Risk based approach towards more sustainability in European pig production

Major aim of this thesis was to demonstrate how the use of a HACCP concept, a risk based approach, improves the sustainability of added value chains in European pig production. The thesis is featured as a pseudo-cumulative work with general introduction and conclusions and five independent chapters. Sustainability comprises nine themes. This thesis concentrates on animal health, meat quality and meat safety. 127 pig producing farms from five European countries and 15 different farming systems were investigated to design a catalogue proposing checklists for sustainability evaluation. This catalogue was used to assess farm specific risks in relation to animal health and meat safety, using principal component analysis. Only in the case of low risks due to diseases and failures of management, pig producing farms and the whole meat production chain can be categorized as sustainable. High intra- and inter-system differences of present risks were identified by this procedure. A combination of results from audits based on checklists and results from monitoring measures increase the certainty of risk assessment. A method for a continuous control and management of these sustainability aims was developed based on the principles of the HACCP concept. Unspecific and sensitive inflammatory markers take a key role regarding these monitoring measures. The innate immune system is affected by many factors like lesions, diseases, infections and permanent psychological stress and responds by increased concentrations of so called acute phase proteins. During a life cycle study with 99 pigs from rearing to slaughter, resulting in a data set of more than 18000 individual data records, these indicators of increased risks were investigated in detail. The correlation analyses of serum concentrations of these indicators measured at an age of 13 weeks presented the most significant coherences with parameters of meat and carcass quality. A direct coherence of the sustainability themes animal health and meat quality was proved for the first time. The risk of organ abnormalities was 16 times higher in cases of increased serum concentrations of one of these indicators measured directly before slaughter. The results proved these indicators in combination with further information to improve efficiency in terms of risk assessment and attendant measures. Implementation to practice was supported by the development of a rapid measurement method for the indicators based on a biosensor system.Risikobasierter Ansatz zur Steigerung der Nachhaltigkeit der Europäischen Schweineproduktion Das übergeordnete Ziel der Arbeit war es, zu demonstrieren, wie durch das HACCP Konzept als Risiko basierter Ansatz für die gesamte Wertschöpfungskette die Nachhaltigkeit der Europäischen Schweineproduktion gesteigert werden kann. Die Arbeit ist als pseudo-kumulative Schrift gestaltet mit allgemeiner Einleitung und Zusammenfassung sowie fünf in sich geschlossenen Publikationen. Nachhaltigkeit umfasst neun Themengebiete, von denen im Rahmen dieser Arbeit schwerpunktmäßig Tiergesundheit, Fleischqualität und Lebensmittelsicherheit bearbeitet wurden. Zur Gestaltung eines Checklistenkatalogs standen insgesamt 127 Schweine haltende Betriebe aus 5 EU Ländern und 15 verschiedenen Haltungssystemen zur Verfügung. Der entwickelte Checklistenkatalog ermöglichte es, unter Anwendung der Hautkomponentenanalyse, betriebsspezifische Risiken für die Gesundheit der Tiere zu bewerten und die Sicherheit, der von diesen Tieren stammenden Lebensmittel, einzuschätzen. Nur, wenn bezogen auf beide Bewertungskriterien die Risiken für das Auftreten von Krankheiten oder Managementfehler gering sind, können tierhaltende Betriebe sowie die gesamte Wertschöpfungskette Fleisch als nachhaltig eingestuft werden. Sowohl innerhalb als auch zwischen verschiedenen Haltungssystemen konnten sehr unterschiedliche Risikolagen identifiziert werden. Die Sicherheit der Risikobewertung wird durch eine Kombination von Checklisten-gestützten Audits mit Ergebnissen aus Monitoringmaßnahmen erhöht. Hier wurde eine Methode zur kontinuierlichen Überwachung und Steuerung dieser Nachhaltigkeitsziele in Anlehnung an die HACCP-Methode entwickelt. Eine zentrale Rolle im Rahmen eines Monitorings nehmen dabei unspezifische, sensitive Entzündungsmarker ein. Das Immunsystem reagiert auf eine Vielzahl von Faktoren, wie Verletzungen, Infektionen oder andauernden psychischen Stress, durch eine vermehrte Ausschüttung der sogenannten Akute Phase Proteine. Diese Risikoindikatoren wurden, im Rahmen eines Versuchs zur Beschreibung des Lebenszyklus von 99 Schweinen mit mehr als 18.000 erfassten Einzeldaten, weiter untersucht. Korrelationsanalysen der Serumkonzentrationen dieser Indikatoren wiesen im Lebensalter von 13 Wochen die meisten signifikanten Zusammenhänge zu Parametern der Fleisch- und Schlachtkörperqualität, die nach der Schlachtung bewertet wurden, auf. Es wurde erstmals nachgewiesen, dass zwischen den Nachhaltigkeitsaspekten Tiergesundheit und Fleischqualität ein direkter Zusammenhang besteht. Für das Auftreten von Organveränderung lag bei erhöhten Konzentrationen eines Indikators, ermittelt kurz vor der Schlachtung, ein 16fach erhöhtes Risiko vor. Es wurde gezeigt, wie diese Indikatoren in Verbindung mit Informationen aus Audits, zu einer Effizienzsteigerung im Rahmen der Risikobewertung und sich daran anschließenden Folgemaßnahmen genutzt werden können. Die Entwicklung einer Schnellmethode zur Messung der Risikoindikatoren, basierend auf einem Biosensorsystem, unterstützt eine mögliche Implementierung der entwickelten Methode

Extracellular Recordings of Field Potentials from Single Cardiomyocytes

AbstractOpen microfluidic channels were used to separate the extracellular space around a cardiomyocyte into three compartments: the cell ends and a central partition (insulating gap). The microchannels were filled with buffer solution and overlaid with paraffin oil, thus forming the cavities for the cell ends. The central part of the cardiomyocyte rested on the partition between two adjacent microchannels and was entirely surrounded by the paraffin oil. This arrangement increased the extracellular electrical resistance to >20MΩ and facilitated the recording of the time course of the change in extracellular voltage and current during subthreshold and suprathreshold stimuli. The waveform of the extracellular current and voltage in response to an extracellular depolarizing stimulus comprised an initial monophasic signal followed by a biphasic signal with a delay of 2–15ms. The latter was associated with a transient contraction and therefore caused by an action potential. The biphasic signal became monophasic after the depolarization of one cell end by raised extracellular [K+]. This form of differential recording revealed the repolarization phase of the action potential. At rest, the sarcomere length within the gap was 12%±4.8% longer than outside the gap, but intracellular Ca2+ transients occurred to the same extent as that observed in the outer pools. This data demonstrate the feasibility of the use of a microfluidic bath design to limit the extracellular resistance between two ends of an isolated cardiomyocyte

Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e ⩽ [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense

Imaging of Ca2+ transients induced in Paramecium cells by a polyamine secretagogue

In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (1 to 10 mM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent

Frustrated Exocytosis - a novel phenomenon : membrane fusion without contents release, followed by detachment and reattachment of dense core vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca2+]i transient and exocytosis of dense core vesicles (trichocysts'') in Paramecium cells, when applied at usual concentrations (h10 7m) in presence of extracellular Ca2+ ([Ca2+]o = 50 7m). When [Ca2+]o is kept at 30 nm

Caffeine-induced Ca2+ transients and exocytosis in paramecium cells : a correlated Ca2+ imaging and quenched-flow/freeze-fracture analysis

Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti 50 to 80 nm, [Ca2+]acti rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9 28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent ≥2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]ochelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge

Microfluidic systems to examine intercellular coupling of pairs of cardiac myocytes

