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Nutztiere sind aufgrund ihrer Haltungsbedingungen diversen Stressoren ausgesetzt, welche ihre physische und psychische Gesundheit beeinträchtigen können. Im Rahmen von Animal Welfare Monitoring besteht daher ein großes Interesse an der Entwicklung und dem Einsatz minimal-invasiver Methoden und tierbezogener Belastungsindikatoren. Haarcortisol-konzentrationen (HCCs) könnten hierfür ein vielversprechender, retrospektiver Stressindikator sein, da sie die durchschnittlichen systemischen Cortisolkonzentrationen der letzten Wochen bis Monate in nur einer Probe widerspiegeln. Bevor HCCs jedoch als zuverlässiger Indikator eingesetzt werden können, müssen potenzielle Einflussfaktoren darauf ermittelt werden. Das generelle Ziel der vorliegenden Arbeit ist die Untersuchung von Einflussfaktoren auf Haarcortisol und seine Eignung für die Beurteilung von Langzeitstress bei Rindern und Schweinen. Daher zielten die vorliegenden Studien darauf ab, (1) Wissenslücken in der Forschung zu identifizieren, (2) die Wirkung potenzieller Einfluss- und Störfaktoren zu evaluieren und (3) zu untersuchen, ob und wann erhöhte systemische Cortisollevel durch HCCs nachweisbar sind. Alle Haarproben wurde von Holstein-Rindern, Landrasse- und Sattelschweinen bzw. Kreuzungstieren entnommen. Aufgrund der Literaturrecherche (Studie 1) wurden potenzielle Einflussfaktoren identifiziert und in den nachfolgenden Studien untersucht. Für die Evaluierung nicht-stressbedingter Faktoren (Studie 2), wurden insgesamt 614 Tiere verwendet. Hierfür wurden Haarproben in verschiedenen Altersstufen (Neugeborene bis Erwachsene), von beiden Geschlechtern und während der Sommer- und Wintersaison entnommen. Außerdem wurden schwarze und weiße Haarproben, unterschiedliche Körperregionen (Nacken/Schulter, Rücken, Schwanzspitze) und verschiedene Haarsegmente untersucht. Der Einfluss von Kontaminationen auf HCCs wurde in einer in-vitro-Studie (Studie 4) mit Haarproben von 12 Kühen und 12 Sauen evaluiert. Diese Proben wurden für vier Wochen täglich mit Urin, Speichel, Kot oder Wasser behandelt oder blieben unbehandelt. Um die Wirkung von Langzeitstress auf HCCs zu untersuchen (Studie 3), wurde bei 34 Rindern und 38 Jungsauen über vier Wochen jeden zweiten Tag ACTH- oder Kochsalzlösung appliziert. Vor, sowie zu drei Zeitpunkten nach Ende der Behandlung, wurden native und neu gewachsene Haare sowie zusätzlich Haarsegmente entnommen. Alle Haarproben wurden zweimal mit Isopropanol gewaschen, mit einer Kugelmühle gemahlen und Cortisol nach Extraktion mit Methanol mittels ELISA nachgewiesen. Die statistische Auswertung erfolgte mittels SAS/STAT-Software unter Anwendung von ANOVA und paarweisen Vergleichen durch Tukey-Kramer Tests. Die Ergebnisse der Studie 2 zeigen signifikant höhere HCCs bei neugeborenen Kälbern im Vergleich zu Jungrindern, Färsen und Kühen (p < 0,001). Ebenso wiesen 2 Wochen alte Ferkel höhere HCCs auf als Schweine im Alter von 10 oder 27 Wochen oder Sauen (p < 0,001). Das Geschlecht hatte keinen Einfluss auf die HCCs beider Tierarten. Jedoch waren bei beiden Spezies die HCCs in Schwanzhaaren, im Vergleich zu den Schulter-, Nacken- und Rückenhaaren signifikant erhöht (p < 0,001), ebenso in schwarzen Haaren im Vergleich zu weißen Haaren (p < 0,05) und in distalen im Vergleich zu proximalen Haarsegmenten (p < 0,001). Außerdem wiesen Rinder im Winter höhere HCCs als im Sommer auf (p < 0,001). Die Ergebnisse der Studie 4 zeigen, dass die Kontamination mit Urin bei beiden Spezies eine konzentrationsabhängige Zunahme der HCCs bewirkt. Auch die Kontamination mit Speichel und Kot erhöhte die HCCs, jedoch nur bei Rindern (alle p < 0,05). Die Behandlung mit Wasser führte zu einer Auswaschung von Cortisol aus Haaren vom Schwein, jedoch nicht vom Rind. Die Ergebnisse der ACTH-Studie (Studie 3) zeigen