Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development.

The ruminant developmental transition from late foetus to lamb is associated with marked changes in skeletal muscle structure and function that reflect programming for new physiological demands following birth. To determine whether epigenetic changes are involved in this transition, we investigated the genomic architecture of the chromatin modification, histone 3 lysine 27 trimethylation (H3K27me3), which typically regulates early life developmental processes; however, its role in later life processes is unclear. Chromatin immunoprecipitation coupled with next-generation sequencing was used to map H3K27me3 nucleosomes in ovine longissimus lumborum skeletal muscle at 100 days of gestation and 12 weeks post-partum. In both states, H3K27me3 modification was associated with genes, transcription start sites and CpG islands and with transcriptional silencing. The H3K27me3 peaks consisted of two major categories, promoter specific and regional, with the latter the dominant feature. Genes encoding homeobox transcription factors regulating early life development and genes involved in neural functions, particularly gated ion channels, were strongly modified by H3K27me3. Gene promoters differentially modified by H3K27me3 in the foetus and lamb were enriched for gated ion channels, which may reflect changes in neuromuscular function. However, most modified genes showed no changes, indicating that H3K27me3 does not have a large role in late muscle maturation. Notably, promyogenic transcription factors were strongly modified with H3K27me3 but showed no differences between the late gestation foetus and lamb, likely reflecting their lack of involvement in the myofibre fusion process occurring in this transition. H3K27me3 is a major architectural feature of the epigenetic landscape of ruminant skeletal muscle, and it comments on gene transcription and gene function in the context of late skeletal muscle development.

New insights into polar overdominance in callipyge sheep.

The callipyge phenotype in sheep involves substantial postnatal muscle hypertrophy and other changes to carcass composition. A single nucleotide polymorphism in the DLK1-DIO3 imprinted gene cluster alters gene expression of the paternal allele-specific protein-coding genes and several maternal allele-specific long noncoding RNA and microRNA when the mutation is inherited in cis. The inheritance pattern of the callipyge phenotype is polar overdominant because muscle hypertrophy only occurs in heterozygous animals that inherit a normal maternal allele and the callipyge SNP on the paternal allele (+/C). We examined the changes of gene expression of four major transcripts from the DLK1-DIO3 cluster and four myosin isoforms during the development of muscle hypertrophy in the semimembranosus as well as in the supraspinatus that does not undergo hypertrophy. The homozygous (C/C) animals had an intermediate gene expression pattern for the paternal allele-specific genes and two myosin isoforms, indicating a biological activity that was insufficient to change muscle mass. Transcriptome analysis was conducted by RNA sequencing in the four callipyge genotypes. The data show that homozygous animals (C/C) have lower levels of gene expression at many loci relative to the other three genotypes. A number of the downregulated genes are putative targets of the maternal allele-specific microRNA with gene ontology, indicating regulatory and cell signaling functions. These results suggest that the trans-effect of the maternal noncoding RNA and associated miRNA is to stabilize the expression of a number of regulatory genes at a functional, but low level to make the myofibers of homozygous (C/C) lambs less responsive to hypertrophic stimuli of the paternal allele-specific genes.

The sheep genome reference sequence: a work in progress.

Until recently, the construction of a reference genome was performed using Sanger sequencing alone. The emergence of next-generation sequencing platforms now means reference genomes may incorporate sequence data generated from a range of sequencing platforms, each of which have different read length, systematic biases and mate-pair characteristics. The objective of this review is to inform the mammalian genomics community about the experimental strategy being pursued by the International Sheep Genomics Consortium (ISGC) to construct the draft reference genome of sheep (Ovis aries). Component activities such as data generation, sequence assembly and annotation are described, along with information concerning the key researchers performing the work. This aims to foster future participation from across the research community through the coordinated activities of the consortium. The review also serves as a 'marker paper' by providing information concerning the pre-publication release of the reference genome. This ensures the ISGC adheres to the framework for data sharing established at the recent Toronto International Data Release Workshop and provides guidelines for data users.

Molecular cytogenetics and gene mapping in sheep (Ovis aries, 2n = 54).

The development of a completely annotated sheep genome sequence is a key need for understanding the phylogenetic relationships and genetic diversity among the many different sheep breeds worldwide and for identifying genes controlling economically and physiologically important traits. The ovine genome sequence assembly will be crucial for developing optimized breeding programs based on highly productive, healthy sheep phenotypes that are adapted to modern breeding and production conditions. Scientists and breeders around the globe have been contributing to this goal by generating genomic and cDNA libraries, performing genome-wide and trait-associated analyses of polymorphism, expression analysis, genome sequencing, and by developing virtual and physical comparative maps. The International Sheep Genomics Consortium (ISGC), an informal network of sheep genomics researchers, is playing a major role in coordinating many of these activities. In addition to serving as an essential tool for monitoring chromosome abnormalities in specific sheep populations, ovine molecular cytogenetics provides physical anchors which link and order genome regions, such as sequence contigs, genes and polymorphic DNA markers to ovine chromosomes. Likewise, molecular cytogenetics can contribute to the process of defining evolutionary breakpoints between related species. The selective expansion of the sheep cytogenetic map, using loci to connect maps and identify chromosome bands, can substantially contribute to improving the quality of the annotated sheep genome sequence and will also accelerate its assembly. Furthermore, identifying major morphological chromosome anomalies and micro-rearrangements, such as gene duplications or deletions, that might occur between different sheep breeds and other Ovis species will also be important to understand the diversity of sheep chromosome structure and its implications for cross-breeding. To date, 566 loci have been assigned to specific chromosome regions in sheep and the new cytogenetic map is presented as part of this review. This review will also summarize the current cytogenomic status of the sheep genome, describe current activities in the sheep cytogenomics research sector, and will discuss the cytogenomics data in context with other major sheep genomics projects.

A high-resolution radiation hybrid map of sheep chromosome X and comparison with human and cattle.

A radiation hybrid (RH) map of sheep X chromosome (Ovisaries; OARX) containing 146 physically anchored loci was generated in this study, providing information for comparative X chromosome analysis between the maps of sheep, human, and cattle. Primers typed on the USUoRH5000 ovine whole-genome radiation hybrid panel were designed from sequences predicted to be on the ovine X chromosome, based on comparative mapping within the virtual sheep genome browser (v1.2). The resulting RH map for the ovine X chromosome consists of 4 linkage groups composed of 76 BAC end sequences (BES), 28 gene loci that were confirmed within ovine BAC clones in the CHORI-243 ovine BAC library, 28 additional gene loci from the ovine comparative map and 14 polymorphic sequence tagged sites (STS) from the OARX linkage map. This first-generation RH map of OARX contributes to the expansion of a comprehensive ovine genome map for sheep and provides evidence of rearrangements in loci order compared to the human and cattle orders.

Polar overdominance at the ovine callipyge locus.

An inheritable muscular hypertrophy was recently described in sheep and shown to be determined by the callipyge gene mapped to ovine chromosome 18. Here, the callipyge phenotype was found to be characterized by a nonmendelian inheritance pattern, referred to as polar overdominance, where only heterozygous individuals having inherited the callipyge mutation from their sire express the phenotype. The possible role of parental imprinting in the determinism of polar overdominance is envisaged.

A radiation hybrid comparative map of ovine chromosome 1 aligned to the virtual sheep genome.

Ovis aries chromosome one (OAR1) is the largest submetacentric chromosome in the sheep genome and is homologous to regions on human chromosomes 1, 2, 3 and 21. Using the USUoRH5000 ovine whole-genome radiation hybrid (RH) panel, we have constructed a RH map of OAR1 comprising 102 framework and 75 placed/binned markers across five linkage groups spanning 3759.43 cR5000, with an average marker density of 21.2 cR5000/marker. The alignment of our OAR1 RH map shows good concordance with the recently developed virtual sheep genome, with fewer than 1.86% discrepancies. A comparative map of OAR1 was constructed by examining the location of RH-mapped orthologues in sheep within the genomes of cow, human, horse and dog. Analysis of the comparative map indicates that conserved syntenies within the five ovine RH linkage groups underwent internal chromosomal rearrangements which, in general, reflect the evolutionary distances between sheep and each of these four species. The ovine RH map presented here integrates all available mapping data and includes new genomic information for ovine chromosome 1.

A map of the class III region of the sheep major histocompatibilty complex.

BACKGROUND: The central, or class III, region of the major histocompatibility complex (MHC) is an important gene rich sub-region of the MHC of mammals and contains many loci implicated in disease processes and potential productivity traits. As a prelude to identifying MHC loci associated with productivity traits in sheep, we have used BAC and cosmid libraries of genomic DNA to generate a physical map of the sheep MHC class III region. This map will facilitate association studies and provide insights into the distribution of recombination events in this chromosomal segment. RESULTS: Twenty eight sheep genes were identified in 10 BAC clones which spanned approximately 700 kbp of a chromosomal region adjacent to the class I region of the sheep MHC and which therefore covers most, if not all, of the class III of the sheep MHC. The relative positions of 17 of these genes was established as well as two additional groups of genes for which the intragroup order was not known. Cosmid mapping permitted a more detailed mapping of the complement genes present in the class III and showed a local inversion (relative to humans) of one pair of the duplicated complement C4 and CYP21 loci. A panel of 26 single nucleotide polymorphisms (SNPs) was identified in 10 loci, covering approximately 600 kbp of the mapped region. CONCLUSION: This report provides a physical map covering approximately 700 kbp of the class III of the sheep MHC together with a SNP panel which will facilitate disease and productivity association studies. The presence of a local inversion (relative to humans) of one pair of the duplicated C4 and CYP21 loci and a previously described dinucleotide tandem repeat locus (BfMs) has been located within an intron of the SK12VL gene.

A comparative radiation hybrid map of sheep chromosome 10.

Comparative radiation hybrid (RH) maps of individual ovine chromosomes are essential to identify genes governing traits of economic importance in sheep, a livestock species for which whole genome sequence data are not yet available. The USUoRH5000 radiation hybrid panel was used to generate a RH map of sheep chromosome 10 (OAR10) with 59 markers that span 1,422 cR over an estimated 92 Mb of the chromosome, thus providing markers every 2 Mb (equivalent to every 24 cR). The markers were derived from 46 BAC end sequences (BESs), a single EST, and 12 microsatellites. Comparative analysis showed that OAR10 shares remarkable conservation of gene order along the entire length of cattle chromosome 12 and that OAR10 contains four major homologous synteny blocks, each related to segments of the homologous human chromosome 13. Extending the comparison to the horse, dog, mouse, and chicken genome showed that these blocks share conserved synteny across species.

A high-resolution comparative radiation hybrid map of ovine chromosomal regions that are homologous to human chromosome 6 (HSA6).

In this study, we constructed high-resolution radiation hybrid (RH) and comparative maps of ovine chromosomes or chromosomal segments that are homologous to human chromosome 6 (HSA6). A total of 251 markers were successfully genotyped across the recently developed USUoRH5000 whole-genome panel; 208 of these markers were assigned to five RH linkage groups distributed on three ovine chromosomes (OAR8, 9 and 20). The RH maps have good correspondence with previous chromosome painting data, although a small centromeric region on OAR9 that is homologous to HSA6 had not been previously detected using human chromosome paints on ovine chromosomal spreads. High percentages of the ovine markers were identified as orthologues in the bovine (86.3%), dog (85.8%), horse (69.3%) and human (88.7%) genomes. These maps contribute to investigations in mammalian chromosome evolution and the search for economic trait loci in sheep.

The influence of breeding intensity on above- and below-average sexual performance rams in single- and multiple-sire breeding environments.

Two studies were conducted to evaluate the relationship between serving capacity scores and breeding performance of rams. The first study was conducted to determine whether rams with above or below mean serving capacity scores could perform equally in greater and lesser breeding intensity, single-sire mating schemes. The second study was conducted to determine whether rams with above and below mean serving capacity scores could perform equally well when only one or two ewes were in estrus daily in a multiple-sire breeding scheme (two rams/pen). Rams (n=68) were ranked according to average number of ejaculations recorded in serving capacity tests. Sixteen rams with the greatest scores (above-average) and 16 rams with least scores (below-average) were identified for breeding. Half of above-average and half of below-average rams were used in the two studies. For study 1, each ram was individually introduced to 23 estrus-synchronized ewes for 9 d to simulate high breeding intensity. Rams were given a 5-d rest before they were individually introduced to 23-24 naturally cyclic ewes for 17 d (low breeding intensity). For study 2, 16 rams were paired across ram types, and each pair competed for 20 ewes for 18 d (8 pens). For study 1, ewe fertility (ewes lambing/ewes present at lambing) and number of lambs born were greater (P<0.001) for above-average (0.67+/-0.03 and 27.6+/-1.2, respectively) than for below-average rams (0.39+/-0.07 and 15.3+/-2.7) with greater breeding intensity. Ewe fertility and lambs born did not differ for above-average (0.91+/-0.03 and 37.8+/-1.9, respectively) and below-average rams (0.86+/-0.03 and 39.0+/-1.9) with less breeding intensity. For study 2, number of ewes lambing (99+/-8.0 compared with 72+/-13.6; P=0.12) and number of lambs sired (149+/-18.5 compared with 101+/-22.8; P=0.14) did not differ between above- and below-average rams, respectively, in direct competition. Sexual classifications based on serving capacity tests are related to breeding performance of rams in certain breeding environments. When breeding intensity is greater, above-average rams impregnate more ewes and sire more lambs than below-average rams. When only a small number of ewes are in estrus daily, below-average rams for serving capacity scores perform as well as above-average rams in multiple-sire and single-sire breeding environments. We suggest that above-average rams should be used to reduce number of rams required when breeding intensity is greater.

Segregation of natural and experimental gastrointestinal nematode infection in F2 progeny of susceptible Suffolk and resistant Gulf Coast Native sheep and its usefulness in assessment of genetic variation.

Gastrointestinal nematode parasitism is a concern to small ruminants worldwide. Productivity has been compromised because such nematodes, particularly Haemonchus contortus, have developed resistance to available anthelmintics. Some sheep breeds and lines within breeds are relatively resistant to infection, a trait that may be useful for developing control strategies. Suffolk sheep, which are susceptible to infection, were crossed with Gulf Coast Native sheep, which are more resistant to infection, to produce F1 progeny. F1 rams were bred to F1 ewes which produced 227 F2 offspring. These F2 offspring were evaluated for variability in infection levels, based on fecal egg count (FEC) and blood packed cell volume (PCV), under two natural infection conditions (one at weaning and another after a summer grazing period) and one experimental infection. The range of both FEC and PCV was large for all three infection periods with annual variation. Overall, the range for the three infection periods, respectively, were 167-149,933, 0-31,400 and 17-114,667 eggs per gram (EPG) of feces and 8.7-37.0%, 7.3-33.0% and 8.3-36.0%. This segregation of infection is what would be expected of F(2) progeny from susceptible and resistant parent breeds. Heritabilities of FEC and PCV for the three infection periods, respectively, were 0.15, 0.29 and 0.12, and 0.11, 0.22 and 0.12. Based on segregation of infection, larger heritabilities and maternal environment effects that declined after weaning, the summer natural infection was probably the best model for assessing genetic variation.

Investigation of the Booroola (FecB) and Inverdale (FecX(I)) mutations in 21 prolific breeds and strains of sheep sampled in 13 countries.

Twenty-one of the world's prolific sheep breeds and strains were tested for the presence of the FecB mutation of BMPR1B and the FecX(I) mutation of BMP15. The breeds studied were Romanov (2 strains), Finn (2 strains), East Friesian, Teeswater, Blueface Leicester, Hu, Han, D'Man, Chios, Mountain Sheep (three breeds), German Whiteheaded Mutton, Lleyn, Loa, Galician, Barbados Blackbelly (pure and crossbred) and St. Croix. The FecB mutation was found in two breeds, Hu and Han from China, but not in any of the other breeds. The 12 Hu sheep sampled were all homozygous carriers of FecB (FecB(B)/FecB(B)) whereas the sample of 12 Han sheep included all three genotypes (FecB(B)/FecB(B), FecB(B)/FecB+, FecB+/FecB+) at frequencies of 0.33, 0.58 and 0.08, respectively. There was no evidence of FecX(I) in any of the breeds sampled.

Genetic variation of major histocompatibility complex and microsatellite loci: a comparison in bighorn sheep.

Examining and comparing genetic variation for major histocompatibility complex (MHC) and micro-satellite (MS) loci in the same individuals provides an opportunity to understand the forces influencing genetic variation. We examined five MHC and three MS loci in 235 bighorn sheep (Ovis canadensis) from 14 populations and found that both types of loci were highly variable and were in Hardy-Weinberg proportions. Mean FST values for both markers were very similar and MHC and MS genetic variability was predominantly distributed within rather than among populations. However, analyses of genetic distances and tree topologies revealed different spatial patterns of variation for the two types of loci. Collectively, these results indicated that neutral forces substantially influenced MS and MHC variation, and they provided limited evidence for selection acting on the MHC.