In this paper we describe a microfluidic environment that enables us to explore cell-to-cellsignalling between longitudinally linked primary heart cells. We have chosen to use pairs (or doublets) of cardiac myocyte as a model system, not only because of the importance of cell–cellsignalling in the study of heart disease but also because the single cardiomyocytes are both mechanically and electrically active and their synchronous activation due to the intercellular coupling within the doublet can be readily monitored on optical and electrical recordings. Such doublets have specialised intercellular contact structures in the form of the intercalated discs, comprising the adhesive junction (fascia adherens and macula adherens or desmosome) and the connecting junction (known as gap junction). The latter structure enables adjacent heart cells to share ions, second messengers and small metabolites (<1 kDa) between them and thus provides the structural basis for the synchronous (syncytical) behaviour of connected cardiomyocytes. Using the unique environment provided by the microfluidic system, described in this paper, we explore the local ionic conditions that enable the propagation of Ca<sup>2+</sup> waves between two heart cells. We observe that the ability of intracellular Ca<sup>2+</sup> waves to traverse the intercalated discs is dependent on the relative concentrations of diastolic Ca<sup>2+</sup> in the two adjacent cells. These experiments rely upon our ability to independently control both the electrical stimulation of each of the cells (using integrated microelectrodes) and to rapidly change (or switch) the local concentrations of ions and drugs in the extracellular buffer within the microfluidic channel (using a nanopipetting system). Using this platform, it is also possible to make simultaneous optical recordings (including fluorescence and cell contraction) to explore the effect of drugs on one or both cells, within the doublet

Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca2+/(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca2+-influx via unspecific cation channels

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca2+] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) Extracellular La3+ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca2+]i transient (while injected La3+ causes a sustained fluorescence signal). (iii) Simply increasing [Ca2+]o causes a similar rapid, short-lived [Ca2+]i transient. All these phenomena, (i¨Ciii), are compatible with activation of an extracellular ¡°Ca2+/(polyvalent cation)-sensing receptor¡± known from some higher eukaryotic systems, where this sensor (responding to Ca2+, La3+ and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (¡°alveolar sacs¡±) are physically linked to the cell membrane and they can also be activated by the Ca2+ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca2+ signal also in absence of Ca2+o we largely exclude a ¡°Ca2+-induced Ca2+ release¡± (CICR) mechanism. Our finding of increased cortical Ca2+ signals after store depletion and re-addition of extracellular Ca2+ can be explained by a ¡°store-operated Ca2+ influx¡± (SOC), i.e., a Ca2+ influx superimposing store activation. AED stimulation in presence of Mn2+o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOCtype Ca2+ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca2+/polyamine-sensing receptor, (ii) release of Ca2+ from subplasmalemmal stores, (iii) and Ca2+ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca2+] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca2+] microdomains (¡Ü0.5 mm, ¡Ü33 msec) upon stimulation

Stimulation of Single Isolated Adult Ventricular Myocytes within a Low Volume Using a Planar Microelectrode Array

Microchannels (40-μm wide, 10-μm high, 10-mm long, 70-μm pitch) were patterned in the silicone elastomer, polydimethylsiloxane on a microscope coverslip base. Integrated within each microchamber were individually addressable stimulation electrodes (40-μm wide, 20-μm long, 100-nm thick) and a common central pseudo-reference electrode (60-μm wide, 500-μm long, 100-nm thick). Isolated rabbit ventricular myocytes were introduced into the chamber by micropipetting and subsequently capped with a layer of mineral oil, thus creating limited volumes of saline around individual myocytes that could be varied from 5 nL to 100 pL. Excitation contraction coupling was studied by monitoring myocyte shortening and intracellular Ca(2+) transients (using Fluo-3 fluorescence) . The amplitude of stimulated myocyte shortening and Ca(2+) transients remained constant for 90 min in the larger volume (5 nL) configuration, although the shortening (but not the Ca(2+) transient) amplitude gradually decreased to 20% of control within 60 min in the low volume (100 pL) arrangement. These studies indicate a lower limit for the extracellular volume required to stimulate isolated adult cardiac myocytes. Whereas this arrangement could be used to create a screening assay for drugs, individual microchannels (100 pL) can also be used to study the effects of limited extracellular volume on the contractility of single cardiac myocytes