TRIENNIAL GROWTH AND DEVELOPMENT SYMPOSIUM: Factors influencing bovine intramuscular adipose tissue development and cellularity.

Appearance, distribution, and amount of intramuscular fat (IMF), often referred to as marbling, are highly variable and depend on environmental and genetic factors. On the molecular level, the concerted action of several drivers, including hormones, receptors, transcription factors, etc., determines where clusters of adipocytes arise. Therefore, the aim of future studies remains to identify such factors as biological markers of IMF to increase the ability to identify animals that deposit IMF early in age to increase efficiency of high-quality meat production. In an attempt to unravel the cellular development of marbling, we investigated the abundance of markers for adipogenic differentiation during fattening of cattle and the transcriptome of muscle and dissected IMF. Markers of different stages of adipogenic differentiation are well known from cell culture experiments. They are usually transiently expressed, such as delta-like homolog 1 (DLK1) that is abundant in preadipocytes and absent during differentiation to mature adipocytes. It is even a greater challenge to detect those markers in live animals. Within skeletal muscles, hyperplasia and hypertrophy of adipocytes can be observed throughout life. Therefore, development of marbling requires, on the cellular level, recruitment, proliferation, and differentiation of adipogenic cells to store excess energy in the form of lipids in new cells. In a recent study, we investigated the localization and abundance of early markers of adipogenic differentiation, such as DLK1, in bovine muscle tissue. An inverse relationship between IMF content and number of DLK1-positive cells in bovine muscle was demonstrated. Considering the cellular environment of differentiating adipocytes in muscle and the secretory action of adipocytes and myocytes, it becomes obvious that cross talk between cells via adipokines and myokines may be important for IMF development. Secreted proteins can act on other cells, inhibiting or stimulating their function via autocrine and paracrine actions. Such factors with potential influence on IMF, among them, agouti signaling protein and thrombospondin 4, were identified in transcriptome analyses and further investigated. Furthermore, results from transcriptome analysis indicate involvement of genes that are not directly related to adipogenesis and lipid metabolism, providing new candidates for future research.

Prenatal muscle fiber development and bundle structure in beef and dairy cattle.

Muscle fiber development during gestation determines the muscle structure at birth and establishes the conditions for muscle development in growing cattle. Differences in muscle structure among beef cattle breeds and between beef and dairy cattle are obvious already shortly after birth. The objective of the study was to investigate the development of muscle fibers and muscle fiber bundle structure in semitendinosus muscle of divergent cattle breeds from 3 mo of gestation until birth. Fetuses of German Angus (GA), Galloway (GW), Belgian Blue (BB), and Holstein Friesian (HF) were harvested at 3, 4.5, 6, or 9 mo of gestation. Muscle sections were analyzed for fiber size and types as well as for bundle structure. The results confirmed that primary muscle fiber development occurs mainly during the first trimester of gestation. All fibers were initially positive for fetal fast myosin. Slow myosin as a marker for fiber maturation was detected in primary fibers at 3 mo of gestation showing a weak immunostaining. During the second trimester, the intensity of immunostaining strongly increased indicating increased slow myosin protein expression. Concurrently, the shape of primary fibers changed from myotubes to myofibers whereas the size stayed nearly constant. The main increase in muscle mass during the second trimester was caused by secondary fiber development. As an example, the ratio between secondary and primary fibers increased in Holstein Friesian fetuses from 5.9 at 4.5 mo of gestation to 21.6 at 6 mo of gestation. Primary and secondary fibers continued to growth during the third trimester. Regional differences in the density of slow muscle fibers were detected leading to greater variation within the muscle than among breeds. Structural organization of muscle fibers in muscle fiber bundles developed early in fetal life. At first, large main bundles were visible. Smaller structural units defined as primary bundles were measurable at 6 mo of gestation when most fibers were developed. The size of primary bundles nearly doubled from 6 mo of gestation to birth in all breeds. In summary, differences among breeds in the early fetal muscle fiber development were detected in contractile differentiation and partly in muscle fiber bundle structure. A prolonged secondary fiber generation and altered contractile differentiation may be involved in breed differences of postnatal muscle development.

