Precision diagnostics in transplantation: from bench to bedside.

The Canadian and American Societies of Transplantation held a symposium on February 22, 2012 in Quebec City focused on discovery, validation and translation of new diagnostic tools into clinical transplantation. The symposium focused on antibody testing, transplantation pathology, molecular diagnostics and laboratory support for the incompatible patient. There is an unmet need for more precise diagnostic approaches in transplantation. Significant potential for increasing the diagnostic precision in transplantation was recognized through the integration of conventional histopathology, molecular technologies and sensitive antibody testing into one enhanced diagnostic system.

Single-center kidney paired donation: the Methodist San Antonio experience.

Many potential kidney transplant recipients are unable to receive a live donor transplant due to crossmatch or blood type incompatibility. Kidney paired donation increases access to live donor transplantation but has been significantly underutilized. We established a kidney paired donation program including consented incompatible donor/recipient pairs as well as compatible pairs with older non-human leukocyte antigen identical donors. Over a 3-year period, a total of 134 paired donor transplants were performed, including 117 incompatible pairs and 17 compatible pairs. All transplants were done with negative flow cytometry crossmatches and five were done with desensitization combined with paired donation. Kidney paired donation transplants included two-way and three-way exchanges as well as three chains initiated by nondirected donors. Of the sensitized recipients transplanted by paired donation, 44% had calculated panel reactive antibody levels greater than 80%. Transplantation of females and prior transplant recipients was significantly higher with paired donation. Only three episodes of rejection occurred and no transplants were lost due to rejection. These data highlight the potential of kidney paired donation and suggest that all transplant centers should be actively engaged in paired donation to increase access to live donor transplantation.

Second trimester maternal plasma levels of cytokines IL-1Ra, Il-6 and IL-10 and preterm birth.

OBJECTIVE: To examine the interaction of the cytokines interleukin-1 receptor antagonist (IL)-1Ra, IL-6 and IL-10 to predict preterm birth (PTB) in pregnant Hispanic women (n=470). STUDY DESIGN: In this prospective study, demographic data were obtained prenatally and birth outcome data were obtained from the medical chart. Cytokines were measured from plasma obtained at 22 to 24 weeks gestation. Data analysis utilized logistic regression. RESULT: PTB was predicted by level of IL-1Ra (odds ratio (OR)=2.55; 95% confidence interval (CI)=1.24, 5.24). The interaction between IL-1Ra and IL-6 and between IL-1Ra and IL-10 was significant (Wald=4.01, P=0.04 and Wald=8.84, P<0.003, respectively) and was also predictive of PTB. As IL-1Ra levels increased while IL-10 levels were low, the probability of PTB greatly increased. CONCLUSION: The interactions of select cytokines and cytokine receptor antagonists were associated with PTB. Future research should focus on the changes in cytokines during pregnancy to identify critical periods of change, and examine predictors of the cytokine response.

Pharmacological characterization of a novel positive modulator at alpha 4 beta 3 delta-containing extrasynaptic GABA(A) receptors.

The in vitro and in vivo pharmacological effects of [2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504), which is a close analogue of retigabine, have been investigated. AA29504 induced a rightward shift of the activation threshold at cloned KCNQ2, 2/3 and 4 channels expressed in Xenopus oocytes, with a potency 3-4fold lower than retigabine. AA29504 (1 muM) had no agonist activity when tested at alpha(1)beta(3)gamma(2s) or alpha(4)beta(3)delta GABA(A) receptors expressed in Xenopus oocytes, but left-shifted the EC(50) for GABA and gaboxadol (THIP) at both receptors. The maximum GABA response at alpha(1)beta(3)gamma(2s) receptors was unchanged by AA29504 (1 muM), but increased 3-fold at alpha(4)beta(3)delta receptors. In slices prepared from the prefrontal cortex of adult rats AA29504 had no effect alone on the average IPSC or the tonic current in layer II/III pyramidal neurons, but potentiated the effect of gaboxadol on both phasic and tonic currents. Thus, the effects of gaboxadol could be positively modulated by AA29504. Systemic administration of AA29504 at doses relevant for modulating GABA transmission produced anxiolytic effects and reduced motor coordination consistent with activity at GABA(A) receptors. We conclude that AA29504 exerts a major action via alpha(4)beta(3)delta-containing GABA(A) receptors, which will be important for interpreting its effect in vivo.

