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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Cooperative Research Centre for Cattle and Beef Quality, Australia; CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Rd, St. Lucia, Queensland 4067, Australia;
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South Australian Research & Development Institute, Livestock Systems Alliance, Roseworthy, South Australia 5371, Australia; and
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Beef Industry Centre of Excellence, NSW Department of Primary Industries, JSF Barker Building, University of New England, Armidale NSW 2351, Australia
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Abstract |
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Key Words: cattle • gene expression • microarray • muscle • nutrition
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Compensatory growth after a period of BW loss can improve beef tenderness (Allingham et al., 1998). Detailed molecular knowledge of the regulation of muscle growth, fiber type, and extracellular matrix deposition is important not only for animal production but also for human medicine in the context of diseases that result in muscle wastage and also important for athletic performance.
Relatively little is known about the response of skeletal muscle to nutritional restriction in large animal species. A recent microarray study by da Costa et al. (2004) examined the molecular changes induced by moderate dietary restriction in young pigs, whereas Byrne et al. (2005) studied the response of bovine skeletal muscle to short-term dietary restriction.
In this study, we measured global changes in the gene expression profile of bovine LM during BW loss and subsequent realimentation using a bovine muscle-and fat-derived cDNA microarray (Lehnert et al., 2004). The LM was chosen as a postural muscle that is commercially important.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In brief, Belmont Red steers (n = 24) approximately 8 mo of age, with initial BW of 205 ± 34 kg, were randomly allocated to 2 groups. To achieve divergent growth paths, 2 diets were fed. The BW loss (WL) group was fed a low-quality grass hay (Angleton grass, Dichanthium aristatum) ad libitum, with average intake of 13 g of DM·kg of BW–1·d–1, and the rapid growth (RG) group was fed alfalfa hay ad libitum, resulting in intakes of 28 g of DM·kg of BW–1·d–1. These diets were fed for 120 d to achieve a 10% BW loss, or rapid growth (
0.6 kg of BW/d), respectively. This treatment period was followed by a 110-d realimentation period, during which both groups were fed on the RG diet. Tomkins et al. (in press) described the longer-term effects of the treatment.
Samples of tissue from the LM were collected at 3 times from every animal: 6 d before the commencement of the experiment (d –6, T1), near the end of the nutritional treatment (d 114, T2), and after 84 d of realimentation (d 204, T3; Figure 1). For muscle biopsy, steers were isolated, immobilized in a cattle chute, and sedated with 0.3 mg of Rompun (Bayer AG, Leverkusen, Germany)/kg of BW. Muscle samples of 1 to 2 g were removed using a scalpel and immediately snap-frozen using liquid nitrogen for RNA analyses.
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. A biopsy of approximately 1 cm2 x 1.5 cm was excised, and subcutaneous fat, fascia, and approximately 5 mm of muscle were removed from the superficial surface, leaving a cube approximately 1 cm3 for myofiber analysis. Serial cross-sections were cut from each sample and immunostained to identify myosin heavy chain (MHC) isoforms and the basal lamina surrounding each myofiber, as detailed by Greenwood et al. (2006a, b).
Microscopic image analysis was used to classify myofibers as types 1, 2C (type1-type2A intermediate), 2A, 2AX, or 2X and to measure the size of myofibers of each type, using the immunohistochemical staining procedures described by Greenwood et al. (2006a). Classification of myofibers was based on the staining characteristics of 3 antibodies against MHC (Picard et al., 1998), with modifications for brightfield microscopy (Greenwood et al., 2006a,b), and classification of myofiber types previously described as 2B and 2AB by Picard et al. (1998) as types 2X and 2AX, respectively. This nomenclature was used on the basis that antibody S5-8H2 binds to type 2B and type 2X MHC (Reggiani and Mascarello, 2004) but that only type 2X is expressed in limb and trunk muscles of cattle (Maccatrozzo et al., 2004). For each sample classified, the total area of each myofiber type relative to the total myofiber area was calculated from the percentage and average size of the myofiber types.
Statistical Assessment of Myofiber Data
Myofiber characteristics were analyzed by comparing data for WL and RG animals at each time point using REML in Genstat (Release 7.1, Lawes Agricultural Trust, Rothamstead Experimental Station, UK).