Technical note: Determination of cell-specific gene expression in bovine skeletal muscle tissue using laser microdissection and reverse-transcription quantitative polymerase chain reaction.

Skeletal muscle is a very heterogeneous tissue consisting of diverse cell types with specific transcription profiles. Therefore, the measured mRNA abundance of a certain cell type marker is influenced by the transcriptional activity as well as by the usually unknown number of contributing cells in the sample. In studies on the transcriptional activity of adipogenic genes, as indicators for the development of intramuscular adipocytes, an altered number of adipocytes or respective progenitor cells can mask changes in transcriptional activity. To overcome this problem, we started to use laser microdissection to isolate RNA of adipocytes and muscle fibers separately for downstream analysis. Even muscle fiber types can be collected and analyzed separately. Laser microdissection in combination with biopsy techniques enables gene expression studies of particular cell types during the life cycle of an animal. First experiences using laser microdissection for adipogenic gene expression studies in bovine skeletal muscle are described, and the influence of sample preparation and future challenges are discussed.

Cellular conditions for intramuscular fat deposition in Japanese Black and Holstein steers.

The experiment was conducted to study the development of intramuscular fat in Japanese Black (JB) compared to Holstein (HS) steers and to find breed differences for fat depot development and distribution in the carcass under equal feeding conditions. Additional to slaughter samples, biopsy samples of longissimus muscle (LM) and subcutaneous fat, taken at 10, 14, 18, and 22 months of age, were used for histological and molecular investigations. Japanese Black steers stored about 14% more fat in the LM (P = 0.001), resulting in larger marbling flecks (P < 0.001). Muscle fibers and intramuscular adipocytes in both breeds responded to the high energy feeding with significant enlargement, which was faster in JB. Histograms of intramuscular adipocytes size showed a shift toward larger cells during growth, but also the abundance of small, developing adipocytes. This development was accompanied by a correlated up-regulation of adipogenic genes until 22 months of age.

Expression of the porcine adrenergic receptor beta 2 gene in longissimus dorsi muscle is affected by cis-regulatory DNA variation.

The beta-2 adrenergic receptor (AR) mediates metabolic actions of catecholamines, including glycogenolysis, lipolysis and proteolysis, in muscle and adipose tissue. Factors influencing the density of beta-2 ARs thus might affect carcass composition and meat quality. One such factor might represent cis-regulatory DNA variation affecting mRNA expression of the adrenergic receptor beta 2 (ADRB2) gene in relevant tissues. To identify potential cis-regulatory DNA variation of porcine ADRB2, we comparatively sequenced part of the 5' flanking region and identified 10 single nucleotide polymorphisms (SNPs). The SNP at position g.673C>T (AF000134) resides in an evolutionarily conserved region (ECR) in an in silico predicted androgen response element. Quantification of total transcript levels and allelic expression imbalance (AEI) revealed significant variability in mRNA expression of ADRB2 in longissimus dorsi muscle of slaughter pigs, partly attributable to cis-regulatory DNA variation. However, the g.673C>T SNP has, in the given temporo-spatial context, no significant effect but is apparently in linkage disequilibrium with the causal cis-regulatory DNA variant. We used the g.673C>T SNP as a marker to study the association of ADRB2 variation with carcass and meat quality in four commercial lines. We found association with the pH of loin at 45 min and 24 h postmortem (p.m.) and with the pH of ham at 24 h p.m. Supporting evidence for ADRB2 as a candidate gene for pork quality is provided by our assignment of the gene to the telomeric end of the q arm of porcine chromosome 2, where several quantitative trait loci for meat quality were reported.

Evaluation of MAFbx expression as a marker for congenital splay leg in piglets.

Splay leg is a hereditary syndrome observed in highly varying frequency in newborn piglets. Although the phenotype indicates a muscular weakness, the etiology is still poorly understood. Only recently, the gene expression of muscle atrophy F-box (MAFbx; FBXO32) was proposed as being of diagnostic value for splay leg in piglet. In this study, total RNA from three healthy and three affected male piglets was isolated. Samples were collected from M. gracilis, Mm. adductores, and M. sartorius. Further samples were taken for histological and biochemical analyses. Expression of MAFbx was analysed by real-time RT-PCR and with the GeneChip" Porcine Genome Array (Affymetrix). No significant differences (p>0.05) were observed in relative MAFbx expression, either between the three muscles or between splay leg and healthy piglets for each muscle. The expression of further atrophy-related genes was unchanged, indicating that splay leg is not characterized by general muscular atrophy in the affected hind limbs. This is supported by histological and biochemical data that does not demonstrate signs of atrophy in splay leg muscles. We conclude that the diagnostic value of MAFbx expression for congenital splay leg in piglets is doubtful and that the disease is characterized by heterogeneous alterations in skeletal muscle.

Simulation of giant fibre development in biopsy samples from pig longissimus muscle.

The incidence of hyper-contracted giant fibres in pig postmortem skeletal muscle is closely related to poor meat quality in terms of pale, soft, and exudative pork. Detection of a predisposition to develop giant fibres in live pigs could help to predict pork quality and to exclude affected pigs from genetic selection. The abundance and proportion of giant fibres in longissimus muscle were highest in Piétrain followed by Landrace, Large White, and Leicoma pigs of market weight. The postmortem development of giant fibres could be successfully simulated in vitro incubating biopsy samples from longissimus muscle at 37°C for 60min. For repeated measurements on three samples the intraclass correlation coefficient for the number of giant fibres/cm(2) was ϑˆ(3)=0.69 for biopsy and ϑˆ(3)=0.87 for carcass samples. "Simulated" giant fibres exhibited ultrastructural changes in plasma membrane, myofibrils, mitochondria, and sarcoplasmatic reticulum as shown previously for giant fibres in carcass samples.

Exclusion of NFYB as candidate gene for congenital splay leg in piglets and radiation hybrid mapping of further five homologous porcine genes from human chromosome 12 (HSA12).

Nuclear transcription factor Y, beta (NFYB) was evaluated as candidate gene for congenital splay leg in piglets based on data from differential display and QTL analysis. We mapped NFYB to pig chromosome 5 (SSC5). By assigning further five porcine genes from the corresponding region on human chromosome (HSA) 12q23.3--> q24.11 to SSC5 and 14 we could confine an evolutionary breakpoint from an interval of more than 10 Mb to less than 400 kb. Comparative sequence analysis of the coding region of NFYB in healthy and splay leg piglets revealed no polymorphism. Inter-species conservation of the codons ranges from 87% to 95% between pig, human, cow, dog, rat and mouse, respectively. The expression of NFYB in M. biceps femoris was not different between healthy and splay leg piglets. However, healthy male piglets had a significantly higher expression than females. Our results exclude NFYB as candidate gene for congenital splay leg but provide a basis for selection of further candidates for the disease from SSC5.

Identification and analysis of putative regulatory sequences for the MYF5/MYF6 locus in different vertebrate species.

The myogenic factors (MYF) 5 and 6 are integral to the initiation and development of skeletal muscle and to the maintenance of its phenotype. Thus, they are candidate genes for growth- and meat quality-related traits. We performed a comparative sequence analysis of the MYF5/MYF6 locus in swine, cattle, dog, chicken and zebrafish on the basis of structural and functional information from human and mouse. Beside the characterization of upstream regulatory elements recently identified in mice, we demonstrate the existence of further highly conserved elements (E1 to E4) which may play a role in the regulation of MYF5 and MYF6 expression. Comparative sequence analysis of putative regulatory sequences in swine revealed a total of 21 single nucleotide polymorphisms (SNP) including 1 and 6 SNPs new for the promoters of MYF5 and MYF6, respectively. The conserved organization of the locus in vertebrates indicates a common basic mechanism of muscle development. However, the existence of numerous regulatory elements at large distances to MYF5 and MYF6 points to a very complex pattern of the gene regulation with significant differences between species.

Association of a melanocortin 4 receptor (MC4R) polymorphism with performance traits in Lithuanian White pigs.

