Transcriptomic Response of Differentiating Porcine Myotubes to Thermal Stress and Donor Piglet Age.

Climate change is a current concern that directly and indirectly affects agriculture, especially the livestock sector. Neonatal piglets have a limited thermoregulatory capacity and are particularly stressed by ambient temperatures outside their optimal physiological range, which has a major impact on their survival rate. In this study, we focused on the effects of thermal stress (35 °C, 39 °C, and 41 °C compared to 37 °C) on differentiating myotubes derived from the satellite cells of Musculus rhomboideus, isolated from two different developmental stages of thermolabile 5-day-old (p5) and thermostable 20-day-old piglets (p20). Analysis revealed statistically significant differential expression genes (DEGs) between the different cultivation temperatures, with a higher number of genes responding to cold treatment. These DEGs were involved in the macromolecule degradation and actin kinase cytoskeleton categories and were observed at lower temperatures (35 °C), whereas at higher temperatures (39 °C and 41 °C), the protein transport system, endoplasmic reticulum system, and ATP activity were more pronounced. Gene expression profiling of HSP and RBM gene families, which are commonly associated with cold and heat responses, exhibited a pattern dependent on temperature variability. Moreover, thermal stress exhibited an inhibitory effect on cell cycle, with a more pronounced downregulation during cold stress driven by ADGR genes. Additionally, our analysis revealed DEGs from donors with an undeveloped thermoregulation capacity (p5) and those with a fully developed thermoregulation capacity (p20) under various cultivation temperature. The highest number of DEGs and significant GO terms was observed under temperatures of 35 °C and 37 °C. In particular, under 35 °C, the DEGs were enriched in insulin, thyroid hormone, and calcium signaling pathways. This result suggests that the different thermoregulatory capacities of the donor piglets determined the ability of the primary muscle cell culture to differentiate into myotubes at different temperatures. This work sheds new light on the underlying molecular mechanisms that govern piglet differentiating myotube response to thermal stress and can be leveraged to develop effective thermal management strategies to enhance skeletal muscle growth.

Effect of muscle fibre types and carnosine levels on the expression of carnosine-related genes in pig skeletal muscle.

It is generally accepted that carnosine (ß-alanyl-L-histidine) content is higher in glycolytic than in oxidative muscle fibres, but the underlying mechanisms responsible for this difference remain to be elucidated. A first study to better understand potential mechanisms involved was undertaken (1) to determine whether differences in the expression of carnosine-related enzymes (CARNS1, CNDP2) and transporters (SLC6A6, SLC15A3, SLC15A4, SLC36A1) exist between oxidative and glycolytic myofibres and (2) to study the effect of carnosine on myoblast proliferative growth and on carnosine-related gene expression in cultured myoblasts isolated from glycolytic and oxidative muscles. Immunohistochemistry analyses were conducted to determine the cellular localization of carnosine-related proteins. Laser-capture microdissection and qPCR analyses were performed to measure the expression of carnosine-related genes in different myofibres isolated from the longissimus dorsi muscle of ten crossbred pigs. Myogenic cells originating from glycolytic and oxidative muscles were cultured to assess the effect of carnosine (0, 10, 25 and 50 mM) on their proliferative growth and on carnosine-related gene expression. The mRNA abundance of CNDP2 and of the studied carnosine transporters was higher in oxidative than in glycolytic myofibres. Since carnosine synthase (CARNS1) mRNA abundance was not affected by either the fibre type or the addition of carnosine to myoblasts, its transcriptional regulation would not be the main process by which carnosine content differences are determined in oxidative and glycolytic muscles. The addition of carnosine to myoblasts leading to a dose-dependent increase in SLC15A3 transcripts, however, suggests a role for this transporter in carnosine uptake and/or efflux to maintain cellular homeostasis.

Endocannabinoid administration affects taste preference and the expression of cannabinoid and opioid receptors in the amygdala of early lactating cows.