Target Labeling and Microarray Procedures
The process for RNA extraction and purification was performed as described by Reverter et al. (2003). Anti-sense RNA (aRNA) amplification was performed using the MessageAmp aRNA Kit (Ambion, Austin, TX). Indirect labeling procedures and hybridizations were performed as described by Lehnert et al. (2004), except that the complementary DNA (cDNA) was generated using a modified amino C6 dT random primer (Xiang et al., 2002; Geneworks, Adelaide, Australia) and the reverse transcription Superscript III (Invitrogen, Carlsbad, CA). The cDNA (prelabeling) and the postlabeled cDNA were purified using the QIAquick PCR purification columns (Qiagen, Valencia, CA). The hybridization mixture used a reduced detergent concentration of 0.2% SDS. We used a cDNA microarray of approximately 10,000 probes, printed in duplicate, which had been constructed from 2 cattle cDNA libraries: LM and subcutaneous fat tissue derived from a 24-mo-old Angus steer (Lehnert et al., 2004).
Microarray Experimental Design
The main objective of this gene expression experiment was to identify which genes were differentially expressed between the RG and the WL groups among the growth treatments (T1, T2, and T3). Total RNA collected from the treatment groups at each time point (n = 12) was pooled into 2 subgroups (n = 6) on the basis of the BW of the animals at the commencement of the study. In some instances, an individual animal extraction yielded insufficient RNA, and so only 5 of the 6 animals were used in that particular RNA subgroup pool. Equal amounts of total RNA from individual samples within each group were pooled, and amplified aRNA was generated and used as the template for indirect Cy5 and Cy3 dye-labeling of the target for the microarray experimentation. Data from 24 comparison slides were generated. The experimental design, which was balanced for dye swap considerations within diet treatments and across time points is shown in the Appendix, Figure A1.
Microarray Data Analysis
The GenePix 4000A optical scanner (Molecular Devices, Sunnyvale, CA) and the GenePixPro 5.1 image analysis software (Molecular Devices) were used to quantify the gene expression levels. Data acquisition criteria are detailed in Tan et al. (2006). These criteria were applied separately for the red (Cy5) and for the green (Cy3) channels, so that a different number of observations for each channel was obtained. These resulted in a total of 887,706 gene expression readings (443,292 red and 444,414 green) on 8,856 genes (or probes or clones) that were background-corrected and base-2 log-transformed. The arithmetic mean and SD (in parentheses) for the red and green intensities were 11.93 (2.02) and 11.97 (1.95), respectively.
Data normalization was achieved from the most optimal method reported by Reverter et al. (2005), and differentially expressed genes were identified by model-based clustering via mixtures of distributions on the normalized expression of each gene for each treatment (growth group and time point), as detailed in Reverter et al. (2004). In brief, a mixed-model was fitted to the intensity readings, which included the combined fixed effect of array slide, printing block, and dye channel (2,304 levels), and the random effects of gene (8,856 levels), gene x array slide-printing block interaction (231,281 levels), gene x dye channel interaction (17,712 levels), gene x subgroup interaction (17,712 levels), gene x group x time interaction (53,136 levels), and residual.
Synthesis of cDNA for Quantitative Reverse Transcription-PCR (qRT-PCR)
TRIzol-extracted total RNA (Invitrogen) from LM samples collected at the 3 times (T1, T2 and T3) was further purified on RNeasy mini-columns and treated on-column with DNase I (Qiagen), according to the manufacturer’s instructions. Total RNA concentrations and relative purity were determined spectrophotometrically by measurement of the UV absorbance at 260 and 280 nm (GE Healthcare, Little Chalfont, UK). Reverse transcription of 1.5 µg of total RNA was performed with the Omniscript cDNA synthesis kit (Qiagen), using a mixture of oligonucleotides [100 µM OligodTVN (5'-TTTTTTTTTTTTTTVN-3', where V = A,C,G, and N = A,C,G,T) and 1 µM 18S primer (5'-CACACGCTGAGCCAGTCAGT-3')]. The cDNA was diluted 1:10 in 10 mM Tris-HCl (pH 8.0) for target gene measurements, whereas the 18S rRNA reference gene measurements were performed on a 1:200 dilution of cDNA.
