Short communication: The orientation of cubicles plays a role - greater deviation from the north-south direction, more technopathies in dairy cows.

Magnetic alignment (MA) is a biological phenomenon denoting spontaneous orientation of an animal's body at rest, when fleeing a threat, hunting, etc. in relation to the Earth's magnetic field lines, often parallel to the field lines, i.e. in a north-south (NS) direction. MA has been demonstrated in several animal species, including grazing and resting domestic mammals. Based on the assumption that cows prefer to orientate their bodies in the NS direction when resting and sleeping, we hypothesised that MA could contribute to the incidence of technopathies in dairy cows, i.e. various disorders caused by the stable equipment that pose a serious animal welfare problem. It was hypothesised that the incidence of technopathies increases when the cubicles (resting places) orientation deviates from the NS position. The incidence of technopathies was recorded in 34 free-stall dairy farms. The type (hairless patch, scratch, swelling, wound), location and size of the lesions were recorded in almost 1 200 dairy cows. Based on the number of cubicles available and the number of cows, the cubicle-per-cow index (CC index) was calculated and divided into two groups: CC index ≥ 1 for stables with at least one cubicle per cow and CC index < 1 if the number of cows exceeded the number of cubicles available. The orientation of the cubicles was determined by the azimuth (the angle between north and the chosen clockwise direction). The farms were categorised into two groups according to azimuth: NS (azimuth deviating ± 15° from north or south, i.e. 345-15° and 165-195°) and non-NS (azimuth 15-165° and 195-345°). We found that the frequency of cows with technopathies was 30% lower in the NS-orientated cubicles and the number of technopathies per cow was 40% lower in the NS-orientated cubicles than in the non-NS-orientated cubicles. In addition, a higher number of technopathies per cow was observed when the CC index was ≥ 1, with a significant difference in the non-NS-aligned cubicles. According to our results, cubicle orientation has some influence on the incidence of technopathies. Although biological phenomena such as MA are seemingly unimportant and usually overlooked, they should be considered in livestock production when planning the positioning of stable equipment/ cubicle.

Beneficial effects of water-soluble chestnut (Castanea sativa Mill.) tannin extract on chicken small intestinal epithelial cell culture.

Feed and water supplementation with powdered hydrolyzable tannins from chestnut represents a valuable alternative strategy to antibiotics in animal nutrition. In this study, we evaluated the effects and safety of a water-soluble form of chestnut tannin (WST) in an in vitro model of chicken small intestinal epithelial cells (CSIEC). A chicken cell culture was established, and WST in concentrations of 0.025, 0.05, 0.1, and 0.2% were tested for cytotoxicity, cell proliferation, metabolic activity, production of reactive oxygen species, intracellular antioxidative potential, genotoxicity, and influence on the epithelia cell cycle. The tested concentrations showed a significant (P < 0.05) greater proliferative effect on CSIEC than the control medium (maximal proliferation at 0.1% WST as determined by optical density measurements). The 0.2% concentration of WST was cytotoxic, causing significantly higher (P < 0.05) nitric oxide and hydrogen peroxide production but with no short-term genotoxicity. Although increasing the concentration caused a decline in the metabolism of challenged cells (the lowest at 0.1% WST), metabolic activity remained higher than that in control cells. The antioxidant potential was 75% better and significantly (P < 0.05) higher in the 0.1% WST cultured cells compared to control. In conclusion, the cultured CSIEC are useful tools in basic and clinical research for the study of intestinal physiology, as they retain physiological and biochemical properties and epithelial morphology close to the original tissue and, in many ways, reflect the in vivo state. Our results indicate that WST exert a beneficial effect on intestinal epithelia, since they: i) stimulate proliferation of enterocytes; ii) increase antioxidative potential; iii) have no genotoxic effect; and iv) do not affect cellular metabolism. Our results reinforce the importance of WST as promising candidates for further evaluation and use in commercial broiler farm production.

Dynamics of myosin heavy chain isoform transition in the longissimus muscle of domestic and wild pigs during growth: a comparative study.

Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with domestication, breeding and rearing conditions. This study was aimed to comparatively elucidate the age-dependent myosin heavy chain (MyHC) isoform expression and transition pattern in domestic and wild pig (WP) skeletal muscle from birth until adulthood. Domestic pigs (DPs) of Large White breed raised in conventional production system were compared with WPs reared in a large hunting enclosure. Muscle samples for immuno/enzyme histochemistry were taken from the longissimus dorsi muscle within 24 h postmortem at 24 to 48 h, 21 to 23 days, 7 months and ~2 years postpartum. Based on the antibody reactivity to MyHCs (NCL-MHCs, A4.74, BF-F3) and succinate dehydrogenase activity, myofibers were classified into I, I/IIa, IIa, IIx and IIb types. In addition, foetal MyHC expression was determined with the use of F158.4C10 antibody. Maturation of the longissimus dorsi muscle in the WP was characterized by an accelerated transformation of the fast to slow MyHC during the first hours postpartum, followed by differentiation towards oxidative myofibers in which type I, IIa and IIx MyHCs predominated. In the DP, the transformation shifted towards glycolytic myofibers that expressed MyHC-IIb. The expression of foetal MyHC was higher in the DP than in the WP at 1 day of age, and the decline in the foetal MyHC during the first 3 weeks was more rapid in the WP than in the DP denoting an accelerated early postnatal muscle maturation in WP than DP piglets. All foetal MyHC-positive myofibers co-expressed IIa isoform, but not vice versa. The intense myofiber hypertrophy was evident from 3 weeks until 7 months of age. In this period, the myofiber cross-sectional area increased up to 10- and 20-fold in the WP and the DP, respectively. In the DP, the hypertrophy of all myofiber types was more pronounced than in the WP, particularly the hypertrophy of IIx and IIb myofibers. To summarize, the comparison between growing DP with wild ancestors showed that genetic selection and rearing conditions lead to substantial changes in the direction and intensity of postnatal MyHC transformation as evidenced by different proportion of individual myofiber types and differences in their hypertrophic potential.

Classification of dry-cured hams according to the maturation time using near infrared spectra and artificial neural networks.

An attempt to classify dry-cured hams according to the maturation time on the basis of near infrared (NIR) spectra was studied. The study comprised 128 samples of biceps femoris (BF) muscle from dry-cured hams matured for 10 (n=32), 12 (n=32), 14 (n=32) or 16 months (n=32). Samples were minced and scanned in the wavelength range from 400 to 2500 nm using spectrometer NIR System model 6500 (Silver Spring, MD, USA). Spectral data were used for i) splitting of samples into the training and test set using 2D Kohonen artificial neural networks (ANN) and for ii) construction of classification models using counter-propagation ANN (CP-ANN). Different models were tested, and the one selected was based on the lowest percentage of misclassified test samples (external validation). Overall correctness of the classification was 79.7%, which demonstrates practical relevance of using NIR spectroscopy and ANN for dry-cured ham processing control.

Effects of divergent selection for 8-week body weight on postnatal enzyme activity pattern of 3 fiber types in fast muscles of male broilers (Gallus gallus domesticus).

A divergent selection experiment was conducted for 8-wk BW in chickens. At 3, 6, 9, and 12 wk of age, samples of pectoralis profundus (PP) and biceps femoris (BF) muscles from fast-growing and slow-growing lines were used to estimate the enzyme activities and muscle fiber diameter. Microphotometric measurements made in situ of succinate dehydrogenase (SDH, EC 1.3.99.1) and glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.99.5) were completed on serial sections of PP and BF muscles from male chickens, in order to examine the ratio of SDH:GPDH activity in single fibers. On the basis of the SDH:GPDH activity ratios, muscle fibers were divided using cluster analysis into 3 populations of different fiber types (O = oxidative, OG = oxidative-glycolytic, and G = glycolytic). Cockerels of the SGL attained an 8.1-fold increase and those of the FGL a 6.8-fold increase in BW at 12 wk compared with that at 3 wk of age. The O, OG, and G type fibers of the BF muscles of the SGL had significantly (P ≤ 0.001) lower SDH:GPDH activity ratios than those of the FGL. A step decrease in the SDH:GPDH activity of O, OG, and G fibers in the PP of both lines occurred, and this differed significantly between SGL and FGL (P ≤ 0.001). Age and line effects influenced the diameter of the 3 fiber types in the BF muscle only. In contrast to this response, all 3 fiber types of the PP muscles reached similar diameters in both lines during the growth process from wk 3 to 12. From the results of this study, we concluded that the activities of metabolic enzymes in skeletal muscle fibers are under the influence of muscle type, age, and selection pressure. Microphotometry is a suitable method for the evaluation of enzyme activity measured in a single muscle fiber. The method enables precise estimation of enzyme activities, especially in muscles composed of populations of different metabolic fiber types.