signifikant erhöhte HCCs bei ACTH-Tieren zum Ende der Behandlung in nativen Haaren (p < 0,001), nachgewachsenen Haaren (p < 0,01) und in Haarsegmenten (p < 0,05). Die höchsten HCCs wurden innerhalb von vier Wochen nach Behandlungsende gefunden. Bei Schweinen wurde ein Anstieg der HCCs sowohl in ACTH- als auch in Kontrolltieren beobachtet ohne dass Unterschiede zwischen den Behandlungen auftraten. Die Ergebnisse zeigen, dass die Cortisolkonzentrationen im Haar bei Schweinen und Rindern durch Alter, Körperregion, Haarfarbe, Haarsegment und Jahreszeit beeinflusst werden. Es gibt erste Belege, dass die Kontamination von Schweine- und Rinderhaaren mit cortisolhaltigen Körperflüssigkeiten, wie Urin und Speichel, die Aufnahme von externem Cortisol in den Haarschaft verursacht. Bei der Verwendung von HCC als Stressindikator sollten daher diese Einflussfaktoren standardisiert und Kontaminationseffekte vermieden werden, z.B. durch die Verwendung von Aufwuchsproben oder nur proximaler Haarsegmente. Darüber hinaus konnte gezeigt werden, dass Langzeitstress durch wiederholte Aktivierung der HPA-Achse zu erhöhten Cortisolkonzentrationen im Haar führt. Bei Rindern zeigt sich dies in den HCCs verschiedener Haarprobentypen. Insgesamt erweist sich die Analyse von Cortisol im Haar als eine geeignete Methode zum Nachweis von Langzeitstress bei Rindern und Schweinen und könnte daher eine wichtige Komponente bei der Beurteilung von Animal Welfare sein.:1 General introduction 2 Review of the literature 2.1 The relevance of stress assessment in animal welfare 2.2 Stress response in mammals 2.2.1 Structure and function of the hypothalamic-pituitary-adrenal axis 2.2.2 Characteristics and effects of cortisol 2.2.3 Conventional biological matrices for cortisol analysis 2.3 Hair as a matrix for cortisol analysis 2.3.1 Hair structure and hair types 2.3.2 Hair growth cycle 2.3.3 Incorporation of cortisol into the hair 2.3.3.1 Passive diffusion 2.3.3.2 Multi-compartment model 2.3.4 Elimination of cortisol from the hair 2.3.5 Specific characteristics and applications of hair cortisol 3 Research focuses and aims 4 Results 4.1 Study 1: Hair cortisol for the assessment of stress (review) 4.2 Study 2: Effects of animal-based, seasonal and hair-specific factors on hair cortisol concentrations 4.3 Study 3: Effects of long-term stress on hair cortisol concentrations 4.4 Study 4: Effects of contamination and elimination on hair cortisol concentrations 5 General discussion 5.1 Influencing factors on hair cortisol concentrations in cattle and pigs 5.1.1 Impact of animal-based, seasonal and hair-specific factors 5.1.2 Impact of contamination and elimination by washout 5.1.3 Implications 5.2 Hair cortisol concentration as an indicator of long-term stress in cattle and pigs 5.2.1 Model for the increased release of systemic cortisol 5.2.2 Models for the time course of cortisol incorporation into the hair shaft 5.2.3 Impact of hair sample type and sampling time 5.2.4 Implications Table of contents 5.3 Future perspectives 5.4 Conclusions 6 Summary 7 Zusammenfassung 8 References 9 Danksagung / Farm animals can be exposed to various stressors due to their husbandry conditions, which can impair their health and welfare. Thus, there is interest in the use of minimally invasive methods and animal-based stress indicators as part of welfare assessment. Cortisol in hair is a promising retrospective stress indicator, as a sample reflects systemic cortisol levels of the past weeks or months. Previous studies have shown that long-term stress with elevated cortisol release can be related to increased cortisol incorporation into the hair shaft. However, potential influencing factors that may affect hair cortisol concentrations (HCCs) must be determined before HCC can be applied as a reliable indicator of stress. The general objectives of this