Quantitative RT-PCR Analyses
To measure mRNA transcript levels, qRT-PCR using SYBR Green I was carried out according to Nattrass et al. (2006). Real-time PCR measurements were performed in triplicate on all available cDNA samples (n = 64) or pools of cDNA (n = 6) in a RotorGene3000 (Corbett Life Science, Sydney, Australia). The triplicate measurements were carried out at least twice for each cDNA sample or pool of cDNA. Primer sequences are listed in Table 1.
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Statistical Assessment of qRT-PCR Gene Expression Data
For the RT-PCR validation trials, gene expression data were obtained from individual RNA samples (n = 64) for stearoyl-CoA desaturase (SCD) and lipoprotein lipase (LPL) at T1, T2, and T3, and from pooled RNA samples (n = 6) for MYOD1, also at the 3 time points. Statistical analyses were performed using the MIXED procedure (SAS Inst. Inc., Cary, NC) with animal included as the random term when individual samples were available. All statistical assessments were conducted on log-transformed, real-time PCR data, as none of the gene expression profiles exhibited a normal distribution. First- and second-order interactions were examined and sequentially removed if nonsignificant (P > 0.05). Target gene expression data were normalized against 18S rRNA expression measured at the same time.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Tomkins et al., 2006). During the realimentation phase (d 121 to 230), the animals in the WL group grew faster than during treatment, as would be expected with nutritional repletion. Nonetheless, an average of 50-kg difference in BW between the 2 groups remained until the slaughter date 536 d after the start of treatment (data not shown; Tomkins et al., 2006). Comparing the growth rates in the first 60 d of realimentation, it appears that the WL animals were exhibiting compensatory growth (Sainz et al., 1995), though this was insufficient to recover the BW lost during treatment.
Immunohistochemical Analysis of Muscle Fiber Type
Myofiber characteristics did not differ between the WL and RG groups before commencement of the nutritional treatments (Table 2 and Figure 2). After 114 d of WL (T2), the cross-sectional area of all myofiber types was significantly reduced compared with RG cattle, the magnitude of the effect being greatest in type 2X (69% reduction), followed by type 2A (64% reduction) and type 1 (48% reduction) myofibers. The net effect was an increase in the percentage of the total myofiber area comprising type 1 myofibers (P < 0.001) and a decrease in type 2X myofibers (P = 0.022; Figure 2), with no significant effect on the relative area of type 2A myofibers (RG 17.3 vs. WL 20.6%, SED = 2.48%, P = 0.193). The cross-sectional area of myofibers remained smaller in the WL compared with RG cattle at T3, with most myofiber growth occurring in type 2X compared with types 1 and 2A within the WL group (Table 2). The relative area of the myofiber types did not differ between the WL and RG groups at T3 (Figure 2).
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) were differentially expressed (Appendix, Table A6). For a number of array elements that were identified as differentially expressed, sequence information of sufficient length and quality was not available. These elements are omitted from further discussion. Forty-six of the differentially expressed genes identified in the comparison of LM RNA from animals in the WL and RG groups showed 2-fold or greater gene expression differences. These 46 genes and their expression values are listed in Tables 3 to 5.
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).
For 7 differentially expressed genes (CSRP3, SCD, CYR61, KBTBD5, SESN1, SQSTM1, and TM4SF2) the opposite trend was observed, where there was a 2- to 4-fold increase in expression at T2 in the muscles from the WL group at T2, followed by a return to comparable gene expression levels to the RG group by T3 (Table 4). The gene for myosin regulatory light chain (MRCL3) is the only differentially expressed gene identified as having a 2-fold difference between the treatment groups at T3 only (Table 5).
Significant differences between the treatment groups were observed in 2 genes (FOS and HSPA1A) at T1, before the start of the nutritional treatment (Table 5). In addition, a number of other stress response genes also showed less expression in the WL group at T1 (Appendix, Table A4). Quantitative RT-PCR expression analyses of individual animal RNA showed that, compared with the interanimal variation in expression levels for CRYAB and HSPA8, the cumulative expression differences between the 2 treatment groups were not significant (Appendix, Figure A2).
Quantitative RT-PCR Assay of Lipogenic Enzymes and Myogenic Growth Factors
Quantitative RT-PCR analysis was undertaken to verify SCD and LPL differential expression (Table 3 and 4). The qRT-PCR analysis was carried out on individual RNA samples, across all 3 time points. The qRT-PCR results confirmed the findings from the microarray study. The SCD expression in the WL group at T2 was greater (P < 0.001) than the levels observed at all the other time points (Figure 3A). When measured in the T3 sample, SCD gene expression in the WL group had returned to comparable levels with those measured at T1. Appendix Figure A3 plots the qRT-PCR data for individual animals and documents the magnitude of the gene expression changes between consecutive biopsies from the same individual. In all but one animal, the WL treatment led to an increase in SCD gene expression levels from 3- to 15-fold at T2.
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). The LPL expression for the WL group at T2 was less than the levels observed for the RG treatment at T1 (P = 0.006), T2 (P = 0.01), and T3 (P = 0.003) and lower than the level observed in the WL group at T1 (P = 0.003). The LPL expression in the T3 sample from the WL group was not significantly different from that of preceding time points (P > 0.05).
Quantitative RT-PCR analysis of myogenic growth factor MYOD1 was carried out on pooled RNA samples (Figure 3C). The MYOD1 expression was slightly greater in the muscle of the RG animals at the start of the treatment. At T2, significant differences with respect to the expression of MYOD1 were seen between the WL and the RG groups. The WL treatment resulted in elevated levels of MYOD1 expression. At T3, the gene expression differences between the 2 treatment groups were still significant.
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DISCUSSION |
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In summary, the observation that transcription related to fast-twitch glycolytic myofibers was more profoundly downregulated than that for slow-twitch oxidative fibers during BW loss points to a specific atrophic muscular phenotype in the bovine aimed at preserving slow-twitch fibers. This finding is further corroborated by the results of histological fiber typing, which clearly show the loss of fast-twitch, type 2X myofibers during BW loss.
At the gross myofiber level, the response of mammalian muscle to starvation (as represented by humans, pigs, hamsters, and mice) has been well characterized, with a clear preferential atrophy of the fast, glycolytic fibers (Goldspink and Ward, 1979; Essen et al., 1981; White et al., 2000). The data presented here and those from an earlier study examining a shorter duration of BW loss (Byrne et al., 2005) support this trend, indicating a common muscle phenotype associated with starvation in mammals.
It is unclear whether the preferential atrophy of glycolytic muscle fibers in mammals is an adaptation to better cope with nutritional deprivation. However, proponents of this hypothesis have argued that the fast glycolytic fibers are less commonly used and so are more dispensable as a protein reserve and that the resultant muscle phenotype might then perform more efficiently because slow, oxidative fibers expend less energy per unit tension and produce more ATP per unit glucose than glycolytic fibers (Wendt and Gibbs, 1973). An additional adaptive aspect of preferential atrophy of glycolytic fibers could be that a muscle that contains fewer of these fibers will directly conserve glucose, as ruminant glycolytic fibers have higher densities of insulin receptors and glucose transporters (Hocquette et al., 1995).
Nutritional restriction, be it caloric or protein restriction, has often been associated with changes in protein synthesis and particularly extracellular matrix proteins such as collagen (Laurent, 1987). The important observation from this study and others from our laboratory (Reverter et al., 2003) is the coordinated downregulation of transcripts coding for extracellular matrix proteins that, in turn, are involved in supramolecular complexes with collagen in the matrix. Interestingly, coordinated upregulation of expression of these genes was not seen during compensatory growth, though this experimental design does not allow delineation of the mechanism.
Transcriptional Changes Associated with Compensatory Growth
This study aimed to identify molecular processes that may characterize compensatory growth in bovine muscle. As a first approximation, we compared our data to those collected during muscle regeneration, and expression profiling studies in mice, which showed significant upregulation of extracellular matrix-related gene expression as well as myogenic factors (Goetsch et al., 2003). In contrast, our microarray study identified only 1 differentially expressed gene MRCL3 specifically due to a change in expression at T3, during compensatory growth. It is likely that the choice of this time point, 84 d after the start of realimentation, emphasized long-lived effects of nutrient restriction, realimentation, and the compensatory growth response. By this time, the animals had entered a normal growth trajectory, even though previous studies have suggested persistent effects of nutrient restriction (Allingham et al., 1998). The timing of future gene expression studies of compensatory growth may need to be targeted at the early days and weeks after realimentation to characterize more acute adaptations to better nutrition after severe, prolonged BW loss.