The aim of the study was to investigate the influence of intraperitoneal N-arachidonoylethanolamide (AEA) on taste preference for feed and water, tongue taste receptor signalling (TAS1R2, GNAT3), and endocannabinoid (CNR1, CNR2, GPR55) and opioid (OPRD1, OPRK1, OPRM1, OPRL1) receptors in the amygdala and nucleus accumbens in periparturient cows. We conducted taste preference tests using unaltered, umami-tasting, and sweet-tasting water and feed, before and after calving. After calving, eight cows received AEA injections (3 µg/(kg bodyweight × day), 25 days), whereas eight control (CON) cows received saline injections. Tissue was sampled 30 days after calving. Before calving, both cow groups preferred sweet-tasting feed and umami-tasting water. After calving, only the AEA-treated group preferred sweet-tasting feed, whereas the CON group showed no clear taste preference. In the amygdala, the mRNA expression of CNR1, OPRD1 (left hemisphere) and OPRK1 (right hemisphere) was lower in AEA animals than in CON animals, whereas no differences were found in the nucleus accumbens and tongue taste receptor expression. In conclusion, AEA administration enhanced existing taste preferences and reduced the expression of specific endocannabinoid and opioid receptors in the amygdala. The results support endocannabinoid-opioid interactions in the control of taste-dependent feed preference in early lactating cows.

The effects of temperature and donor piglet age on the transcriptomic profile and energy metabolism of myoblasts.

Rapid climate change is associated with frequent extreme heat events and the resulting thermal stress has consequences for the health, welfare, and growth of farm animals. The aim of this study was to characterize the transcriptional changes and the effects on energy metabolism in proliferating porcine myoblasts derived from piglets of different ages, representing differences in thermoregulatory abilities, and cultivated below (35°C) and above (39°C, 41°C) the standard cultivation temperature (37°C). Satellite cells originating from Musculus rhomboideus of piglets isolated on days 5 (P5, thermolabile) and 20 (P20, thermostable) of age were used. Our expression analyses highlighted differentially expressed genes in porcine myoblasts cultures under heat or cold induced stress. These gene sets showed enrichment for biological processes and pathways related to organelle fission, cell cycle, chromosome organization, and DNA replication. Culture at 35°C resulted in increased metabolic flux as well as a greater abundance of transcripts of the cold shock protein-encoding gene RBM3 and those of genes related to biological processes and signaling pathways, especially those involving the immune system (cytokine-cytokine receptor interaction, TNF and IL-17 signaling pathways). For cultivation at 39°C, differences in the expression of genes related to DNA replication and cell growth were identified. The highest glutathione index ratio was also found under 39°C. Meanwhile, cultivation at 41°C induced a heat stress response, including the upregulation of HSP70 expression and the downregulation of many biological processes and signaling pathways related to proliferative ability. Our analysis also identified differentially expressed genes between cells of donors with a not yet (P5) and already fully developed (P20) capacity for thermoregulation at different cultivation temperatures. When comparing P5 and P20, most of the changes in gene expression were detected at 37°C. At this optimal temperature, muscle cells can develop to their full capacity. Therefore, the most diverse molecular signaling pathways, including PI3K-Akt signaling, Wnt signaling, and EGFR tyrosine kinase inhibitor, were found and are more pronounced in muscle cells from 20-day-old piglets. These results contribute to a better understanding of the mechanisms underlying the adaptation of skeletal muscle cells to temperature stress in terms of their thermoregulatory ability.

Are n-3 PUFAs from Microalgae Incorporated into Membrane and Storage Lipids in Pig Muscle Tissues?-A Lipidomic Approach.

For the study of molecular mechanisms of to lipid transport and storage in relation to dietary effects, lipidomics has been rarely used in farm animal research. A feeding study with pigs (German Landrace sows) and supplementation of microalgae (Schizochytrium sp.) was conducted. The animals were allocated to the control group (n = 15) and the microalgae group (n = 16). Shotgun lipidomics was applied. This study enabled us to identify and quantify 336 lipid species from 15 different lipid classes in pig skeletal muscle tissues. The distribution of the lipid classes was significantly altered by microalgae supplementation, and ether lipids of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidic acid (PA) were significantly decreased. The total concentration of triacylglycerides (TAGs) was not affected. TAGs with high degree of unsaturation (TAG 56:7, TAG 56:6, TAG 54:6) were increased in the microalgae group, and major abundant species like TAG 52:2 and TAG 52:1 were not affected by the diet. Our results confirmed that dietary DHA and EPA are incorporated into storage and membrane lipids of pig muscles, which further led to systemic changes in the lipidome composition.

Replacing Needle Injection by a Novel Waterjet Technology Grants Improved Muscle Cell Delivery in Target Tissues.

