Immediate and long-term transcriptional response of hind muscle tissue to transient variation of incubation temperature in broilers.

BACKGROUND: In oviparous species accidental variation of incubation temperatures may occur under natural conditions and mechanisms may have evolved by natural selection that facilitate coping with these stressors. However, under controlled artificial incubation modification of egg incubation temperature has been shown to have a wide-ranging impact on post-hatch development in several poultry species. Because developmental changes initiated in-ovo can affect poultry production, understanding the molecular routes and epigenetic alterations induced by incubation temperature differences may allow targeted modification of phenotypes. RESULTS: In order to identify molecular pathways responsive to variable incubation temperature, broiler eggs were incubated at a lower or higher temperature (36.8 °C, 38.8 °C) relative to control (37.8 °C) over two developmental intervals, embryonic days (E) 7-10 and 10-13. Global gene expression of M. gastrocnemius was assayed at E10, E13, and slaughter age [post-hatch day (D) 35] (6 groups; 3 time points; 8 animals each) by microarray analysis and treated samples were compared to controls within each time point. Transcript abundance differed for between 113 and 738 genes, depending on treatment group, compared to the respective control. In particular, higher incubation temperature during E7-10 immediately affected pathways involved in energy and lipid metabolism, cell signaling, and muscle development more so than did other conditions. But lower incubation temperature during E10-13 affected pathways related to cellular function and growth, and development of organ, tissue, and muscle as well as nutrient metabolism pathways at D35. CONCLUSION: Shifts in incubation temperature provoke specific immediate and long-term transcriptional responses. Further, the transcriptional response to lower incubation temperature, which did not affect the phenotypes, mediates compensatory effects reflecting adaptability. In contrast, higher incubation temperature triggers gene expression and has long-term effects on the phenotype, reflecting considerable phenotypic plasticity.

Molecular changes in mitochondrial respiratory activity and metabolic enzyme activity in muscle of four pig breeds with distinct metabolic types.

Skeletal muscles are metabolically active and have market value in meat-producing farm animals. A better understanding of biological pathways affecting energy metabolism in skeletal muscle could advance the science of skeletal muscle. In this study, comparative pathway-focused gene expression profiling in conjunction with muscle fiber typing were analyzed in skeletal muscles from Duroc, Pietrain, and Duroc-Pietrain crossbred pigs. Each breed type displayed a distinct muscle fiber-type composition. Mitochondrial respiratory activity and glycolytic and oxidative enzyme activities were comparable among genotypes, except for significantly lower complex I activity in Pietrain pigs homozygous-positive for malignant hyperthermia syndrome. At the transcriptional level, lactate dehydrogenase B showed breed specificity, with significantly lower expression in Pietrain pigs homozygous-positive for malignant hyperthermia syndrome. A similar mRNA expression pattern was shown for several subunits of oxidative phosphorylation complexes, including complex I, complex II, complex IV, and ATP synthase. Significant correlations were observed between mRNA expression of genes in focused pathways and enzyme activities in a breed-dependent manner. Moreover, expression patterns of pathway-focused genes were well correlated with muscle fiber-type composition. These results stress the importance of regulation of transcriptional rate of genes related to oxidative and glycolytic pathways in the metabolic capacity of muscle fibers. Overall, the results further the breed-specific understanding of the molecular basis of metabolic enzyme activities, which directly impact meat quality.

Altered incubation temperatures between embryonic Days 7 and 13 influence the weights and the mitochondrial respiratory and enzyme activities in breast and leg muscles of broiler embryos.