thesis are to investigate influencing factors on HCC, and to examine the potential of hair cortisol concentration as an indicator of long-term stress in cattle and pigs. Thus, the present studies aimed to (1) identify knowledge gaps in hair cortisol research, (2) evaluate the impact of animal-based, seasonal and hair-specific factors as well as contamination and elimination on HCC, and (3) investigate whether and when long-term increased systemic cortisol levels are reflected in elevated HCCs. Hairs were sampled from Holstein Friesian cattle, Landrace or Saddleback pigs and crossbreeds. The findings of the literature review (Study 1) identified potential animal-based, seasonal, hair-specific and stress-related factors on HCCs, which our experimental studies considered. To examine the impact of influencing factors (Study 2), a total of 614 animals were used. Hair samples were taken at different ages (newborn to adult), from different sexes and during both summer and winter. Variations by hair-specific factors were determined by studying black and white hair samples, varying body regions (neck/shoulder, back and tail tip) and different hair segments. In general, female animals were used. The effect of contamination on HCCs was examined in an in vitro study (Study 4) using hair samples from 12 cows and 12 sows. Samples were treated daily with urine, saliva, faeces or water for four weeks or remained untreated. To investigate long-term stress (Study 3), 34 cattle and 38 gilts were injected intramuscularly either with ACTH solution or saline every second day for four weeks. Natural and regrown hair samples were taken before and three times after the end of treatment, and hair segments were collected. All the hair samples were shaved with electric clippers, washed twice with isopropanol and ground with a ball mill. Cortisol was detected by ELISA after extraction with methanol. Statistical analyses were performed using ANOVA and pairwise comparisons of the least square means by Tukey-Kramer tests with the MIXED procedure in SAS/STAT software. The results of Study 2 showed significantly higher HCCs in newborn calves than in young cattle, heifers and cows (p < 0.001). Likewise, 2-week-old piglets had higher HCCs than pigs aged 10 or 27 weeks and sows (p < 0.001). Sex had no effect on HCCs in pigs or cattle. In both species, HCCs were also significantly higher in samples obtained from the tail tip than from the shoulder, neck and back regions (p < 0.001), in black hair than in white hair (p < 0.05) and in distal hair than in proximal hair segments (p < 0.001). Season had an impact on HCC only in cattle, which exhibited higher levels in winter than in summer (p < 0.001). The results of Study 4 showed that contamination with urine caused a considerable concentration-dependent increase in HCCs in both species. Contamination with saliva and faeces also raised HCCs, but only in cattle (all p < 0.05). Treatment with water washed cortisol out from porcine hair but not from bovine hair. In cattle, repeated ACTH application (Study 3) revealed significantly higher HCCs after the end of treatment in natural hair (up to eight weeks, p < 0.001), regrown hair (up to four weeks, p < 0.01) and segmental hair (eight weeks, p < 0.05) than in the control animals. The highest HCCs were found four weeks after the end of treatment. In pigs, elevated HCCs were observed in both ACTH and control animals in all hair sample types after the application period, with no differences between treatments. These results show that hair cortisol concentrations in pigs and cattle are affected by age, body region, hair colour, hair segment and season. There is first evidence that contamination of porcine and bovine hair with cortisol-containing body fluids, such as urine and saliva, may cause the incorporation of external cortisol