Analysis of the sheep genome.

The identification of genes controlling several traits of interest in sheep has been accomplished by positional candidate cloning. In these studies, the trait is first mapped to a specific chromosomal region by linkage analysis, which requires families that are segregating for the trait and for polymorphic markers. Microsatellite markers are usually used for these analyses because of their extensive genetic variability. Once the location of a trait is determined by linkage to the markers, possible candidate genes controlling the trait can be inferred because of their proximity to linked markers. It is not necessary to map all possible genes in sheep for this strategy to be effective. Rather, a subset of genes that are mapped in humans and mice have also been mapped in sheep; these genes serve as "anchors" across the comparative maps of the different species. Further study of these positional candidates has revealed naturally occurring mutations that produce phenotypes that are unique to sheep. Thus the genetic analysis of sheep traits advances knowledge not only in this species but provides critical information for understanding biological pathways in mammalian species.

Current status of the ovine genome map.

Genome maps in livestock species have been under development for the last decade. While the sheep map is one of the least advanced for livestock, the amount of available information is noteworthy, in light of the paucity of funding and personnel devoted to this project. These limited resources have been strategically aligned to take advantage of information from the human, mouse and bovine mapping and sequencing efforts. The resulting ovine linkage and physical maps have greatly enhanced the search for genes controlling important traits in sheep. In order to improve the efficiency of these investigations, it is imperative that efforts on the sheep comparative map be continued.

Effect of the callipyge gene on muscle growth, calpastatin activity, and tenderness of three muscles across the growth curve.

Changes in muscle growth, calpastatin activity, and tenderness of three muscles were assessed in 20 callipyge and 20 normal wether lambs slaughtered at live weights (LW) of 7, 20, 36, 52, and 69 kg. At 24 h postmortem, the longissimus (LM), semimembranosus (SM), and supraspinatus (SS) muscles were removed and weighed and samples were obtained for calpastatin activity (CA; 24 h) and Warner-Bratzler shear force (WBS; aged 6 d). For muscle weights and calpastatin activity, the weight group x muscle x phenotype interaction was significant (P < 0.05). Muscle weights were similar (P > 0.05) between phenotypes for all three muscles at 7 kg LW. At 20 kg LW, the LM and SM muscles from the callipyge lambs were heavier (P < 0.05) than those from normal lambs; however, the SS did not differ (P > 0.05) between phenotypes at 7, 20, or 52 kg. From 20 to 69 kg LW, the LM and SM weights were 42 and 49% heavier (P < 0.05) for callipyge than for normal lambs. Calpastatin activity of the callipyge LM was greater (P < 0.05) than that of normal LM at 36, 52, and 69 kg. In the callipyge LM, CA was similar (P > 0.05) at 20, 36, and 52 kg LW and did not differ (P > 0.05) from 7-kg or 69-kg values. Calpastatin activity declined (P < 0.05) across the growth curve for the SM and SS, but values were higher (P < 0.05) in the SM in callipyge than in normal lambs. Shear force values of the LM were lower (P < 0.05) for normal lambs at 36, 52, and 69 kg LW than for callipyge lambs. In the SM and SS, WBS values decreased (P < 0.05) across the growth curve, but values were higher (P < 0.05) for callipyge lambs in the SM only. These data indicate that the selective muscular hypertrophy of the callipyge phenotype develops during the postnatal growth period between 7 and 20 kg LW (19 and 100 d of age). Longissimus and semimembranosus muscles in the callipyge lambs were over 40% heavier from 20 to 69 kg LW; however, they also had higher levels of calpastatin activity and Warner-Bratzler shear force during this time period, indicating the need for postmortem tenderization treatments to improve palatability.

Evaluation of melatonin receptor 1a as a candidate gene influencing reproduction in an autumn-lambing sheep flock.

The effect of the melatonin receptor 1a (MTNR1A) gene on fertility and litter size in autumn lambing was studied with 373 ewes from a population of sheep selected for 10 yr for fertility in May and June matings. Animals were from a composite line of 50% Dorset, 25% Rambouillet, and 25% Finnsheep. Two restriction fragment length polymorphisms were present in the population, with allelic frequencies of 0.42 and 0.58 for an MnII polymorphism (alleles M and m, respectively) and of 0.34 and 0.66 for an RsaI polymorphism (alleles R and r, respectively). Genotypic frequencies for the polymorphisms were not independent, suggesting an association between them in foundation animals. Effects of MTNR1A genotype on fertility and litter size were evaluated using mixed linear model or REML procedures, but were not significant for matings involving ewes of all ages. However, in adult ewes (3 yr old and older), fertility of ewes of genotype mm was 10.0 (mixed model; P < 0.09) to 11.2% (REML; P = 0.03) less than that of other ewes. Genotypic effects associated with MTNR1A in adult ewes accounted for 23.8% of the estimated total additive genetic variance in fertility. Litter size was not significantly associated with MTNR1A genotype; adult ewes of genotype mm had approximately 0.11 fewer lambs per ewe lambing than ewes of other genotypes. Fertility in autumn lambing is lowly heritable, expressed only in females, and manifested relatively late in life and only in some management systems. Access to genetic markers would thus be advantageous in selection programs.

Prediction of live lamb chemical composition utilizing electromagnetic scanning (ToBEC).

Electromagnetic scanning was investigated to determine its accuracy in predicting chemical composition in live lambs. Forty-seven Rambouillet wether lambs were scanned with an electromagnetic instrument (ToBEC Model HA-2). Lambs were serially scanned and slaughtered over the weight range of 29.5 to 63.5 kg. Each lamb was scanned twice: before and immediately after 24 h of food deprivation. Chemical composition was determined from whole-animal ground samples by AOAC methods for percentage of DM, CP, ether extract (EE), and Ash. Percentage of fat-free mass (FFM) was calculated from the percentage of moisture and CP. Correlation and stepwise regression procedures were used to identify the most reliable independent variables for predicting chemical composition. Independent variables included electromagnetic scan data and live animal measures for weight, body length, and chest girth circumference. Electromagnetic data included the average scan response curve (PH0) and Fourier transformations (P1T, P1R, P2T, and P2R). Repeatability of the HA-2 model was extremely high (r = .98). Reliable prediction equations were obtained for DM, CP, EE, and FFM (R2 > .66). The percentage of ash could not be predicted from the independent variables. Electromagnetic scan responses contributed little to the model sum of squares. Body weight accounted for the majority of the model sum of squares. Depriving lambs of food for 24 h slightly improved the R2 value and significantly decreased scan responses (P < .01). Body weight was a better predictor of chemical composition over a large weight range than any of the scan responses. Further investigation of the HA-2 is needed to determine whether it is effective in determining differences in live body composition between animals of equal weight.

Histology and composition of muscles from normal and callipyge lambs.

The histology and composition of muscles from normal (n = 10) and callipyge (n = 11) wether lambs was compared. Normal Rambouillet ewes were mated with callipyge Dorset rams, and their progeny were visually classified as callipyge or normal based on muscle definition in the loin and hind quarters. The muscles examined included three muscles that hypertrophy in callipyge lambs (semitendinosus, longissimus, and gluteus medius) and one muscle believed not to hypertrophy (supraspinatus). The hypertrophy-responsive muscles from callipyge lambs had a higher (P < .001) percentage of fast-twitch glycolytic (FG) fibers and lower (P < .001; P < .02 for SO in gluteus medius) percentages of slow-twitch oxidative (SO) and fast-twitch oxidative glycolytic (FOG) fibers. The diameters of the FG and FOG fibers were larger (P < .005 and P < .04, respectively) in hypertrophy-responsive muscles from callipyge lambs, but the SO fiber diameter was smaller (P < .05). Also, the protein:DNA ratio, an indicator of cell size, was greater (semitendinosus, P < .05); longissimus, P < .002; gluteus medius, P < .008) in the hypertrophy-responsive muscles from callipyge lambs. Thus, hypertrophy in callipyge lambs was, at least in part, due to fiber type changes and muscle cell enlargement. Hypertrophy was strongly associated with changes in the FG fibers, the only fiber type that increased in both proportion and average diameter in callipyge muscles. The protein:RNA ratio and RNA:DNA ratio, which are indicators of translational and transcriptional activity in the muscle cells, were not different between callipyge and normal muscles. This indicated that the accumulation of protein necessary for myofiber enlargement occurred without differences in the translational or transcriptional activity of callipyge muscle.