Stearoyl-CoA Desaturase Gene Expression
Stearoyl-CoA desaturase is considered to be a key regulatory enzyme in the lipogenic pathway (Ntambi, 1999; Al-Hasani and Joost, 2005). In rat liver, SCD activity decreased during starvation and was rapidly induced to high levels upon refeeding high carbohydrate diets (Oshino and Sato, 1972). The reduction of SCD gene activity in the liver in response to leptin-mediated BW loss has recently been proposed as one of the main mechanisms through which leptin exerts its effects on body composition (Cohen et al., 2002). In contrast to rodents, bovine SCD is expressed at high levels in subcutaneous fat and muscle but not in liver (Cameron et al., 1994).
The microarray analysis of lean skeletal muscle during BW loss shows that SCD gene transcripts were more highly represented in the muscle of WL animals than their RG contemporaries. This result appears to contradict existing data on SCD regulation in the liver under BW-loss conditions (Sprecher, 1981) and other findings on the response of skeletal muscle lipid metabolism to diet (Cameron-Smith et al., 2003). However, in contrast to the upregulation of SCD gene expression, LPL gene expression followed the expected pattern of a slight downregulation in the muscle of WL animals.
It is possible that SCD transcripts may be more highly represented in the WL muscle due to the reduction in size or number of cells that do not express SCD. Specifically, it may be the case that SCD gene transcription in muscle is associated mainly with type 1 oxidative and type 2A oxidative-glycolytic myofiber types. Evidence from a study of skeletal muscle membrane lipid composition shows that more oxidative fiber types have a greater percentage of unsaturated fatty acids (Storlien et al., 1996). Recent data on the effect of SCD1-deficiency on skeletal muscle metabolism in the mouse showed that "red" muscles with a greater proportion of oxidative fibers were much more profoundly affected than "white" muscles (Dobrzyn et al., 2005).
On the other hand, the increase in SCD gene expression may indicate that the muscle in the nutritionally restricted animals synthesized unsaturated fatty acids as a specific response. da Costa et al. (2004) reported an accumulation of intramuscular lipid and an upregulation of lipid metabolism gene expression, including SCD, in the muscle tissues of young pigs fed a moderately restricted diet. Similar findings were reported in a study of murine skeletal muscles under dietary restriction (Lee et al., 1999). Lipid composition of muscle biopsies was not measured in the current study, although a crude indicator of muscle fat deposition, marbling, was not greater in meat from the WL animals than from the control animals (Tomkins et al., 2006).
The lipid content and lipid profile of bovine muscle has important implications for meat quality (Pethick et al., 2004). If moderate growth restriction could lead to the accumulation of unsaturated fatty acids in bovine muscle, this offers a potentially novel way to modify the quality of beef. More detailed studies of cellularity, lipid profiles and enzyme activities of bovine growth-restricted muscle are necessary to evaluate this possibility.
Elevated Gene Expression in Response to BW Loss
In addition to SCD, 6 other genes also showed greater levels of expression in the muscle of WL animals. The significance of these differential gene expression signals has to be considered in the light of the comments made in relation to SCD gene expression, namely that these transcripts may be found to be more highly represented as a consequence of proportional changes in muscle fiber types, rather than displaying a specific transcriptional response of skeletal muscle tissue to nutrient deprivation.
In studies of muscle atrophy in mice and humans, elevated expression of the ubiquitin-protein ligases atrogin-1 (MAFbx) and muscle RING finger (MuRF1) has been identified as part of the molecular signature of atrophic muscle (Jagoe et al., 2002; Glass, 2003). Byrne et al. (2005) observed evidence of specific regulation of different aspects of the ubiquitin/proteasome pathway, as well as of ribosomal proteins in bovine muscle after 27 d of BW loss. Despite a much larger number of genes detected as significantly differentially expressed in the current study, the ubiquitin-associated sequestosome (SQSTM1) protein (Lange et al., 2005) was the only element of the ubiquitin/proteasome system that appeared to be differentially regulated in this study of cattle muscle remodelling. This result may indicate that the major reprogramming events in muscle in response to nutritional deprivation were no longer evident by 84 d. Furthermore, our findings suggest that the muscle exhibited a homeorhetic response resulting in altered homeostasis (Bauman and Currie, 1980) to support increased efficiency (greater mass of oxidative and reduced mass of glycolytic myofibers) that would maximize the conservation of protein and energy in the face of prolonged nutritional restriction.