Mitochondrial enzyme defects in normal and low-frequency-stimulated muscles of young and aging rats.

The age-related increase in cytochrome c-oxidase-deficient (COX(-)) muscle fibers has been suggested to be positively correlated with mitochondrial content (Müller-Höcker, Brain Pathol. 1992; 2:149-158). As a way to test this relationship, tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway rats were exposed to chronic low-frequency stimulation (CLFS) for 50 days, an experimental protocol known to induce marked increases in mitochondrial content. CLFS produced elevated activity levels of COX and succinate dehydrogenase (SDH) in most fibers of young and aging rats. Some fibers low or deficient in COX and a few fibers low or deficient both in COX and SDH (COX(-)/SDH(-)) were detected in unstimulated muscles of young and, more frequently, aging rats. According to their myosin complement, these fibers were immunohistochemically identified as type I fibers. CLFS increased their number in young muscles, but reduced it in aging muscles. Stimulated aging muscles contained some very small, most likely newly formed COX(+) and SDH(+) type I fibers. Thus, the fraction of COX(-) fibers was reduced in aging muscle by enhanced contractile activity.

Fiber-type transitions and satellite cell activation in low-frequency-stimulated muscles of young and aging rats.

We examined satellite cell content and the activity of satellite cell progeny in tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway (BN) rats, after they were exposed for 50 days to a standardized and highly reproducible regime of chronic low-frequency electrical stimulation. Chronic low-frequency electrical stimulation was successful in inducing fast-to-slow fiber-type transformation, characterized by a 2.3-fold increase in the proportion of IIA fibers and fourfold and sevenfold decreases in the proportion of IID/X and IIB fibers in both young and aging BN rats. These changes were accompanied by a twofold increase in the satellite cell content in both the young and aging groups; satellite cell content reached a level that was significantly higher in the young group (p <.04). The total muscle precursor cell content (i.e., satellite cells plus progeny), however, did not differ between groups, because there was a greater number of satellite cell progeny passing through the proliferative and differentiative compartments of the aging group. The resulting 1.5-fold increase in myonuclear content was similar in the young and aging groups. We conclude that satellite cells and satellite cell progeny of aging BN rats possess an unaltered capacity to contribute to the adaptive response.

Adaptive potentials of skeletal muscle in young and aging rats.

We compared responses of the fast extensor digitorum longus (EDL) and tibialis anterior (TA) muscles in young (15-week) and aging (101-week) male Brown Norwegian rats to 50 days of chronic low-frequency stimulation (CLFS, 10 Hz, 10 hours/day). After 50 days of CLFS, the EDL muscles of the young (22-week) and aging (108-week) rats displayed similar increases in type IIA fibers, relative concentration of myosin heavy chain MHCIIa, elevations in mitochondrial citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities, and similar decreases in glycolytic enzyme activities (glyceraldehydephosphate dehydrogenase, lactate dehydrogenase). TA muscle in young rats contained a few cytochrome c oxidase negative (COX-) type I fibers. Their number was approximately 2-fold elevated by CLFS. Conversely, aging muscle, which contained a slightly higher amount of COX- fibers than young TA muscle, responded to CLFS with a significant decrease in COX- fibers. The appearance of small COX-positive type I fibers in stimulated aging muscle indicated that regenerating type I fibers "diluted" the COX-deficient fiber population.

Identical responses of fast muscle to sustained activity by low-frequency stimulation in young and aging rats.

To investigate effects of sustained activity on major phenotypic properties, the left extensor digitorum longus muscle of young (15 wk) and aging (101 wk) male Brown Norway rats was subjected to 50 days of chronic low-frequency stimulation (CLFS; 10 Hz, 10 h/day). The contralateral muscle served as control. Changes in metabolic enzymes were analyzed by using glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase as reference enzymes of glycolysis and by using citrate synthase and 3-hydroxyacyl-CoA dehydrogenase as mitochondrial enzymes representative of aerobic-oxidative metabolism. Myosin heavy chain (MHC) isoforms were analyzed by SDS-PAGE. No differences existed between the enzyme activity profiles of control muscles from young and aging rats. CLFS induced similar increases in mitochondrial enzymes, as well as similar decreases in glycolytic enzymes. Although the MHC composition of the control muscles in the aging rats displayed a shift toward slower isoforms, the ultimate changes induced by CLFS led to nearly identical MHC phenotypes in both young and aging rats. These results demonstrate an unaltered adaptability of skeletal muscle to increased neuromuscular activity in the aging rat.