Current regimen to treat patients suffering from stress urinary incontinence often seems not to yield satisfactory improvement or may come with severe side effects. To overcome these hurdles, preclinical studies and clinical feasibility studies explored the potential of cell therapies successfully and raised high hopes for better outcome. However, other studies were rather disappointing. We therefore developed a novel cell injection technology to deliver viable cells in the urethral sphincter complex by waterjet instead of using injection needles. We hypothesized that the risk of tissue injury and loss of cells could be reduced by a needle-free injection technology. Muscle-derived cells were obtained from young male piglets and characterized. Upon expansion and fluorescent labeling, cells were injected into cadaveric tissue samples by either waterjet or injection needle. In other experiments, labeled cells were injected by waterjet in the urethra of living pigs and incubated for up to 7 days of follow-up. The analyses documented that the cells injected by waterjet in vitro were viable and proliferated well. Upon injection in live animals, cells appeared undamaged, showed defined cellular somata with distinct nuclei, and contained intact chromosomal DNA. Most importantly, by in vivo waterjet injections, a significantly wider cell distribution was observed when compared with needle injections (P < .05, n ≥ 12 samples). The success rates of waterjet cell application in living animals were significantly higher (≥95%, n = 24) when compared with needle injections, and the injection depth of cells in the urethra could be adapted to the need by adjusting waterjet pressures. We conclude that the novel waterjet technology injects viable muscle cells in tissues at distinct and predetermined depth depending on the injection pressure employed. After waterjet injection, loss of cells by full penetration or injury of the tissue targeted was reduced significantly in comparison with our previous studies employing needle injections.

Effects of temperature on proliferation of myoblasts from donor piglets with different thermoregulatory maturities.

BACKGROUND: Climate change and the associated risk for the occurrence of extreme temperature events or permanent changes in ambient temperature are important in the husbandry of farm animals. The aim of our study was to investigate the effects of permanent cultivation temperatures below (35 °C) and above (39 °C, 41 °C) the standard cultivation temperature (37 °C) on porcine muscle development. Therefore, we used our porcine primary muscle cell culture derived from satellite cells as an in vitro model. Neonatal piglets have limited thermoregulatory stability, and several days after birth are required to maintain their body temperature. To consider this developmental step, we used myoblasts originating from thermolabile (five days of age) and thermostable piglets (twenty days of age). RESULTS: The efficiency of myoblast proliferation using real-time monitoring via electrical impedance was comparable at all temperatures with no difference in the cell index, slope or doubling time. Both temperatures of 37 °C and 39 °C led to similar biochemical growth properties and cell viability. Only differences in the mRNA expression of myogenesis-associated genes were found at 39 °C compared to 37 °C with less MYF5, MYOD and MSTN and more MYH3 mRNA. Myoblasts grown at 35 °C are smaller, exhibit higher DNA synthesis and express higher amounts of the satellite cell marker PAX7, muscle growth inhibitor MSTN and metabolic coactivator PPARGC1A. Only permanent cultivation at 41 °C resulted in higher HSP expression at the mRNA and protein levels. Interactions between the temperature and donor age showed that MYOD, MYOG, MYH3 and SMPX mRNAs were temperature-dependently expressed in myoblasts of thermolabile but not thermostable piglets. CONCLUSIONS: We conclude that 37 °C to 39 °C is the best physiological temperature range for adequate porcine myoblast development. Corresponding to the body temperatures of piglets, it is therefore possible to culture primary muscle cells at 39 °C. Only the highest temperature of 41 °C acts as a thermal stressor for myoblasts with increased HSP expression, but it also accelerates myogenic development. Cultivation at 35 °C, however, leads to less differentiated myoblasts with distinct thermogenetic activity. The adaptive behavior of derived primary muscle cells to different cultivation temperatures seems to be determined by the thermoregulatory stability of the donor piglets.

Characterisation of intracellular molecular mechanisms modulated by carnosine in porcine myoblasts under basal and oxidative stress conditions.