into the hair shaft. Thus, when using HCC as a potential stress indicator, these influencing factors should be standardised and contamination effects should be avoided, such as by using the shave-reshave procedure, clean sampling regions and only the most proximal hair segments. The results also demonstrated that long-term stress by repeated activation of the HPA axis increases hair cortisol concentrations. In cattle, HCCs in different hair sample types reliably reflected the preceding period with increased systemic cortisol levels. In conclusion, the analysis of HCC appears to be a suitable method to evaluate long-term stress in cattle and pigs and can therefore be an important component in the assessment of animal welfare.:1 General introduction 2 Review of the literature 2.1 The relevance of stress assessment in animal welfare 2.2 Stress response in mammals 2.2.1 Structure and function of the hypothalamic-pituitary-adrenal axis 2.2.2 Characteristics and effects of cortisol 2.2.3 Conventional biological matrices for cortisol analysis 2.3 Hair as a matrix for cortisol analysis 2.3.1 Hair structure and hair types 2.3.2 Hair growth cycle 2.3.3 Incorporation of cortisol into the hair 2.3.3.1 Passive diffusion 2.3.3.2 Multi-compartment model 2.3.4 Elimination of cortisol from the hair 2.3.5 Specific characteristics and applications of hair cortisol 3 Research focuses and aims 4 Results 4.1 Study 1: Hair cortisol for the assessment of stress (review) 4.2 Study 2: Effects of animal-based, seasonal and hair-specific factors on hair cortisol concentrations 4.3 Study 3: Effects of long-term stress on hair cortisol concentrations 4.4 Study 4: Effects of contamination and elimination on hair cortisol concentrations 5 General discussion 5.1 Influencing factors on hair cortisol concentrations in cattle and pigs 5.1.1 Impact of animal-based, seasonal and hair-specific factors 5.1.2 Impact of contamination and elimination by washout 5.1.3 Implications 5.2 Hair cortisol concentration as an indicator of long-term stress in cattle and pigs 5.2.1 Model for the increased release of systemic cortisol 5.2.2 Models for the time course of cortisol incorporation into the hair shaft 5.2.3 Impact of hair sample type and sampling time 5.2.4 Implications Table of contents 5.3 Future perspectives 5.4 Conclusions 6 Summary 7 Zusammenfassung 8 References 9 Danksagung

Despite an abundance of archaeological material recovered from sites in Island Southeast Asia, the timing and route by which cultigens first arrived in Wallacea remains unclear. Many of the staple crops now grown on these islands were domesticated in mainland Asia, and were deliberately introduced by humans at an unknown point during the Holocene, through several possible routes. In this study, the δ 13C, δ15N and δ18O values of subfossil bones and teeth attributed to Sus celebensis and Sus scrofa are analyzed. These materials, which span the last 5160 years at Liang Bua, Flores, Indonesia are used to determine if and when there was a shift towards agricultural intensification, and whether this intensification included the integration of domesticated C4 crops. The δ13C and δ15N values of the bone and dentin collagen samples indicate an abrupt shift towards enrichment in 13C and depletion in 15N at some time between 5160 and 2750 yBP. This hints at changes in human subsistence patterns that may have included the clearing of forests, and the integration of nonendemic C4 cultigens such as foxtail millet (Setaria italica) onto the island. No statistically significant variation in the δ 18O values of the enamel carbonate samples over time is observed, suggesting that once they appeared on Flores, semidomesticated pigs became an important part of the island ecosystem, and were bred and raised on Flores instead of being continuously imported from elsewhere.