Parameter estimation methods for single neuron models.

With the advancement in computer technology, it has become possible to fit complex models to neuronal data. In this work, we test how two methods can estimate parameters of simple neuron models (passive soma) to more complex ones (neuron with one dendritic cylinder and two active conductances). The first method uses classical voltage traces resulting from current pulses injection (time domain), while the second uses measures of the neuron's response to sinusoidal stimuli (frequency domain). Both methods estimate correctly the parameters in all cases studied. However, the time-domain method is slower and more prone to estimation errors in the cable parameters than the frequency-domain method. Because with noisy data the goodness of fit does not distinguish between different solutions, we suggest that running the estimation procedure a large number of times might help find a good solution and can provide information about the interactions between parameters. Also, because the formulation used for the model's response in the frequency domain is analytical, one can derive a local sensitivity analysis for each parameter. This analysis indicates how well a parameter is likely to be estimated and helps choose an optimal stimulation protocol. Finally, the tests suggest a strategy for fitting single-cell models using the two methods examined.

A model of the action potential and underlying membrane currents in a rabbit atrial cell.

We have developed a mathematical model of the rabbit atrial myocyte and have used it in an examination of the ionic basis of the atrial action potential. Available biophysical data have been incorporated into the model to quantify the specific ultrastructural morphology, intracellular ion buffering, and time- and voltage-dependent currents and transport mechanisms of the rabbit atrial cell. When possible, mathematical expressions describing ionic currents identified in rabbit atrium are based on whole cell voltage-clamp data from enzymatically isolated rabbit atrial myocytes. This membrane model is coupled to equations describing Na+, K+, and Ca2+ homeostasis, including the uptake and release of Ca2+ by the sarcoplasmic reticulum and Ca2+ buffering. The resulting formulation can accurately simulate the whole cell voltage-clamp data on which it is based and provides fits to a family of rabbit atrial cell action potentials obtained at 35 degrees C over a range of stimulus rates (0.2-3.0 Hz). The model is utilized to provide a qualitative prediction of the intracellular Ca2+ concentration transient during the action potential and to illustrate the interactions between membrane currents that underlie repolarization in the rabbit atrial myocyte.

Quantitative analysis of electrotonic structure and membrane properties of NMDA-activated lamprey spinal neurons.

Parameter optimization methods were used to quantitatively analyze frequency-domain-voltage-clamp data of NMDA-activated lamprey spinal neurons simultaneously over a wide range of membrane potentials. A neuronal cable model was used to explicitly take into account receptors located on the dendritic trees. The driving point membrane admittance was measured from the cell soma in response to a Fourier synthesized point voltage clamp stimulus. The data were fitted to an equivalent cable model consisting of a single lumped soma compartment coupled resistively to a series of equal dendritic compartments. The model contains voltage-dependent NMDA sensitive (INMDA), slow potassium (IK), and leakage (IL) currents. Both the passive cable properties and the voltage dependence of ion channel kinetics were estimated, including the electronic structure of the cell, the steady-state gating characteristics, and the time constants for particular voltage- and time-dependent ionic conductances. An alternate kinetic formulation was developed that consisted of steady-state values for the gating parameters and their time constants at half-activation values as well as slopes of these parameters at half-activation. This procedure allowed independent restrictions on the magnitude and slope of both the steady-state gating variable and its associated time constant. Quantitative estimates of the voltage-dependent membrane ion conductances and their kinetic parameters were used to solve the nonlinear equations describing dynamic responses. The model accurately predicts current clamp responses and is consistent with experimentally measured TTX-resistant NMDA-induced patterned activity. In summary, an analysis method is developed that provides a pragmatic approach to quantitatively describe a nonlinear neuronal system.

Localization and interaction of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of lamprey spinal neurons.

Small volumes of N-Methyl-D-Aspartate (NMDA) and non-NMDA excitatory amino acid receptor agonists were applied to localized regions of the dendritic trees of lamprey spinal neurons along their medial-lateral axis to obtain a spatial map of glutamate receptor distribution. Voltage clamp and frequency domain methods were used to obtain quantitative kinetic data of the voltage dependent ionic channels located both on the soma and on highly branched dendritic membranes. Pressure pulses of NMDA applied to the most peripheral regions of the dendritic tree elicited large somatic impedance increases, indicating that the most peripheral dendrites are well supplied with NMDA receptors. Experiments done with kainate did not elicit somatic responses to agonist applications on peripheral dendrites. The data obtained are consistent with the hypothesis that the activation of NMDA receptors by exogenous glutamate is significantly modified by the simultaneous activation of non-NMDA receptors, which shunts the NMDA response. The non-NMDA shunting hypothesis was tested by a combined application of kainate and NMDA to mimic the action of glutamate showing that the shunting effect of non-NMDA receptor activation virtually abolished the marked voltage dependency typical of NMDA receptor activation. These data were interpreted with a compartmental neuronal model having both NMDA and non-NMDA receptors.

A mathematical model of a rabbit sinoatrial node cell.

A mathematical model for the electrophysiological responses of a rabbit sinoatrial node cell that is based on whole cell recordings from enzymatically isolated single pacemaker cells at 37 degrees C has been developed. The ion channels, Na(+)-K+ and Ca2+ pumps, and Na(+)-Ca2+ exchanger in the surface membrane (sarcolemma) are described using equations for these known currents in mammalian pacemaker cells. The extracellular environment is treated as a diffusion-limited space, and the myoplasm contains Ca(2+)-binding proteins (calmodulin and troponin). Original features of this model include 1) new equations for the hyperpolarization-activated inward current, 2) assessment of the role of the transient-type Ca2+ current during pacemaker depolarization, 3) inclusion of an Na+ current based on recent experimental data, and 4) demonstration of the possible influence of pump and exchanger currents and background currents on the pacemaker rate. This model provides acceptable fits to voltage-clamp and action potential data and can be used to seek biophysically based explanations of the electrophysiological activity in the rabbit sinoatrial node cell.

A model of slow conduction in bullfrog atrial trabeculae.

The success or failure of the propagation of electrical activity in cardiac tissue is dependent on both cellular membrane characteristics and intercellular coupling properties. This paper considers a linear arrangement of individual bullfrog atrial cells that are resistively coupled end to end to form a cylindrical strand. The strand, in turn, is encased by an endothelial sheath that provides a restricted extracellular space and an ion diffusion barrier to the outer bathing medium. This encased strand serves as an idealized model of an atrial trabeculum. Excitable membrane characteristics of the atrial cell are specified in terms of a Hodgkin-Huxley type of model that is quantitatively based on single-microelectrode voltage clamp data from bullfrog atrial myocytes. This membrane model can simulate the behavior of normal cells as well as of ischemic cells that exhibit depressed electrophysiological behavior (e.g., decreased resting potential, upstroke velocity, peak height, and action potential duration). Depressed activity can be easily simulated with variation of a single model parameter, the gain of the Na+/K+ pump current (INaK). Intercellular coupling properties are specified in terms of a lumped resistive T-type network between adjacent cells. The atrial strand model provides a means for studying the theoretical aspects of slow conduction in a "hybrid" strand that consists of a central region of cells having abnormal membrane or coupling properties, flanked on either side by normal atrial cells. Both uniform and discontinuous conduction are simulated by means of appropriate changes in the coupling resistance between cells. In addition, by varying either the degree of depressed electrical activity or the intercalated disc resistance in the central zone of the strand, slow conduction or complete conduction block in that region is demonstrated. Since the cellular model used in this study is based on experimental data and closely mimics both the atrial action potential and the underlying membrane currents, it has the potential to (1) accurately represent the current and voltage wave-forms occurring in the region of intercalated discs and (2) provide detailed information regarding the mechanisms in intercellular current spread in the region of slow conduction.

Conduction in bullfrog atrial strands: simulations of the role of disc and extracellular resistance.

A number of fundamental properties of intercellular conduction in simulated cylindrical strands of cardiac tissue are examined. The paper is based on recent biophysical information describing the transmembrane ionic currents in bullfrog atrial cells as well as anatomical data on the structures (gap junctions) responsible for the coupling between cells in that tissue. A mathematical model of the single bullfrog atrial cell based on suction microelectrode single-cell voltage clamp data is employed, as well as a modified version of the well-known model of Heppner and Plonsey, to characterized the resistive connections between adjacent cells in a cardiac strand. In addition, the simulated cellular strand is assumed to be encased in a cylindrical, resistive endothelial sheath, thus forming an idealized atrial trabeculum; the trabeculum is immersed in an extensive volume conductor. It is possible to simulate both uniform and discontinuous conduction in this atrial strand model by appropriately changing the resistance of the intercalated discs that occur at cell boundaries. The conduction velocity achieved in the normal or control case is within the range of conduction velocities that have been measured for bullfrog atrial trabeculae using optical methods. Extracellular resistance is shown to have a significant effect on both conduction velocity and the critical value of disc resistance at which discontinuous conduction first occurs. Since the atrial cell model employed in this study is based on experimental data and can accurately simulate the atrial action potential, the transmembrane ionic currents generated by the model are capable of providing detailed information concerning the mechanisms of intercellular current spread, particularly in the region of the intercalated disc.

Characterization of cutability and palatability attributes among different slaughter groups of beef cattle.

Fifteen slaughter cattle from five groups (Charolais crossbred bulls, Brahman crossbred steers, Holstein steers, mixed-Exotic crossbred heifers and Hereford-Angus crossbred steers) were randomly selected from a commercial feedlot. Time-on-feed was 108, 114, 102, 108 and 145 days for the aforementioned groups, respectively. Carcasses from Charolais crossbred bulls had the highest percentage yield of chuck and round, but the lowest percentage yield of loin; hereford-Angus crossbred steers had the lowest percentage yield of round and Holstein steers had the lowest percentage yield of rib. Carcasses from Charolais crossbred bulls had the highest percentage yield of major retail-ready subprimals and lean trim, Hereford-Angus crossbred steers had the highest percentage yield of fat trim and Holstein steers had the highest percentage yield of bone from the major wholesale cuts. Mean percentage yields of closely trimmed, boneless retail cuts were highest for carcasses from the bulls and lowest for carcasses from the Hereford-Angus steers. Although Holstein steer carcasses had less fat trim than Brahman-crossbred steers mixed-Exotic heifers, this advantage was largely offset by their higher percentage of bone. Loin steaks from carcasses of Charolais crossbred bulls, Holstein, mixed-Exotic heifers were comparable (P > 0·05)with those of Hereford-Angus steers for shear force and sensory panel tenderness ratings. However, loin steaks from carcasses of Brahman-crossbred steers had significantly higher (P < 0·05) shear force values (least tender) and lower (P < 0·05) tenderness and overall palatability ratings. No diffeerences (P > 0·05) were found for flavor desirability of loin steaks among any of the groups. For bottom round steaks, Hereford-Angus steers received the highest palatability ratings while those from Holstein steers received the lowest palatability ratings.

Carcass characteristics and composition of Brahman, angus and Brahman x Angus steers fed for different times-on-feed.

Twenty-five steers of each of three breedtypes (Angus, Brahman and F(1) Brahman x Angus) were sorted by frame size and muscle thickness, assigned to groups (five steers of each breedtype) to be fed for 0, 56, 112, 168 or 224 days, slaughtered and compared for various carcass traits. Steers of each breedtype had similar dressing percentages. Carcasses from all three breedtypes merited similar USDA quality and yield grades; breedtypes differences in quality grade were slight. Differences were found in the fat deposition patterns exhibited by the three breedtypes. Brahman steers tended to deposit more of their total fat as subcutaneous fat early in the feeding period. Angus steers had more (P < 0·05) seam fat as a percentage of carcass weight at all five feeding periods and more (P < 0·05) kidney, pelvic and heart fat at two of the five feeding periods than Brahman steers. Brahman steers had a higher percentage of their separable lean in the muscles of the round than did steers of the other breedtypes.

USDA yield grades and various carcass traits as predictors of carcass composition.

Twenty-five carcasses from each of three breedtypes (Brahman, Angus and Brahman × Angus) were physically separated into fat, lean and bone. Several muscles from the round and the femur were used to derive equations to predict carcass composition and muscle-to-bone ratio. The femur (as a percentage of the carcass) was shown to predict percentage carcass bone with 90% accuracy. All of the muscles studied were highly related to total carcass lean but the percentage of carcass as M. biceps femoris was the best single muscle indicator of carcass lean of the muscles studied. More variation in carcass lean could be accounted for by a multiple regression equation, involving all four muscles studied, than by any single muscle. M. biceps femoris-to-femur ratio was found to predict carcass muscle-to-bone ratio with a high degree of accuracy. The USDA yield grades were found to be reliable indicators of carcass composition. A two-variable equation involving adjusted fat thickness and biceps femoris accounted for 88·6% of the variation (RSD = 1/·64) in percentage of carcass as separable lean.

Predicting cutability of pork carcasses and hams using the Hennessy and Chong Fat Depth Indicator.

Hot and cold Fat Depth Indicator (FDI) readings were taken on seventy-four pork sides to examine the relative precision of different measurement locations. Fat measurements taken over the M. longissimus opposite the fourteenth thoracic vertebra (CD = 47·5; RSD = 1·63) and the last lumbar vertebra (CD = 61·1; RSD = 1·40) were the measurement locations most closely associated with percentages of four trimmed lean cuts. Using certain combinations of carcass weight and/or hot and cold FDI readings in multiple regression equations, 71·0% (RSD = 1·35) and 63·3% (RSD = 1·55), respectively, of the variability in percentage yields of four lean cuts could be explained; this was as much as 40 percentage points more than that explained by average backfat thickness (first rib, last rib, last lumbar vertebra) taken on the split surface in the dorsal midline. When added to an unpublished equation, the addition of up to four hot or cold FDI readings made dramatic increases in the explained variation in carcass yields of four lean cuts. In each of two studies involving green, skinned hams, FDI readings explained a low percentage of the variability in percentage of boneless, defatted, deseamed lean. However, it was determined that these low relationships were primarily due to the site selections at which the hams were probed-not because of an inadequacy of the FDI to measure fatness. A third study, involving different FDI probe sites, taken on intact sides along the ham to loin-belly juncture, determined that the best of these FDI readings could account for a maximum of 66·1% of the variability (RSD = 2·75) in yield of bone-in, skinned, defatted hams; a four-variable prediction equation developed using two FDI readings, untrimmed ham weight and muscling score explained 83·9% of the variation (RSD = 2·04).

Muscle: Bone ratios in beef rib sections.

Thirty-eight steers and thirty heifers (14 to 17 months of age, from F(1) Hereford × Brahman cows bred to Angus or Hereford bulls), were either forage-fed for 123 days on millet-bermudagrass pasture or grain-fed for 90 days on a high-concentrate diet and were then commercially slaughtered. Warm carcass weights ranged from 167·8 kg to 324·3 kg. At 24 h post mortem, Texas Agricultural Experiment Station personnel (1) assigned scores or took measurements on each carcass for all factors used in yield grading and quality grading, (2) measured the length of hind leg (HL) and carcass length (CL) and (3) assigned a score for carcass muscling (MS) and, as appropriate, made an adjusted longissimus muscle area (ALA) evaluation. The 9th-10th-11th rib section from one side of each carcass was physically separated into longissimus muscle, fat, 'other soft tissue' and bone and ether extract determinations of the longissimus muscle and 'other soft tissue' components were made and used to adjust the yields of each of these components to a fat-free basis. Muscle to bone ratios ranged from 2·38 to 4·37. With both age and carcass weight held constant, diet, breed and sex explained only 35·8% of the variation in muscle to bone ratio. The best simple correlation with muscle to bone ratio was ALA/CL (r = ·59). Other measures significantly correlated with muscle to bone ratio included ALA (r = 0·55), MS (r = 0·50) and carcass weight (r = 0·49). Multiple regression analyses identified a three-variable subset comprised of ALA, carcass weight and CL which was related (P < 0·01) to muscle to bone ratio R(2) = 0·41). Data suggest that muscle to bone ratios differ widely among beef carcasses of similar genetic-management history and that there are carcass measures useful for predicting muscle to bone ratio.

Enhancement of lean characteristics of veal carcasses by electrical stimulation.