The melanocortin 4 receptor is expressed in virtually all brain regions of mammals and plays an important role in energy homeostasis. Polymorphisms in this gene may thus be related to growth and obesity. In pigs, a non-synonymous polymorphic site was described (Asp298Asn) and demonstrated to affect cAMP production and to alter adenylyl cyclase signalling. Association studies revealed significant linkage of this mutation with production trait in pigs. In this study, 207 Lithuanian White pigs were genotyped at the MC4R locus and analysed on relationships between genotype and breeding values for several performance traits. The observed allele and genotype frequencies did not deviate significantly from Hardy-Weinberg equilibrium (wildtype allele 0.59; mutant allele 0.41) and are comparable with those described in other Large White populations. The mutant Asn298 allele of the MC4R gene was significantly associated with increased test daily gain, higher lean meat percentage and lower backfat thickness. There was a trend towards an improved feed conversion ratio (p = 0.065) in animals with the mutant allele whereas no significant effect was found on lifetime daily gain. These results indicate that the MC4R polymorphism should be integrated in selection programmes in the Lithuanian White to improve carcass composition.

Genetic spatial structure of European common hamsters (Cricetus cricetus)--a result of repeated range expansion and demographic bottlenecks.

The spatial genetic structure of common hamsters (Cricetus cricetus) was investigated using three partial mitochondrial (mt) genes and 11 nuclear microsatellite loci. All marker systems revealed significant population differentiation across Europe. Hamsters in central and western Europe belong largely to two allopatric mitochondrial lineages south and northwest of the Carpathian and Sudetes. The southern group, 'Pannonia', comprises populations inside the Carpathian basin (Czech Republic, Hungary) while the second group, 'North', includes hamsters from Belgium, the Netherlands, France, and Germany. Isolation of the lineages is maintained by a combination of geographical and ecological barriers. Both main phylogeographical groups show signs of further subdivision. North is separated into highly polymorphic central German and less polymorphic western populations, which most likely split during late glacial expansion (15,000-10,000 bp). Clock estimates based on haplotype distributions predict a divergence of the two major lineages 85,000-147,000 bp. Expansion times fall during the last glaciation (115,000-10,000 bp) corroborating fossil data, which identify Cricetus cricetus as characteristic of colder climatic phases. Despite the allopatry of mt haplotypes, there is an overlap of nuclear microsatellite alleles between phylogeographical units. Although there are strong evidence that Pannonian hamsters have persisted inside the Carpathian basin over the last 50,000 years, genetic differentiation among European hamsters has mainly been caused by immigration from different eastern refugia. Possible source populations are likely to be found in the Ukrainian and the southern Russian plains--core areas of hamster distribution. From there, hamsters have repeatedly expanded during the Quaternary.

Effect of long-term heat exposure on peripheral concentrations of heat shock protein 70 (Hsp70) and hormones in laying hens with different genotypes.

1. The effect of long-term, moderate heat stress (30 to 32 degrees C) on heat shock protein 70 (Hsp70) concentration in mononuclear blood cells and plasma concentrations of 3,5,3'-triiodothyronine (T3) and corticosterone in laying hens was investigated. 2. Three groups of 48 hens each (Ethopian line [Angete Melata, Na], New Hampshire [NH], F1 cross [Na x NH]) were divided into an experimental group (24 each) and a control group (24 each, ambient temperature 18 to 20 degrees C), respectively. All hens were kept in individual cages up to an age of 68 weeks and performance data were recorded. 3. Blood samples were taken from the wing vein of 12 hens from each group at weeks 22, 38, 51 and 65 (12 hens x 3 lines x 2 treatments). Mononuclear blood cells were isolated and Hsp70 concentrations were determined by Western Blot analysis with a monoclonal anti-Hsp70 antibody. T3 and corticosterone were measured with commercially available ELISA and RIA kits, respectively. 4. The moderate heat stress caused significantly increased Hsp70 levels compared with the control groups in weeks 51 and 65. However, the responses of the lines were not uniform at different ages. 5. In contrast, T3 levels were significantly decreased in stressed birds regardless of line and age. There was no effect of treatment and line on corticosterone levels during the experimental period. 6. Our results indicate that Hsp70 and T3 levels are affected by mild heat stress applied over a long period but are both involved in independent mechanisms of acquisition of thermotolerance. Further investigations are necessary to clarify whether the observed differences in Hsp70 response between the genotypes are indicators for differences in thermotolerance.