Accessing the inner ear presents a significant challenge for the diagnosis and treatment of inner ear diseases. Many existing techniques to access the inner ear are invasive and can cause permanent damage to the cochlea. Recently, a novel microneedle has been fabricated to perforate the round window membrane (RWM) – a membrane sealing one of the two openings in the cochlea. These perforations enhance drug delivery into the inner ear, potentially improving the efficacy of therapeutics. Furthermore, they allow for the aspiration of perilymph samples, which is essential for diagnosing inner ear diseases. However, owing to limited knowledge about the mechanical properties of the RWM, certain technical aspects remain unexplored. Specifically, the interaction between the RWM and the microneedle during perforation is yet to be examined. This investigation is pivotal for the optimal design of microneedles — those robust enough to perforate RWMs yet delicate enough to minimize damage. In this thesis, we conduct a thorough examination of the guinea pig RWM, encompassing its geometry and its mechanical responses to pressures from the middle ear and inner ear. Additionally, we also formulate a comprehensive constitutive law for the guinea pig RWM. Our exploration begins with the creation of a U-Net model tailored to automatically segment the RWM. Despite the presence of other structures in the same image—such as bone, the basilar membrane, and ambient noise—the model proved invaluable for efficiently and automatically segmenting the RWM. To enhance accuracy, post-processing techniques like connected component analysis and majority voting were incorporated. Using this 3D model, we proceeded to study the RWM’s geometry. Recognizing the shrinkage observed in fixed RWMs, we integrated fresh RWM data to estimate the shrinkage ratio. Subsequently, we analyzed both the overall RWM thickness and that of the middle connective tissue layer—crucial metrics for future RWM modeling. Next, we proposed a method to evaluate the in-plane deformation of the RWM due to applied pressure. This involved using a bulge test system to pressurize and deform the RWM, combined with confocal microscopy to track stained nuclei or pre-introduced fluorescent beads on the RWM. We then utilized the coherent point drift (CPD) algorithm to measure the displacement of beads and nuclei. Results indicated that both markers could be successfully used to measure the RWM’s displacement. Further analysis revealed the in-plane Lagrangian strain of the RWM, with a significant observation being that the direction of maximum in-plane Lagrangian strain is perpendicular to the fiber direction. This underscores the crucial role of collagen fibers in determining the RWM’s mechanical properties. To conclude our study, we devised a constitutive law for the RWM, conceptualizing it as a combination of the ground substance and a family of dispersed fibers. This model was integrated into a FEBioStudio plugin, facilitating simulations of the RWM’s mechanical reactions to different pressures. Although our simulations closely aligned with experimental findings, some discrepancies were noted, likely stemming from an incomplete understanding of fiber dispersions. Nevertheless, our constitutive law reinforces the notion that fibers primarily govern the RWM’s mechanical characteristics.