Forty Holstein vealers (approximately 16 weeks of age) were slaughtered, skinned, split longitudinally and one side was electrically stimulated (700 V, 2 A and seventeen impulses) for 1 min. Internal temperatures of the rib and round were recorded at 1 h and 24 h post mortem. Approximately 24 h post mortem, lean colour scores were assigned to the cross-section of the longissimus muscle at the 11th-12th rib interface, to the outside surface of the biceps femoris muscle and to the rectus abdominis and transverse abdominis muscles of the flank; lean texture and firmness scores were also assigned to the cut surface of the longissimus muscle. 'House grades' were assigned to each side of each carcass by plant personnel. Initial (1 h) rib and round temperatures were significantly higher for the electrically stimulated sides; however, at 24 h, neither round nor rib temperatures differed between control and electrically stimulated sides. Lean colour, firmness and texture scores for longissimus muscles were significantly (P < 0·0001) improved by electrical stimulation. Electrically stimulated sides also had lighter lean colour scores in the rectus and transverse abdominis and biceps femoris muscles. Electrical stimulation increased the number of carcasses receiving the top 'house grade' and reduced the number of carcasses in the lowest 'house grade'. These data indicate that electrical stimulation will improve lean colour, firmness and texture of veal carcasses when ribbed and evaluated after a 24 h chill.

Singular and combined effects of electrical stimulation, post-mortem ageing and blade tenderisation on the palatability attributes of beef from young bulls.

Thirty Santa Gertrudis bulls (approximately 15-18 months old) were slaughtered, dressed and split into siides. The right side of each carcass was electrically stimulated (ES) with seventeen impulses (1·8s impulse duration; 1·8s interval between impulses) of 550 V (AC) and 5 A while the left side served as a non-stimulated control (not-ES). At 24h post morten, USDA quality and yield grade data were obtained from each side. On the second day post mortem, all sides were fabricated and strip loins, top sirloin butts and ribeyes were obtained from each side for post-mortem ageing and blade tenderisation studies. Steaks were removed after a post-mortem ageing period of 4 or 18 days and before (not-BT) or after blade tenderisation (BT) for sensory panel evaluations or shear force determinations. ES sides had more youthful lean maturity (P < 0·0001), higher marbling (P < 0.·002), higher USDA quality grades (P < 0·0.0001) and finer-textured lean (P < 0·002) than did not-stimulated (not-ES) sides. ES significantly improved (P < 0·05) palatability traits in two of twenty-four comparisons; BT significantly improved palatability traits in twelve of twenty-four comparisons and 18-day post-mortem ageing significantly improved palatability traits in seven of twelve comparisons. No significant reductions (P < 0·05) in shear force values were observed for steaks from ES versus not-ES sides while significant reductions (P < 0·05) were observed for steaks from BT versus non-BT cuts (four of six comparisons) and for steaks from cuts aged for 18 versus 4 days (ten of twelve comparisons). BT and 18-day post-mortem ageing were more effective for increasing palatability or for decreasing shear force requirements than was ES; however, ES greatly improved lean colour of meat from bulls.

Feeder cattle frame size, muscle thickness and subsequent beef carcass characteristics.

Sixty feeder steers were assigned scores for frame size (small, medium or large) and muscle thickness (No. 1, No. 2 or No. 3), fed for 112 days and slaughtered. Grade data were collected for all 60 carcasses; 12 sides (four from each muscle thickness group) were fabricated into boneless, closely trimmed retail cuts and the 12 rounds from each of these sides were also physically separated into muscle, fat and bone. Marbling score and USDA quality grade varied inversely (P < 0·05) with frame size. Carcass quality grades were: 33·3% Choice; 67·7% Good and 0·0% Standard for small-framed cattle; 30·3% Choice, 42·4% Good and 27·3% Standard for medium-framed cattle and 5·5% Choice, 66·7% Good and 27·8% Standard for large-framed cattle. Analysis of variance showed significant (P < 0·05) differences among all muscle thickness groups in the longissimus muscle area and carcass weight but no difference in yield grade between the No. 1 and No. 3 muscle thickness groups; the larger mean longissimus muscle area of carcasses from steers in the No. 1 muscle thickness group was offset by their heavier carcass weight and their greater thickness of fat over the longissimus muscle. However, when analysis of covariance was used to hold fatness or fatness and frame size constant, the difference in yield grade between muscle thickness groups No. 1 and No. 3 was significant (P < 0·05). Also, carcasses from cattle assigned muscle thickness scores of No. 1, as feeders, had the highest (P < 0·05) muscle to bone ratio of the round (4·1 to 1) while carcasses from cattle assigned thickness scores of No. 3, as feeders, had the lowest (P < 0·05) muscle to bone ratio of the round (3·4 to 1).