Functional promoter variants of highly conserved inducible hsp70 genes significantly affect stress response.

Promoter variants differing in potential cis-acting elements and showing impaired binding of corresponding transcription factors are described in the inducible porcine hsp70.2 gene (GC box; initiator-like region, InR) and in its bovine homologue (AP2 site), respectively. Interestingly, all these promoter variants are stable established in the analysed populations in spite of significant negative effects on transcriptional activity and indications of not neutral behaviour in response to breeding selection. Moreover, in pigs they are likely to be evolutionary fixed as suggested by their presence in recent breeds and in wild boars as well. Compensation of the effect of the observed 'down'-mutations by further yet unknown gene variants is assumed.

Isolation of expressed sequence tags of skeletal muscle of neonatal healthy and splay leg piglets and mapping by somatic cell hybrid analysis.

We have isolated 14 differentially displayed and 10 further expressed sequence tags (ESTs) from Musculus biceps femoris of newborn healthy and splay leg piglets. By comparison with EMBL/GenBank data we could identify nine porcine homologues to human genes (TATA box binding protein associated factor B TAF1B; B-cell CLL/lymphoma 7B BCL7B; pyruvate dehydrogenase kinase, isoenzyme 4 PDK4; ribosomal protein S10 RPS10; SPARC-like 1 SPARCL1; epithelial protein lost in neoplasm beta EPLIN; N-myc downstream-regulated gene 2 NDRG2; pleiomorphic adenoma gene like 2 PLAGL and, BCL-2 associated transcription factor short form BTFS). Eight fragments correspond to uncharacterized ESTs and 7 ESTs had no significant match with database sequences. These data provide the first expression profiles in skeletal muscle of neonatal piglets and are a basis for candidate gene investigations for congenital splay leg in piglets. Eleven ESTs were physically mapped to porcine chromosomes 1, 3, 4, 5, 7, 8, 9 and 10 and contribute to the comparative map of humans and pigs.

Low microsatellite variation in laboratory gerbrils.

The Mongolian gerbil has become a model organism of increasing importance for the understanding of aging, epilepsy, the process of domestication or sociobiological questions. We report the development and characterization of the first nine polymorphic dinucleotide repeat loci in this species. Average observed heterozygosity and allele number of laboratory animals measured 0.136 (SE = +/-0.065) and 1.78 (SE = +/-0.278) compared to 0.761 (SE = +/-0.025) and 9.2 (SE = +/-0.57) found for a reference group of wild gerbils. The extreme low genetic variation observed in laboratory animals is caused by several severe population size bottlenecks due to the initial founder event and the later establishment of subpopulations. Reduced levels of allelic polymorphism in experimental animals hamper genetic mapping or parental studies. Therefore experiments relying on kinship analyses have to be carried out on wild animals. Estimates of genetic identity and parental exclusion were calculated as Pid = 2.8 x 10(-12) and Pex > 0.999 in wild gerbils. Laboratory gerbil strains show the expected high degree of genetic similarity. However, significant allele frequency differences (P < .001) between American and European gerbils at some microsatellite loci may still allow discrimination between breeding lines.

Structural and functional characteristics of muscle fibres in pigs with different malignant hyperthermia susceptibility (MHS) and different meat quality.

In pigs, intensive growth of the musculature is often accompanied by malignant hyperthermia susceptibility (MHS; n gene) and poorer meat quality. Using histological and histochemical methods, different fibre characteristics in the Longissimus muscle were found in Pietrain×German Landrace pigs with this gene defect. Compared to MHS homozygous negative pigs, groups with the n gene had increased diameters of the mean fibre types and increased glycolytic metabolic potential, as shown by a higher frequency of the fast twitch glycolytic type and a lower frequency of the slow twitch oxidative fibre type. Differences between the groups were also found in the number of angular and giant fibre types. Furthermore, a positive correlation was found between the frequency of oxidative fibres and the relative enzyme activity of NADH tetrazolium reductase. The changes correlated with lower pH and higher drip loss in meat from the MHS homozygous positive group. In conclusion, the different muscle fibre characteristics can be interpreted as endogenous factors which influence the physiological condition in the muscle of the live animal and meat quality post mortem.