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Tesis por compendio / [ES] Actualmente, la sostenibilidad del sector porcino depende de su capacidad para responder a la elevada demanda de productos ganaderos derivada del crecimiento de la población, adaptándose a los cambios en los contextos económico y político, y mejorando su rendimiento medioambiental mediante la mitigación de su impacto ambiental. En este contexto, el uso de subproductos agroindustriales ofrece materias primas alternativas en producción animal, con una menor carga ambiental asociada, en forma de piensos para el ganado, fuente de compuestos bioactivos o materias primas útiles en la producción de bioenergía. Esta tesis doctoral pretende evaluar el uso de subproductos agroindustriales mediterráneos como ingredientes en piensos para el ganado porcino o como co-substratos para la producción de biogás. Con este objetivo, se diseñaron y realizaron cuatro ensayos para evaluar el uso de subproductos de la industria del aceite de oliva y del zumo de naranja en alimentación porcina, evaluando su valor nutricional y las consecuencias de su inclusión sobre el rendimiento y la salud de los animales, la calidad del producto final y las emisiones de gases de los purines. Además, se realizó un ensayo para evaluar el efecto de cuatro sustratos agrícolas sobre el potencial bioquímico de metano (BMP) en co-digestión anaerobia con purines. Los resultados obtenidos a partir de los ensayos de valor nutricional indican que las tortas de aceituna y las pulpas de naranja ensayadas pueden ser incluidas en la dieta con cambios asociados en la excreción de nutrientes que conducen a modificaciones en las emisiones potenciales de amoníaco y BMP de los purines. En cuanto a los subproductos de la torta de aceituna, el ensayo de valor nutricional con tortas de aceituna crudas (COC) y parcialmente desgrasadas (PDOC) mostró que ambas tortas son fuentes apreciables de fibra insoluble, pero tienen un valor energético limitado (11.2 y 7.4 MJ/kg MS para COC y PDOC respectivamente) y un bajo valor como fuente de proteínas. En cambio, las pulpas de naranja deshidratadas (DOP) y ensilada secada al sol (ESDOP) ensayadas son una fuente de energía relevante (14.2 y 13.2 MJ/kg MS para DOP y ESDOP respectivamente) con valor añadido debido a su contenido en fibra soluble. En los ensayos de emisiones in vitro, los subproductos ensayados generaron una disminución en la excreción de N en la orina y, en el caso de la pulpa de aceituna, un aumento de la excreción de materia seca en heces. La emisión de amoniaco por kg de purín disminuyó con la inclusión de torta de aceituna y pulpa de naranja, mientras que el BMP por animal y día se vio negativamente afectado por la inclusión de torta de aceituna obteniendo un mayor BMP con estos subproductos. En cuanto a los ensayos de rendimientos productivos, la PDOC y la DOP pueden incluirse en la dieta hasta 120 y 240 g/kg respectivamente, sin efectos negativos en el caso de la PDOC y efectos menores para la DOP sobre los rendimientos productivos, la composición corporal y la calidad de la canal. La inclusión de PDOC y DOP no afectó a los recuentos microbianos ni al volumen, la composición y la emisión global de gases de los purines. Además, se observaron efectos beneficiosos sobre la grasa subcutánea con la inclusión de PDOC, mejorando su concentración en ácido oleico. La co-digestión anaerobia de subproductos agrícolas y purines mejora el BMP de la mezcla de sustratos en comparación con la digestión única de purines. Se obtuvieron mayores valores de BMP con la adición de los sustratos agrícolas, lo que confirma el mejor rendimiento de los sistemas en co-digestión a niveles de inclusión adecuados. Las combinaciones con tomate, pimiento y melocotón al nivel de inclusión 3 (50% de SV) alcanzaron el mayor BMP. Esto supuso un incremento del BMP del 41% con tomate, 44% con pimiento, 28% con melocotón y 12% con caqui. Los sustratos vegetales mostraron un mayor contenido en lípidos, prote / [CAT Actualment, la sostenibilitat del sector porcí depèn de la seua capacitat per a respondre a l'elevada demanda de productes ramaders derivada del creixement de la població, adaptant-se als canvis en els contextos econòmic i polític, i millorant el seu rendiment mediambiental mitjançant la mitigació del seu impacte ambiental. En aquest context, l'ús de subproductes agroindustrials ofereix matèries primeres alternatives en producció animal, amb una menor càrrega