Altering incubation temperature during embryogenesis has an impact on chicken embryo growth, but the underlying molecular mechanisms are not understood; the present study was performed to address these changes. Broiler eggs were incubated at low (36.8°C), control (37.8°C), and high (38.8°C) temperatures between Embryonic Day (ED) 7 and 10 or ED 10 and 13, which cover critical periods of embryonic myogenesis. The embryos were then dissected immediately after treatment on ED 10 or 13 to assess body, liver, and heart weights as well as to analyze breast and leg muscle fibers for their mitochondrial respiratory activity (MRA). Breast muscle samples were additionally used to evaluate the activity of enzymes involved in energy metabolism and cell-cycle progression. ED-10 embryos incubated at 38.8°C showed elevated weights (body, liver, and heart), MRA, and activities of lactate dehydrogenase and cytochrome oxidase compared to the ED-10 embryos incubated at 36.8°C. Similarly, the ED-13 embryos incubated at 38.8°C showed elevated body weight, MRA, and activities of glycogen phosphorylase, phosphofructokinase, and cytochrome oxidase compared to their 36.8°C counterparts. Embryos incubated at the normal temperature (37.8°C), however, showed variable differences from those incubated at 38.8°C versus 36.8°C. Cell-cycle enzyme activities were not impacted by the different temperature treatments. Thus, an increase or decrease in the incubation temperature during embryonic broiler myogenesis results in altered embryo activity, muscle energy metabolism, and activity-dependent muscle growth.

Application of MALDI-TOF-mass spectrometry to proteome analysis using stain-free gel electrophoresis.

The combination of MALDI-TOF-mass spectrometry with gel electrophoretic separation using protein visualization by staining procedures involving such as Coomassie Brilliant Blue has been established as a widely used approach in proteomics. Although this approach has been shown to present high detection sensitivity, drawbacks and limitations frequently arise from the significant background in the mass spectrometric analysis. In this chapter we describe an approach for the application of MALDI-MS to the mass spectrometric identification of proteins from one-dimensional (1D) and two-dimensional (2D) gel electrophoretic separation, using stain-free detection and visualization based on native protein fluorescence. Using the native fluorescence of aromatic protein amino acids with UV transmission at 343 nm as a fast gel imaging system, unstained protein spots are localized and, upon excision from gels, can be proteolytically digested and analyzed by MALDI-MS. Following the initial development and testing with standard proteins, applications of the stain-free gel electrophoretic detection approach to mass spectrometric identification of biological proteins from 2D-gel separations clearly show the feasibility and efficiency of this combination, as illustrated by a proteomics study of porcine skeleton muscle proteins. Major advantages of the stain-free gel detection approach with MALDI-MS analysis are (1) rapid analysis of proteins from 1D- and 2D-gel separation without destaining required prior to proteolytic digestion, (2) the low detection limits of proteins attained, and (3) low background in the MALDI-MS analysis.

Identification of a vibration regime favorable for bone healing and muscle in estrogen-deficient rats.

Numerous whole-body vibration (WBV) devices of various forces are available on the market, although their influence on the musculoskeletal system is not yet understood. The effect of different WBVs on bone healing and muscle function was evaluated in rats ovariectomized at 3 months of age. 2 months after ovariectomy, bilateral metaphyseal tibia osteotomy and T-plate osteosynthesis were performed. Rats were divided into groups: intact, OVX, and OVX exposed to vertical WBVs of 35, 50, 70, or 90 Hz (experiment 1) or horizontal WBVs of 30, 50, 70, or 90 Hz (experiment 2) 5 days after osteotomy (0.5 mm, 15 min/day for 30 days). The tibia and gastrocnemius and soleus muscles were collected. Vertical vibrations (>35 Hz) improved cortical and callus densities, enlarged callus area and width, suppressed the tartrate-resistant acid phosphatase gene, enhanced citrate synthase activity, accelerated osteotomy bridging (35 and 50 Hz), upregulated the osteocalcin (Oc) gene (70 Hz), and increased relative muscle weight (50 Hz). Horizontal vibrations reduced cortical width (<90 Hz) and callus density (30 Hz), enhanced alkaline phosphatase (Alp) gene expression (50 Hz), decreased the size of oxidative fibers (35 and 70 Hz), and increased capillary density (70, 90 Hz). Biomechanical data; serum Oc, Alp, and creatine kinase activities; body weight; and food intake did not change after WBVs. Vertical WBVs of 35 and 50 Hz produced more favorable results than the higher frequencies. Horizontal WBV showed no positive or negative effects. Further studies are needed to elucidate the effects of WBV on different physiological systems, and precautions must be taken when implementing WBV in the treatment of patients.

Musculoskeletal response to whole-body vibration during fracture healing in intact and ovariectomized rats.