Carnosine is a naturally occurring histidine-containing dipeptide present at high concentration in mammalian skeletal muscles. Carnosine was shown to affect muscle contraction, prevent the accumulation of oxidative metabolism by-products and act as an intracellular proton buffer maintaining the muscle acid-base balance. The present study was undertaken to gain additional knowledge about the intracellular mechanisms activated by carnosine in porcine myoblast cells under basal and oxidative stress conditions. Satellite cells were isolated from the skeletal muscles of 3 to 4 day-old piglets to study the effect of 0, 10, 25 and 50 mM carnosine pre-treatments in cells that were exposed (0.3 mM H2O2) or not to an H2O2-induced oxidative stress. Study results demonstrated that carnosine acts differently in myoblasts under oxidative stress and in basal conditions, the only exception being with the reduction of reactive oxygen species and protein carbonyls observed in both experimental conditions with carnosine pre-treatment. In oxidative stress conditions, carnosine pre-treatment increased the mRNA abundance of the nuclear factor, erythroid 2 like 2 (NEF2L2) transcription factor and several of its downstream genes known to reduce H2O2. Carnosine prevented the H2O2-mediated activation of p38 MAPK in oxidative stress conditions, whereas it triggered the activation of mTOR under basal conditions. Current results support the protective effect of carnosine against oxidative damage in porcine myoblast cells, an effect that would be mediated through the p38 MAPK intracellular signaling pathway. The activation of the mTOR signaling pathway under basal condition also suggest a role for carnosine in myoblasts proliferation, growth and survival.

Establishment and validation of cell pools using primary muscle cells derived from satellite cells of pig skeletal muscle.

Primary cell cultures derived from satellite cells of skeletal muscle provide an appropriate in vitro model for proliferating myoblasts and differentiating myotubes for muscle biological research. These cell cultures may consist of harvested cells per animal or of a cell pool made of cells from several animals. However, cell pooling reduces the biological variability of the different cell donors. On the other hand, the use of cell pools offers an opportunity to use less donor tissue and to perform long-term projects with a broad spectrum of analysis and replications. In the literature, information about the donors of cell pools, the procedure used for pooling, and the characterization/validation of cell pools is often lacking. In this study, we established three cell pools consisting of M. rhomboideus or M. longissimus from ten or six piglets, each with one gender and medium birth weight. Real-time impedimetric monitoring was used to evaluate the proliferative growth behavior of myoblasts for the cell pools in comparison to their corresponding unpooled cells over a period of 72 h, with a measurement being taken every 30 min. For each of the tested cell pools, cell index, slope, and doubling time did not differ between the cell pool and the unpooled cells of the donor animals. Differentiation capacity and mRNA expression of PAX7, MYOD and MYOG remained unchanged between the cell pool and the unpooled cells. Current results support that the use of cell pools is an appropriate method to reflect the average proliferative growth behavior of unpooled cells.

Transcript profile of skeletal muscle lipid metabolism genes affected by diet in a piglet model of low birth weight.

Dysregulated skeletal muscle metabolism (DSMM) is associated with increased inter- and intramuscular fat deposition in low birth weight (L) individuals. The mechanisms behind DSMM in L individuals are not completely understood but decreased muscle mass and shifts in lipid and carbohydrate utilisation may contribute. Previously, we observed lower fat oxidation in a porcine model of low birth weight. To elucidate the biological activities underpinning this difference microfluidic arrays were used to assess mRNA associated with lipid metabolism in longissimus dorsi (LD) and semitendinosus (ST) skeletal muscle samples from thirty-six female L and normal birth weight (N) pigs. Plasma samples were collected from a sub-population to measure metabolite concentrations. Following overnight fasting, skeletal muscle and plasma samples were collected and the association with birth weight, diet and age was assessed. Reduced dietary fat was associated with decreased LD intermuscular fat deposition and beta-oxidation associated mRNA, in both birth weight groups. Lipid uptake and intramuscular fat deposition associated mRNA was reduced in only L pigs. Abundance of ST mRNA associated with lipolysis, lipid synthesis and transport increased in both birth weight groups. Lipid uptake associated mRNA reduced in only L pigs. These changes were associated with decreased plasma L glucose and N triacylglycerol. Post-dietary fat reduction, LD mRNA associated with lipid synthesis and inter- and intramuscular fat deposition increased in L, whilst beta-oxidation associated mRNA remains elevated for longer in N. In the ST, mRNA associated with lipolysis and intramuscular fat deposition increased in both birth weight groups, however this increase was more significant in L pigs and associated with reduced beta-oxidation. Analysis of muscle lipid metabolism associated mRNA revealed that profile shifts are a consequence of birth weight. Whilst, many of the adaptions to diet and age appear to be similar in birth weight groups, the magnitude of response and individual changes underpin the previously observed lower fat oxidation in L pigs.

Cross-talk between energy metabolism and epigenetics during temperature stress response in C2C12 myoblasts.

Objective: Environmental stress induces disturbances in cell energy metabolism and may cause epigenetic modifications. This study aimed to understand the possible impact of temperature stress (35 °C, 39 °C and 41 °C, compared to control 37 °C) on energy metabolism and epigenetic modifications, such as DNA methylation and histone H4 acetylation, as well as its effects on the expression of genes responsible for epigenetic changes, in mouse skeletal myoblasts (C2C12 cells). Methods: The results showed significantly reduced maximal respiration and spare respiratory capacity under heat stress (39 °C and 41 °C), suggesting that mitochondrial functions were compromised under these conditions. The glycolytic capacity and glycolysis markedly increased following low-temperature stress (35 °C). The results suggested that, under cold stress, cells prefer glycolysis as a rapid compensatory mechanism to meet energy requirements for adaptive thermogenic response. Results: Epigenetic changes (histone H4 acetylation and global DNA methylation) were observed under both heat and cold stress. Among the genes coding for DNA methyltransferases, the Dnmt3a was significantly increased under high-temperature conditions (39 °C and 41 °C), while Dnmt1 expression was significantly increased at low temperature (35 °C), indicating that under these conditions the cells preferred maintenance of methylation to de novo methylation activity. An expression pattern similar to Dnmt3a was observed for Gcn5, encoding for a histone acetyltransferase. The study revealed that temperature stress induced changes in the metabolic profiles, as well as epigenetic modifications, including the dynamics of the key enzymes. Conclusion: The results indicated the existence of crosstalk mechanisms between energy metabolism and epigenetics during cell stress response.

Effects of long-term microalgae supplementation on muscle microstructure, meat quality and fatty acid composition in growing pigs.

We investigated the effects of long-term microalgae supplementation (7% in a piglet diet and 5% in a fattening diet) on muscle microstructure and meat quality, including fatty acid composition in female Landrace pigs (n = 31). The major effects were muscle-specific increases in n-3 and n-6 polyunsaturated fatty acids (PUFA) concentrations, resulting in increased accumulation of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Carcass traits and meat quality of longissimus thoracis muscle were not affected by the microalgae diet with the exception of reduced drip loss (p = 0.01) and increased protein proportion (p = 0.04). In addition, the microalgae diet resulted in a shift to a more oxidative myofibre type composition in semitendinosus but not longissimus thoracis muscle. In conclusion, microalgae supplementation offers a unique opportunity to enhance essential n-3 PUFA contents in pig meat. The results support small but coordinated changes in skeletal muscle phenotypic appearance and functionality.

Voluntary locomotor activity promotes myogenic growth potential in domestic pigs.

Self-determined physical activity is an essential behavioural need and can vary considerably between individuals of a given species. Although locomotion is suggested as a prerequisite for adequate function of skeletal muscle, domestic pigs are usually reared under limited space allowance. The aim of our study was to investigate if a different voluntary locomotor activity leads to altered properties in the muscle structure, biochemistry and mRNA expression of selected genes involved in myogenesis and skeletal muscle metabolism. Based on a video tracking method, we assigned pigs to three categories according to their total distances walked over five observed time points: long distance, medium distance, and short distance. The microstructure and biochemistry parameters of the M. semitendinosus were unaffected by the distance categories. However, we found distance-dependent differences in the mRNA expression of the genes encoding growth (IGF2, EGF, MSTN) and transcription factors (MRF4, MYOD). In particular, the IGF2/MSTN ratio appears to be a sensitive indicator, at the molecular level, for the locomotor activity of individuals. Our results indicate that the myogenic growth potential of pigs under standard rearing conditions is triggered by their displayed voluntary locomotor activity, but the covered distances are insufficient to induce adaptive changes at the tissue level.

Differentiation of bovine satellite cell-derived myoblasts under different culture conditions.

The aim of this study was to develop adequate in vitro conditions for the differentiation of bovine skeletal muscle cells. Therefore, satellite cells isolated from the left foreleg of a Holstein-Friesian fetus at 4.5 mo of gestation were seeded on 24-well plates coated with extracellular matrix gel. Cells were cultured for 5 d in growth medium containing 10% fetal bovine serum. After reaching confluence, several differentiation media were tested for inducing myotube formation. The highest fusion rate of approximately 30% was achieved with a serum-free medium containing 1 µM dexamethasone, 1 µg/ml linoleic acid, and 0.1 µM insulin after a differentiation phase of 72 h. Two different culture conditions (serum-free and serum-containing) appropriate for bovine skeletal muscle cell differentiation are described in detail which allow the investigation of bovine skeletal muscle cell proliferation and differentiation in general as well as in response to bioactive compounds.

Domestication does not narrow MHC diversity in Sus scrofa.

The Major Histocompatibility Complex (MHC) is a multigene family of outstanding polymorphism. MHC molecules bind antigenic peptides in the peptide-binding region (PBR) that consists of five binding pockets (P). In this study, we compared the genetic diversity of domestic pigs to that of the modern representatives of their wild ancestors, the wild boar, in two MHC loci, the oligomorphic DQA and the polymorphic DRB1. MHC nucleotide polymorphism was compared with the actual functional polymorphism in the PBR and the binding pockets P1, P4, P6, P7, and P9. The analysis of approximately 200 wild boars collected throughout Europe and 120 domestic pigs from four breeds (three pureblood, Pietrain, Leicoma, and Landrace, and one mixed Danbred) revealed that wild boars and domestic pigs share the same levels of nucleotide and amino acid polymorphism, allelic richness, and heterozygosity. Domestication did not appear to act as a bottleneck that would narrow MHC diversity. Although the pattern of polymorphism was uniform between the two loci, the magnitude of polymorphism was different. For both loci, most of the polymorphism was located in the PBR region and the presence of positive selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous substitutions in the PBR. P4 and P6 were the most polymorphic binding pockets. Functional polymorphism, i.e., the number and the distribution of pocket variants within and among populations, was significantly narrower than genetic polymorphism, indicative of a hierarchical action of selection pressures on MHC loci.

Effects of leptin and adiponectin on proliferation and protein metabolism of porcine myoblasts.

The aim of this study was to show the abundance of leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) and to determine the direct effects of leptin and adiponectin on the in vitro growth of porcine skeletal muscle cells. ADIPOR1 and ADIPOR2 were abundant at mRNA and protein level in proliferating and differentiating myoblast cultures derived from semimembranosus and semitendinosus muscles of newborn piglets, whereas LEPR expression was close to the detection limit. Adiponectin (10, 20, 40 µg/ml) attenuated the proliferation of porcine myoblasts, measured as [(3)H]-thymidine incorporation and real-time monitoring of the cells in response to 24- and 48-h exposure, in a dose-dependent manner. This effect resulted from suppressed basic fibroblast growth factor (bFGF)-mediated stimulation of DNA synthesis in serum-free medium (SFM) containing bFGF. No effects of leptin (5, 10, 20, 40, 80 ng/ml) on myoblast proliferation in SFM were detectable. Neither leptin nor adiponectin altered protein synthesis and degradation in differentiating porcine myoblasts cultured in SFM. The results on receptor abundance suggest that porcine skeletal muscle cells may be sensitive to adiponectin and leptin. However, except via inhibitory interaction of adiponectin with bFGF, these adipokines appear not to affect in vitro proliferation and protein metabolism of porcine muscle cells directly under serum-free culture conditions.

Limited and excess protein intake of pregnant gilts differently affects body composition and cellularity of skeletal muscle and subcutaneous adipose tissue of newborn and weanling piglets.

AIM: This study investigated whether dietary protein intake less (50%) or greater (250%) than requirements throughout gestation differently affects offspring body composition and cellular properties of skeletal muscle and subcutaneous adipose tissue (SCAT). METHODS: Primiparous gilts were fed iso-energetic diets containing adequate (22 AP), high (21 HP), or low (19 LP) protein contents. Newborn (n = 166) and weanling piglets cross-fostered to sows fed a standard diet (day 28; n = 83) were examined by morphological, biochemical, histological, and molecular analyses of the body, SCAT, and semitendinosus, longissimus, biceps femoris muscles. RESULTS: Lowered birth weight (BW) in response to the HP and LP diets (p < 0.01) resulted from decreases in all body constituents in LP, and mainly from reduced body fat in HP piglets (p < 0.05). In the light BW class within litters, HP piglets exhibited a greater percentage of muscle tissue (p < 0.05) than LP piglets. Less SCAT mass in HP and LP piglets resulted from reduced (p < 0.05) number, but not the size of adipocytes. The LP diet adversely affected myogenesis and muscular differentiation derived from less (p < 0.01) primary and secondary myofibers, lower creatine kinase activity (p < 0.05), less IGF2 mRNA (p < 0.10), and greater expression of the embryonic myosin heavy chain isoform (p < 0.01). Catch-up growth of LP but not HP pigs until day 28 increased body fat (p = 0.01). Despite compensated muscle growth in LP piglets, the deficit in myofiber number remained. CONCLUSION: Poor intrauterine environment by limited and excess protein supply retards fetal growth, but only limited protein supply impairs myogenesis, persistently restricts muscle growth potential, and favors obesity at infancy.