ambiental associada, en forma de pinsos per al bestiar, font de compostos bioactius o matèries primeres útils en la producció de bioenergia. Aquesta tesi doctoral pretén avaluar l'ús de subproductes agroindustrials mediterranis com a ingredients en pinsos per al bestiar porcí o com co-substrats per a la producció de biogàs. Amb aquest objectiu, es van dissenyar i realitzar quatre assajos per a avaluar l'ús de subproductes de la indústria de l'oli d'oliva i del suc de taronja en alimentació porcina, avaluant el seu valor nutricional i les conseqüències de la seua inclusió sobre el rendiment i la salut dels animals, la qualitat del producte final i les emissions de gasos dels purins. A més, es va realitzar un assaig per a avaluar l'efecte de quatre substrats agrícoles sobre el potencial bioquímic de metà (BMP) en co-digestió anaeròbia amb purins. Els resultats obtinguts a partir dels assajos de valor nutricional indiquen que les trotes d'oliva i les polpes de taronja assajades poden ser incloses en la dieta amb canvis associats en l'excreció de nutrients que condueixen a modificacions en les emissions potencials d'amoníac i BMP dels purins. Quant als subproductes de la torta d'oliva, l'assaig de valor nutricional amb tortes d'oliva crues (COC) i parcialment desengreixades (PDOC) va mostrar que totes dues coques són fonts apreciables de fibra insoluble, però tenen un valor energètic limitat (11.2 i 7.4 MJ/kg MS per a COC i PDOC respectivament) i un baix valor com a font de proteïnes. En canvi, les polpes de taronja deshidratades (DOP) i ensitjada assecada al sol (ESDOP) assajades són una font d'energia rellevant (14.2 i 13.2 MJ/kg MS per a DOP i ESDOP respectivament) amb valor afegit a causa del seu contingut en fibra soluble. Pel que fa als assajos d'emissions in vitro, els subproductes assajats van generar una disminució en l'excreció de N en l'orina i, en el cas de la polpa d'oliva, un augment de l'excreció de matèria seca en femtes. L'emissió d'amoníac per kg de purí va disminuir amb la inclusió de torta d'oliva i polpa de taronja, mentre que el BMP per animal i dia es va veure negativament afectat per la inclusió de torta d'oliva obtenint un major BMP amb aquests subproductes. Quant als assajos de rendiments productius, la PDOC i la DOP poden incloure's en la dieta fins a 120 i 240 g/kg respectivament, sense efectes negatius en el cas de la PDOC i efectes menors per a la DOP sobre els rendiments productius, la composició corporal i la qualitat de la canal. La inclusió de PDOC i DOP no va afectar els recomptes microbians ni al volum, la composició i l'emissió global de gasos dels purins. A més, es van observar efectes beneficiosos sobre el greix subcutani amb la inclusió de PDOC, millorant la seua concentració en àcid oleic. La co-digestió anaeròbia de subproductes agrícoles i purins millora el BMP de la mescla de substrats en comparació amb la digestió única de purins. Es van obtenir majors valors de BMP amb l'addició dels substrats agrícoles, la qual cosa confirma el millor rendiment dels sistemes en co-digestió a nivells d'inclusió adequats. Les combinacions amb tomaca, pimentó i bresquilla al nivell d'inclusió 3 (50% de SV) van aconseguir el major BMP. Això va suposar un increment del BMP del 41% amb tomaca, 44% amb pimentó, 28% amb bresquilla i 12% amb caqui. Els substrats vegetals van mostrar un major contingut en lípids, proteïnes, lignina i cel·lulosa que els substrats de fruita. / [EN] Nowadays the sustainability of the pig sector relies on its capability to respond to the increasing demands for livestock products that are arising from population growth, adapting to changes in the economic and policy contexts, and improving its environmental performance through the mitigation of its impact on climate. In this framework, the use of the agro-industrial by-products offers potential alternative raw materials for animal production with a lower associated environmental burden in the form of feedstuffs for livestock, source of bioactive compounds or raw materials useful in bioenergy production. This PhD thesis aims to evaluate the use of Mediterranean agro-industrial by-products as feed ingredients for pigs or co-substrates for biogas production. To fulfil these objectives, four trials were designed and conducted to evaluate the use of olive oil and orange juice industry by-products in swine nutrition, assessing its nutritional value and the consequences of its inclusion in the diet on animals' performance