The cysteine and glycine-rich protein 3 (CSRP3), also referred to as muscle LIM protein, has been implicated in the regulation of myogenesis (Kong et al., 1997) and in the precise regulation of cardiomyocyte remodelling after myocardial infarction (Heineke et al., 2005). Its upregulation in the WL group at T2 coincides with an upregulation of transcription for myogenic factor MYOD1. The presence of elevated levels of CSRP3and MYOD1 after 114 d of BW loss may indicate that it plays a role in the maintenance of satellite cells in atrophying muscle. Furthermore, the cysteine-rich, angiogenic inducer 61 (CYR61) has recently been detected as a differentially expressed transcript in denervated skeletal muscle (Magnusson et al., 2005). Schütze et al. (2005) have proposed a role for CYR61 in the maintenance of mesenchymal stem cells, a role that may explain its elevated expression in muscle during BW loss.
Muscle atrophy is partly mediated by an accumulation of oxidative damage, where prooxidants such as the hydroxyl radical overwhelm antioxidant defense and tissue repair mechanisms (Kondo et al., 1994). In the current study, the upregulation of the antioxidant sestrin-1 (SESN1; Budanov et al., 2004) may reflect an adaptive response to combat increased oxidative damage, following the release of bound metals from atrophied glycolytic muscle fibers. To our knowledge, this is the first instance of an upregulated antioxidant being documented in atrophic skeletal muscle, although Asayama et al. (1989) observed previously that catalase was upregulated in atrophic rat cardiac muscle.
Kelch repeat and BTB (POZ) domain containing 5 (KBTBD5) belongs to a family of proteins with unknown function (Prag and Adams, 2003). This gene has also been detected as a differentially expressed transcript when comparing different fat depots in the bovine (Hishikawa et al., 2005). Also, the transmembrane 4 L 6 family member (TM4SF2) belongs to a family of membrane proteins of unknown function (Maecker et al., 1997). The importance of increased representation of K5TBD5 and TM4SF2 transcripts in muscle after BW loss remains unclear.
In conclusion, the current study may point to several crucial aspects of BW loss-mediated muscle atrophy in bovine, all of which may contribute to the ability of this mammalian species to cope with and efficiently recover from periods of nutritional deprivation. The molecular profile described in this study confirms that bovine muscle preferentially sacrifices the fast, glycolytic muscle fibers during BW loss conditions. The evidence for increased SCD gene transcription in WL muscle may have implications for the manipulation of fatty acid profiles in skeletal muscle. In addition, we have uncovered evidence for possible growth-factor-mediated maintenance of specific stem cell compartments during atrophy, as well as the possible activation of antioxidative mechanisms. This study also identifies as yet unexplored aspects of the molecular response to BW loss muscle atrophy, with at least 2 transcripts of unknown function having elevated expression. Hence, the knowledge gained in this study has the potential to contribute to future strategies in the management of and recovery from muscular atrophy in mammals
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Footnotes |
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3 Present address: Meat and Livestock Australia, P. O. Box 2363, Fortitude Valley BC QLD 4006.
2 Corresponding author: Sigrid.Lehnert{at}csiro.au
Received for publication March 28, 2006. Accepted for publication July 20, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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  A. Reverter, N. J. Hudson, Y. Wang, S.-H. Tan, W. Barris, K. A. Byrne, S. M. McWilliam, C. D. K. Bottema, A. Kister, P. L. Greenwood, et al. A gene coexpression network for bovine skeletal muscle inferred from microarray data Physiol Genomics, December 13, 2006; 28(1): 76 - 83. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and rapid growth (RG; n = 12;
) treatment groups. Biopsy time points (T1 to T3) are indicated along the x-axis.