Potential sources of early-postnatal increase in myofibre number in pig skeletal muscle.

In pigs, myogenesis is a biphasic phenomenon with the formation of primary and secondary fibres. Hyperplasia was reported to be accomplished around 90 days of gestation. However, some studies suggest a substantial increase in the total fibre number (TFN) from birth to weaning by counting fibre number in the muscle cross sections. The aim of this study was to establish in which way TFN increases after birth and whether this increase is imputable to new (tertiary) myofibres and/or fibre elongation. The semitendinosus muscle of 128 piglets was examined at days 1 (n = 63), 7 (n = 12), 21 (n = 12), and 28 (n = 41) of age. TFN was increased at days 7, 21 and 28 of age when compared with day 1 (P < 0.01). From day 1 to 28, TFN increased from 463 × 10(3) to 825 × 10(3). Microscopy of longitudinal and transversal serial sections revealed that at day 7 of age very small fibres expressing the embryonic myosin heavy chain (MyHC) isoform were apparent all over the muscle. In addition, intrafascicular terminations of normal-sized fibres expressed the embryonic MyHC isoform. These data suggest that the TFN in the pig muscle is not fixed at birth and its postnatal increase may be related to both elongation of existing muscle fibres and genesis of tertiary myofibres, mainly between birth and 3 weeks of age.

Annotation of novel transcripts putatively relevant for bovine fat metabolism.

Two bovine transcripts encoded by the interleukin-1 receptor-associated kinase 1 (IRAK1) gene and the locus LOC618944 predicted as similar to human chromosome 6 open reading frame 52 (C6orf52) gene had indicated divergent expression in bovine skeletal muscle containing different amount of intramuscular fat in a pilot screening experiment. However, for both loci any role in the regulation of energy or fat metabolism is not yet described. In this study, we validated and refined gene structure, screened for mRNA splice variants and analyzed the tissue-specific gene expression patterns of both loci as a prerequisite to elucidate their potential physiological function. Based on comparative sequence analysis, a new full-length gene model for the bovine IRAK1 gene was developed and confirmed experimentally. Expression of IRAK1 mRNA was found in a variety of tissues, and a splice variant was identified in skeletal muscle caused by an in-frame deleted segment of 210 bp affecting regions of intrinsic disorder in the respective protein. For the locus LOC618944, our data contributed to a revised gene model and its assignment to BTA23 (bovine chromosome 23) on the current bovine genome assembly supported by comparative similarity analysis between the bovine and human genomes and experimental data. Furthermore, we identified several splice variants in mammary gland, fat and skeletal muscle tissue and detected a highly similar processed pseudogene on BTA26. All transcript variants of LOC618944 detected in the analyzed tissues represent noncoding RNAs. For both loci, our results suggest yet undetected physiological functions in tissues relevant for fat or energy metabolism in cattle.

Analysis of structure and gene expression of bovine CCDC3 gene indicates a function in fat metabolism.

Our study reports the molecular analysis of the bovine gene encoding the coiled-coil domain-containing protein 3 (CCDC3). Based on comparative sequence analysis and in silico sequence merging of predicted gene models, a new full-length gene model for the bovine CCDC3 gene was predicted and confirmed experimentally. The CCDC3 gene was assigned to bovine chromosome 13. It consists of three exons comprising 2599bp encoding for a respective protein of 274 amino acids. The strong CCDC3 sequence homology on amino acid level between species suggests a conserved universal function of this protein. In mice, the CCDC3 gene had been found to be highly expressed in adipocytes and regulated by hormonal-nutritional alternations and in obesity. The tissue expression pattern of bovine CCDC3 mRNA indicates a ubiquitous physiological function of the gene. Significant differences in CCDC3 mRNA expression in skeletal muscle between individuals characterized by divergent intramuscular fat deposition support the potential function of the gene in fat or energy metabolism, which possibly could also be inferred for other mammalian species. This first report of structural analysis and molecular characterization of the CCDC3 gene in cattle will contribute to a better understanding of the yet unknown physiological role of the respective protein in mammals.