Sequential increases in capillarization and mitochondrial enzymes in low-frequency-stimulated rabbit muscle.

To investigate temporal changes in capillarization and increases in mitochondrial enzyme activity, rabbit tibialis anterior muscles underwent chronic low-frequency stimulation for up to 50 days. Capillary density (CD), capillary-to-fiber ratio (C/F), intercapillary distance (ICD), and mean capillary area (MCA), as well as several other parameters of capillarization, were examined. In addition, tissue levels of mRNA specific to vascular endothelial growth factor (VEGF) were assessed by reverse transcriptase-polymerase chain reaction. Citrate synthase (CS) activity, a marker of aerobic-oxidative metabolic potential, was measured in the same muscles. Significant increases in CD and C/F, respectively, and decreases in ICD and MCA were observed after 2 days. These changes reached stable maxima by 14 days. The increases in capillarization occurred in a fiber-type-specific manner, affecting type IId fibers before types IIda and IIa. VEGF mRNA levels increased in a bimodal time pattern with a first elevation (2.5-fold) after 1 day and a second (9-fold) after 6-8 days. Increases in CS were first noted after 8 days. Obviously, increases in capillarization as induced by enhanced contractile activity precede increases in the aerobic-oxidative potential of energy metabolism.

Microphotometric determination of structure-bound oxidoreductase activities avoiding nothing-dehydrogenase artefacts: succinate dehydrogenase.

A tetrazolium-based microphotometric method has been devised for the determination of structure-bound dehydrogenase activities with correction for nothing-dehydrogenase artefacts. The method is based on the microphotometric recording of maximum reaction rates in a simple incubation chamber and consists of two successive measurements on the same section, the first in the absence and the second in the presence of the substrate. Following the first measurement, the substrate-free medium is quickly exchanged with the substrate-containing medium and a second measurement is taken. Subtraction of the first from the second reaction rate yields the enzyme activity corrected for nothing-dehydrogenase. Measurements of succinate dehydrogenase (SDH) in skeletal muscle fibres, liver, cardiac atrium and ventricle demonstrate the feasibility of the method. Measurements on the extensor digitorum longus muscle of rat reveal a range of up to fivefold differences in SDH activity within the fibre population of this muscle.

Time-dependent increase of succinate dehydrogenase activity in low-frequency stimulated rabbit muscle: a comparison between microphotometric and biochemical methods.

We present an improved method for microphotometric measurement of enzyme activity in muscle fibres by determining maximum reaction rates using computer-assisted image analysis. The method was used to determine absolute and relative activities of succinate dehydrogenase (SDH) in 4801 whole-fibre cross-sections of rabbit tibialis anterior muscles stimulated at low frequency (10 Hz) for different time periods of up to 50 days. Measurements were performed on cross-sections of composite blocks from stimulated and contralateral control muscles. The validity of the method was checked by determining SDH activity in homogenates of the same muscles using a standard photometric assay. Both methods yielded similar results for the time-dependent increases of SDH activity in response to chronic low-frequency stimulation. Significant increases in catalytic activity were detected by the two methods only in muscles stimulated for longer than 8 days. According to homogenate measurements, overall SDH activity was 7.4-fold elevated in 50-day-stimulated total muscles. Depending on whether or not measurements were corrected for the so-called nothing-dehydrogenase activity, the average increase in microphotometrically determined SDH activity amounted to approximately 8-fold or 10-fold, respectively. Microphotometry revealed pronounced scattering of SDH activities within the fibre populations of both normal and stimulated muscles. The heterogeneity of the fibre population with regard to SDH activity increased in long-term stimulated muscles ranging between 5-fold and 15-fold elevations.

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits.

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low-frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy-chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low-frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G1 and G4 molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G1 form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low-frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation.