilisation and impaired animal performance. © 1999 Society of Chemical Industry

The relation between dietary restriction or clenbuterol (a selective β2 agonist) treatment on muscle growth and calpain proteinase ( EC 3.4.22.17) and calpastatin activities in lambs

1. Lamb growth trials were designed to modify growth and protein content of muscle by diet and also by beta-agonist treatment, and to correlate any changes to the activities of calpain proteinases (EC 3.4.22.17) and their inhibitor calpastatin. 2. Wether lambs in a control group were fed on a barley-based diet designed to give a growth rate of 350 g/d; a second group was fed on the same diet but the intake was restricted to give an expected gain of 44 g/d; a third group was fed on the same diet as the first group but the diet included 2 mg clenbuterol/kg. At the end of a 6-week trial, longissimus dorsi wet weights were 635 (n6), 377 (n4) and 788 g (n6) (standard error of difference 53.0) in the three groups respectively. 3. Minced L. dorsi was extracted in low-salt buffers and analysed by a fast protein liquid-chromatographic system for calpain I (low calcium-requiring), calpain II (high Ca2+-requiring) and calpastatin activities. No significant changes in the three activities were associated with reduced muscle weight in the restricted-intake group. The inclusion of clenbuterol in the diet, however, led to highly significant increases (P less than 0.001) in calpain II and calpastatin to approximately double the control values. 4. The results did not support a direct relation between these activities and muscle growth, except when protein accretion was stimulated by a beta-agonist, suggesting a role for this enzyme system in the mechanism by which these agents exert their effect.

Effect of β-agonists on expression of calpain and calpastatin activity in skeletal muscle

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.

Effects of trenbolone acetate and zeranol on protein metabolism in male castrate and female lambs

Tissue composition and skeletal muscle cathepsin D (EC 3.4.23.5) activity were measured in wether lambs treated with trenbolone acetate (TBA) and oestradiol-17 beta (Oe) in combination and female lambs treated with TBA or zeranol. Muscle and liver protein fractional synthesis rates and plasma leucine flux were measured in the female lambs. Male castrate lambs treated with TBA plus Oe showed increased growth rate, improved food conversion efficiency, decreased muscle RNA concentration and decreased total cathepsin D activity in muscle. Female lambs treated with TBA or zeranol showed increased weight gain, improved food conversion efficiency, decreased muscle RNA and DNA concentrations and decreased free cathepsin D activity in muscle. Mixed muscle protein fractional synthesis rate was decreased after TBA treatment. Plasma leucine flux, not corrected for oxidation or food intake, was not increased by TBA or zeranol treatment. Treatment of female lambs with TBA or zeranol caused increased growth rate. This increased growth rate is probably due in part to decreased muscle protein degradation, since evidence was obtained that muscle protein synthesis is decreased by TBA and zeranol treatment.

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles.

The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule×Charolais rams. Samples taken at slaughter were frozen either in liquid N(2) for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically (r(2)=0.612 and 0.366, respectively, p<0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity (r(2)=0.725, p<0.001) and 135 kDa calpastatin (r(2)=0.228, p<0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.

The effect of oestradiol-17 β implantation on the response in voluntary intake, live-weight gain and body composition, to fishmeal supplementation of silage offered to growing calves

The effect of oestradiol-17β on the response to fishmeal supplementation of grass silage was studied in young growing cattle. Voluntary intake and live-weight gain were recorded over 63 days with 36 British Friesian male castrates (initial live weight (LW) 119 kg) offered silage alone (C) or with 50 (FM1), 100 (FM2), or 150 (FM3) g fishmeal/kg silage dry matter. Twelve calves were allocated to each of treatments C and FM3 and six to treatments FM1 and FM2. Half of the calves on each treatment were ear-implanted with oestradiol-17β (Compudose 365) at the start of the experiment. The calves on treatments C and FM3 were slaughtered after 75 days and chemical analysis conducted on half of each carcass. The silage had an organic-matter digestibility in vivo of 0·794 and was well-fermented, with a pH of 3·7. Intake averaged 24·2±0·42 g D.M./kg LW over all the treatments and live-weight gain was 0·77 kg/day on the silage alone. There was a significant ( P P