This study investigated the effect of vibration on bone healing and muscle in intact and ovariectomized rats. Thirty ovariectomized (at 3 months of age) and 30 intact 5-month old female Sprague-Dawley rats underwent bilateral metaphyseal osteotomy of tibia. Five days later, half of the ovariectomized and of the intact rats were exposed to whole-body vertical vibration (90 Hz, 0.5 mm, 4 x g acceleration) for 15 min twice a day during 30 days. The other animals did not undergo vibration. After decapitation of rats, one tibia was used for computed tomographic, biomechanical, and histological analyses; the other was used for gene expression analyses of alkaline phosphatase (Alp), osteocalcin (Oc), tartrate-resistant acid phosphatase 1, and insulinlike growth factor 1. Serum Alp and Oc were measured. Mitochondrial activity, fiber area and distribution, and capillary densities were analyzed in M. gastrocnemius and M. longissimus. We found that vibration had no effect on body weight and food intake, but it improved cortical and callus densities (97 vs. 99%, 72 vs. 81%), trabecular structure (9 vs. 14 trabecular nodes), blood supply (1.7 vs. 2.1 capillaries/fiber), and oxidative metabolism (17 vs. 23 pmol O(2)/s/mg) in ovariectomized rats. Vibration generally increased muscle fiber size. Tibia biomechanical properties were diminished after vibration. Oc gene expression was higher in vibrated rats. Serum Alp was increased in ovariectomized rats. In ovariectomized rats, vibration resulted in an earlier bridging; in intact rats, callus bridging occurred later after vibration. The chosen vibration regimen (90 Hz, 0.5 mm, 4 x g acceleration, 15 min twice a day) was effective in improving musculoskeletal tissues in ovariectomized rats but was not optimal for fracture healing.

Assessment of the sensory quality and shelf stability of selected Horro beef muscles in Ethiopia.

The objective of this work was to assess sensory quality and retail life of Horro beef muscles in Ethiopia. Six muscles: M. rhomboideus (RM), M. infraspinatus (IS), M. longissimus lumborum (LL), M. semimembranosus (SM), M. biceps femoris (BF) and M. rectus femoris (RF) were considered. Sensory quality of the muscles was rated by a 9-member trained panel for palatability, tenderness, juiciness, amount of connective tissue (ACT), lean color and surface discoloration and measured by Warner-Bratzler Shear Force (WBSF). Retail life of the six muscles were evaluated for CIE 1976 L(∗)a(∗)b(∗) color values across 6 days. Significant differences (p<0.05) between muscles for all sensory attributes and WBSF were found. Significant L(∗)a(∗)b(∗) color values of muscles and USDA quality grades by retail life were found (p<0.05). Significant correlations (p<0.05 or p<0.01) of key parameters were also identified.

miRNAs regulate acute transcriptional changes in broiler embryos in response to modification of incubation temperature.

MicroRNAs are post-transcriptional regulators that play critical roles in diverse biological processes. We hypothesize that miRNAs may be involved in regulating transcriptome responses to changes in embryonic incubation temperature in chickens affecting differentiation and proliferation processes during tissue development. Therefore, we conducted comparative transcriptome profiling of miRNAs to examine altered expression in breast and hind muscle of embryos and day 35 chickens experiencing high (38.8 °C), control (37.8 °C), or low (36.8 °C) embryonic incubation temperature during embryonic day (ED) 7-10 or ED10-13. The results revealed differential expression of miRNAs due to modification of embryonic incubation temperature in a muscle type-specific and a developmental stage-specific manner. The immediate effects of thermal change observed in embryos were substantial compared to the subtle long-term effects in chickens at day 35 post-hatch. Upregulation of miR-133 in breast muscle and downregulation of miR-199a-5p, miR-1915, and miR-638 in hind muscle post ED7-10 high-temperature treatment are functionally associated with myogenesis and body size. ED10-13 low-temperature treatment led to downregulation of let-7, miR-93, and miR-130c that are related to proliferation and differentiation. The results provide insight into the dynamics of miRNA expression at variable embryonic incubation temperatures during developmental processes and indicate a major regulatory role of miRNAs in acute responses to modified environmental conditions that affect remodelling of cells and tissues.

Suitability of three commercially produced pig breeds in Germany for a meat quality program with emphasis on drip loss and eating quality.

This study aimed at characterising 606 crossbred pigs of three commercially available breed types in terms of their carcass and meat quality. Breed G and H were German Large White (LW)×German Landrace (LR) sows sired with Pietrain (PI) boars, i.e. PI×(LW×LR). Breed S was 25% Duroc (DU), i.e. PI×(DU×LR). Most of the parameters were affected by breed and/or date of slaughter. The meat of crossbred pigs with 25% Duroc proportion appeared most favourable because of higher intramuscular fat content, lower drip loss and higher sensory liking scores. Conductivity is closely related to drip loss while the data suggests that the relationship is dependent on breed and carcass weight. The application of conductivity and lean meat yield thresholds to select carcasses with uniform and superior meat quality effectively decreased drip loss and increased intramuscular fat content as well as sensory liking scores. The variation of meat quality traits remains high, though.

Transient Shifts of Incubation Temperature Reveal Immediate and Long-Term Transcriptional Response in Chicken Breast Muscle Underpinning Resilience and Phenotypic Plasticity.

Variations in egg incubation temperatures can have acute or long-term effects on gene transcription in avian species. Altered gene expression may, in turn, affect muscle traits in poultry and indirectly influence commercial production. To determine how changes in eggshell temperature affect gene expression, incubation temperatures were varied [36.8°C (low), 37.8°C (control), 38.8°C (high)] at specific time periods reflecting two stages of myogenesis [embryonic days (ED) 7-10 and 10-13]. Gene expression was compared between interventions and matching controls by microarrays in broiler breast muscle at ED10 or ED13 and post-hatch at day 35. Early (ED7-10) high incubation temperature (H10ΔC) resulted in 1370 differentially expressed genes (DEGs) in embryos. Ingenuity pathway analysis revealed temporary activation of cell maintenance, organismal development, and survival ability genes, but these effects were not maintained in adults. Late high incubation temperature (ED10-13) (H13ΔC) had slightly negative impacts on development of cellular components in embryos, but a cumulative effect was observed in adults, in which tissue development and nutrition metabolism were affected. Early low incubation temperature (L10ΔC) produced 368 DEGs, most of which were down-regulated and involved in differentiation and formation of muscle cells. In adults, this treatment down-regulated pathways of transcriptional processes, but up-regulated cell proliferation. Late low temperature incubation (L13ΔC) produced 795 DEGs in embryos, and activated organismal survival and post-transcriptional regulation pathways. In adults this treatment activated cellular and organ development, nutrition and small molecule activity, and survival rate, but deactivated size of body and muscle cells. Thermal interventions during incubation initiate immediate and delayed transcriptional responses that are specific for timing and direction of treatment. Interestingly, the transcriptional response to transiently decreased incubation temperature, which did not affect the phenotypes, prompts compensatory effects reflecting resilience. In contrast, higher incubation temperature triggers gene expression and has long-term effects on the phenotype. These mechanisms of considerable phenotypic plasticity contribute to the biodiversity and broaden the basis for managing poultry populations.

Muscle Transcriptional Profile Based on Muscle Fiber, Mitochondrial Respiratory Activity, and Metabolic Enzymes.

Skeletal muscle is a highly metabolically active tissue that both stores and consumes energy. Important biological pathways that affect energy metabolism and metabolic fiber type in muscle cells may be identified through transcriptomic profiling of the muscle, especially ante mortem. Here, gene expression was investigated in malignant hyperthermia syndrome (MHS)-negative Duroc and Pietrian (PiNN) pigs significantly differing for the muscle fiber types slow-twitch-oxidative fiber (STO) and fast-twitch-oxidative fiber (FTO) as well as mitochondrial activity (succinate-dependent state 3 respiration rate). Longissimus muscle samples were obtained 24 h before slaughter and profiled using cDNA microarrays. Differential gene expression between Duroc and PiNN muscle samples were associated with protein ubiquitination, stem cell pluripotency, amyloid processing, and 3-phosphoinositide biosynthesis and degradation pathways. In addition, weighted gene co-expression network analysis within both breeds identified several co-expression modules that were associated with the proportion of different fiber types, mitochondrial respiratory activity, and ATP metabolism. In particular, Duroc results revealed strong correlations between mitochondrion-associated co-expression modules and STO (r = 0.78), fast-twitch glycolytic fiber (r = -0.98), complex I (r=0.72) and COX activity (r = 0.86). Other pathways in the protein-kinase-activity enriched module were positively correlated with STO (r=0.93), while negatively correlated with FTO (r = -0.72). In contrast to PiNN, co-expression modules enriched in macromolecule catabolic process, actin cytoskeleton, and transcription activator activity were associated with fiber types, mitochondrial respiratory activity, and metabolic enzyme activities. Our results highlight the importance of mitochondria for the oxidative capacity of porcine muscle and for breed-dependent molecular pathways in muscle cell fibers.

How olfactory acuity affects the sensory assessment of boar fat: a proposal for quantification.

Due to animal welfare concerns the production of entire male pigs is one viable alternative to surgical castration. Elevated levels of boar taint may, however, impair consumer acceptance. Due to the lack of technical methods, control of boar taint is currently done using sensory quality control. While the need for control measures with respect to boar taint has been clearly stated in EU legislation, no specific requirements for selecting assessors have yet been documented. This study proposes tests for the psychophysical evaluation of olfactory acuity to key volatiles contributing to boar taint. Odor detection thresholds for androstenone and skatole are assessed as well as the subject's ability to identify odorants at various levels through easy-to-use paper smell strips. Subsequently, fat samples are rated by the assessors, and the accuracy of boar taint evaluation is studied. Considerable variation of olfactory performance is observed demonstrating the need for objective criteria to select assessors.

Sensory evaluation of boar loins: trained assessors' olfactory acuity affects the perception of boar taint compounds.

This study investigated the impact of assessors' varying olfactory acuity on the perceived intensity of androstenone and skatole odour and flavour in boar loins. To discriminate sensitive (SENS) and highly sensitive (SENSHIGH) panellists, two levels of androstenone were used on smell strips. Sensitivity was defined as the correct identification of the androstenone strip in three replicate triangle tests. Judges then assessed loins from boars, castrated pigs and gilts. SENSHIGH assessors scored low-fat boar loins with 1.5 to 2.0µg of androstenone per gram of melted back fat which is significantly different from castrate and gilt loins for androstenone odour and flavour whereas SENS assessors were less discriminating. Panellists' olfactory acuity should thus be considered for selection and training. The presented paper strip system is suggested for objective screening and training purposes and to be used as quantitative references in descriptive analysis.

Consumer perception of boar meat as affected by labelling information, malodorous compounds and sensitivity to androstenone.

This study aimed to assess the influence of two label conditions on the acceptance of boar meat. A central location test was conducted with 145 consumers each assessing 4 pieces of pork loin. Samples varied with respect to two factors: actual meat type (boar vs. standard pork) and label information (young boar meat vs. pork). Androstenone and skatole levels in the tested boar meat ranged from 0.51 to 2.72 µg/g and 0.01 to 0.23 µg/g melted fat, respectively. Consumers' sensitivity to and appreciation of androstenone and skatole odour was determined through a smell experiment. The acceptance of taste, tenderness, juiciness, and overall liking was neither influenced by the label information nor by the meat type. Twenty-seven % of all participants were classified as insensitive to androstenone odour, whereas 52% perceived it as positive and 21% as negative. Consumers who disliked the androstenone odour indicated a higher disliking of boar meat.

Different scalding techniques do not affect boar taint.

The prevention of unpleasant boar taint is the main reason for castration of male piglets. For animal welfare reasons, castration is announced to be banned in the European Community. This study aimed to investigate whether androstenone, skatole and indole in backfat of boars may be reduced by different scalding technologies. To discriminate ante and post mortem effects, carcasses were sampled before and after scalding in two abattoirs using either horizontal (TANK) or vertical (TUNNEL) scalding. Backfat samples were analysed using gas chromatography (androstenone) and liquid chromatography (skatole, indole). Neither TANK nor TUNNEL scalding did significantly reduce malodorous compounds. Skatole and androstenone in backfat obtained after scalding averaged 112 ± 123 ng/g and 1196 ± 885 ng/g melted fat, respectively; significant differences between abattoirs were observed for skatole. Increased skatole levels were tentatively assigned to longer transport duration. Concluding from recent consumer research and subsequent application of suggested sensory rejection thresholds for androstenone (2000 ng/g) and skatole (150 ng/g), nearly 30% of the carcasses may be unacceptably tainted.

Ultrasound velocity and attenuation of porcine soft tissues with respect to structure and composition: I. Muscle.

Ultrasound velocity and attenuation of soft tissues have been widely investigated. However, few studies completely covered considerable variations of both, structure and composition. The aim of this study was to collect acoustic reference data of porcine Longissimus muscle and associate them with compositional traits. In addition, measurements were conducted on fresh, formalin fixed, and frozen-thawed samples to evaluate the effect of processing on ultrasound parameters and comparisons with earlier investigations. Measurement conditions (temperature and fibre orientation) were realised close to hanging carcasses conditions. Sound velocity ranged from 1617 ± 6 to 1622 ± 5 ms(-1), while attenuation mostly ranged from 1.0 ± 0.3 to 1.2 ± 0.3 dB MHz(-1)cm(-1). Only formalin fixed samples showed significantly higher attenuation (2.2 ± 0.6 dB MHz(-1)cm(-1)). Highest correlations have been observed between intramuscular fat and attenuation (up to r = .7). The obtained results are anticipated to improve ultrasound based estimation of the intramuscular fat of pig muscle on intact carcasses.

Ultrasound velocity and attenuation of porcine soft tissues with respect to structure and composition: II. Skin and backfat.

Ultrasound is regarded as a promising method to determine the intramuscular fat content of pork loin. At intact carcasses, the signal passes the backfat whose ultrasound parameters (sound velocity and attenuation) have not been fully investigated. This study intended to collect a dataset of ultrasound parameters for individual backfat layers and to elucidate relationships with structural and compositional characteristics. In-vitro measurements at 10 MHz were conducted on backfat samples of pork carcasses representative for German populations. The average sound velocity ranged from 1436 ± 9 to 1470 ± 37 ms(-1) for the fat layers, and 1682 ± 23 ms(-1) for skin. Velocity of the compound backfat decreased with overall thickness. Attenuation was not affected by thickness ranging between 1.6 ± 0.7 and 2.7 ± 1.5 dB MHz(-1)cm(-1) for all layers. Sound velocity was negatively correlated with fat content and dry matter. The obtained results are anticipated to improve signal correction prior to spectral analysis of ultrasound measurements at intact carcasses.

Influence of intermittent administration of parathyroid hormone on muscle tissue and bone healing in orchiectomized rats or controls.

Influence of human parathyroid hormone (hPTH 1-34) on muscle and bone healing was studied in either orchiectomized (Orx at 8 months of age) or sham-operated male rats. Eleven-month-old Sprague-Dawley rats underwent bilateral transverse metaphyseal osteotomy of tibia and were divided into four groups (n=12): 1) sham-vehicle, 2) sham group-PTH everyday, 3) Orx-vehicle, 4) Orx-PTH everyday, and 5) Orx-PTH every other day. PTH dosage was 40  µg/kg body weight. After 5 weeks, fiber cross-sectional area, capillary density, and enzyme activity (lactate dehydrogenase, citrate synthase, and complex I) were measured in soleus (MS), gastrocnemius (MG), and longissimus (ML) muscles; tibiae were analyzed by computed tomographical, histological, and gene expression analyses. The effect of PTH in all rats was increased serum osteocalcin, cortical and callus densities and callus area. In sham rats capillary density was increased in limb muscles (MS: 1.3-1.7, MG: 1.2-1.4 capillaries/fiber), and rate of osseous bridging of osteotomy was enhanced (67-100%). In Orx rats serum creatine kinase was decreased (6670-2847 U/l), and bone genes (Igf-1, osteoprotegerin, and receptor activator of nuclear factor kB ligand) were up-regulated. Cross-sectional area, enzyme activity, food intake, weight of body, visceral organs, adipose tissue, MG, and MS were not affected by PTH. PTH had a favorable effect on muscle capillary density and improved bone healing being more effective in sham rats and having no adverse systemic effect. The effect was less if PTH was applied every other day. The findings may show up trends for therapeutic treatment of male patients.

Effect of human parathyroid hormone hPTH (1-34) applied at different regimes on fracture healing and muscle in ovariectomized and healthy rats.

Three experiments were conducted to investigate the effect of intermittent administration of parathyroid hormone (PTH) (1-34) applied at different regimes on fracture healing and muscle in healthy and ovariectomized (Ovx at 3 months of age) rats. Five-month old rats underwent bilateral transverse metaphyseal osteotomy of tibia and were divided into groups (12 rats each). In Exp 1, Ovx rats were either treated with PTH (7x/w, 1-35d), with oral estradiol-17beta-benzoate (0.4 mg/kg BW, 1-35d) or untreated. In Exp. 2, there were 3 groups: healthy untreated or treated with PTH (5x/w, 1-35d or 7-35d). In Exp. 3, there were 7 groups: healthy, Ovx, "healthy PTH 5x/w 7-35d", "Ovx PTH 5x/w 7-35d, 14-35d or 14-28d", "Ovx PTH every other day 7-35d". Single dosage of PTH was 40 microg/kg BW. After 35 days of healing one tibia was analyzed by computed tomographical, biomechanical, histological analyses. The other tibia was used in analyses of Alp, Oc, Trap 1, Igf-1, Rankl, Opg genes (Exp.2, 3). Serum Oc and Alp were measured. Body, uterus weight was recorded. M. gastrocnemius was analyzed for weight (Exp. 2), fiber size and mitochondrial respiratory activity (MRA) (Exp.3). Estrogen enhanced uterus weight, prevented body increase, however, did not improve bone healing in Ovx rats (Exp. 1). PTH administration from days 1 and 7 improved bone parameters in all rats regardless of the application frequency (7, 5x/w or every other day) (Exp. 1, stiffness Ovx: 118+13 N/mm, Ovx PTH: 250+/-20 N/mm) being more effective in healthy rats (Exp. 3, stiffness improvement Healthy: 59 to 174 N/mm, Ovx: 52 to 98 N/mm). Serum Oc level was elevated in PTH treated rats. Application from day 14 proved to be less effective (Exp. 3). PTH had no effect (P>0.05) on body, uterus and muscle weight, muscle fiber size, MRA and expression of bone markers. PTH promoted bone healing in Ovx and healthy rats, when it is applied during early stage of healing without having any adverse systemic effect. In perspective, PTH may represent a treatment for enhancement of fracture healing. The findings need to be confirmed by follow-up studies on other animals.

Effect of daidzein, 4-methylbenzylidene camphor or estrogen on gastrocnemius muscle of osteoporotic rats undergoing tibia healing period.

The effect of daidzein (D), 4-methylbenzylidene camphor (4-MBC) or estradiol-17beta-benzoate (E(2)) on muscle of osteoporotic rats during fracture healing was studied. After performing a metaphyseal tibia osteotomy in 96 osteoporotic 5-month-old female Sprague-Dawley rats, they received daily 50 mg D, 200 mg 4-MBC or 0.4 mg E(2) per kg body weight, or soy free (SF) diet up to 36 and 72 days. Mitochondrial activity, fiber area, and capillary density were analyzed in M. gastrocnemius. Osseous callus bridging of fracture was observed in half of the rats after 36 days. By day 72, fracture was healed in most of the animals. State 3 mitochondrial respiration significantly enhanced in E(2), 4-MBC and D groups versus SF after 36 days (30, 32 and 32 vs 23 pmol O(2)/s per mg). It declined after 72 days, however, in E(2) group it was still at a higher level versus SF (25, 23 and 21 vs 20 pmol O(2)/s per mg). Size of fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibers, capillary density did not differ significantly between the groups, however, at day 36 an increase in D and 4-MBC groups was detectable. FOG diameter was 64, 66, 68, and 58 microm and FG diameter was 88, 98, 95, and 89 microm in SF, D, 4-MBC, and E(2) groups. The ratio of capillaries to muscle fiber was 1.1, 1.4, 1.3, and 1.1 in SF, D, 4-MBC and E(2) groups by day 36. D and 4-MBC react similar to estrogen thereby improving oxidative cell metabolism in severe osteoporotic rats. The level of mitochondrial activity was higher, though no significant morphological differences could be shown.