and health, final product quality traits and gas emissions associated to the pig slurry. Additionally, one more trial was conducted to evaluate the effect of four agricultural substrates (tomato, pepper, peach and kaki) on the biochemical methane potential (BMP) in anaerobic co-digestion with pig slurry, focusing on the type of substrate and its inclusion level on the final substrate's mixture. The results presented in this PhD Thesis from the nutritional value assays indicate that the olive cakes and orange pulps tested can be potentially included in pig diets with associated changes in urine and faeces nutrients excretion that leads to modifications in the potential ammonia and BMP emissions from slurries. Concerning olive cake by-products, the nutritional value assay designed to test the crude (COC) and partially defatted (PDOC) olive cakes showed that they are appreciable sources of insoluble fibre but have limited energy value (11.2 and 7.4 MJ/kg DM for COC and PDOC respectively) and a low value as protein source. On the contrary, the dehydrated (DOP) and ensiled sun-dried (ESDOP) orange pulps tested are a relevant energy source (14.2 and 13.2 MJ/kg DM for DOP and ESDOP respectively) with added value in terms of SF concentration. With respect to the in vitro potential ammonia and BMP emissions assays, the by-products tested led to a decreased N excretion in urine and, in the case of the OC, increased DM excretion in faeces. The ammonia emission per kg of slurry decreased with the inclusion of olive cake and orange pulp, whereas the BMP per animal and per day was negatively affected by the inclusion of olive cake obtaining higher BMP with these by-products. Regarding the performance assays, the PDOC and the DOP may be included in balanced pig diets at rates of up to 120 and 240 g/kg respectively, without negative effects in the case of PDOC and minor effects for DOC on growth performance, body composition and carcass quality traits. Contrary to what was expected, the inclusion of PDOC and DOP did not affect microbial counts nor excreta volume, composition and global gas emission from the slurry. Additionally, beneficial effects on subcutaneous fat were observed with the inclusion of PDOC, improving its oleic acid concentration. The anaerobic co-digestion of agricultural by-products and pig slurry improves the BMP from the mixture compared to only pig slurry anaerobic digestion. Higher BMP values were obtained with increasing addition of agricultural substrate, confirming the better performance of co-digestion systems at adequate inclusion levels. In fact, combinations with tomato, pepper and peach at inclusion level 3 (50% of VS) achieved the highest BMP. This resulted in an increase in BMP of 41% with tomato, 44% with pepper, 28% with peach and 12% with kaki. Vegetable substrates (pepper and tomato) showed higher lipid, protein, lignin and cellulose content than fruit substrates (kaki and peach). / Ferrer Riera, P. (2021). Valorisation of Mediterranean agroindustrial by-products in pig production as feed and anaerobic co-digestion of slurry [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171747 / Compendio

The objectives of the research were to examine the effects of high dietary levels of fibrous feeds, and of supplementation with Roxazyme® G2 (RX), on the digestive metabolic and physiological responses of growing pigs fed maize-soybean diets. The nutrient and dietary fibre (DF) composition, the swelling and water-binding capacities of maize (MM), its hominy chop (HC) and cobs (MC), dehulled soybean (dSBM) and the hulls (SH), brewer’s grains (BG), lucerne hay (LH) and wheat bran (WB) were evaluated using standard procedures. Feed fibre fractions were isolated by simulating upper tract digestion in an Ankom® DaisyII Incubator, whereby each feed was digested in pepsin (porcine, 200 FIP-U/g, Merck No, 7190), followed by pancreatin (porcine, grade IV, Sigma No P-1750), with recovery of the fibrous residues. In a third step to complete the simulated pig gastro-intestinal digestion, the pepsin-pancreatin fibre extracts were digested by RX or Viscozyme L ® V2010 (VZ). Enzyme activity was measured as the coefficients of partial degradability (solubilisation) of the washed fibre extracts. The kinetics and products of fermentation of the DF were evaluated in an AnkomRF gas production system, using buffered faecal inoculum. Among the feed ingredients, dissimilar, fibre source-dependent activities between RX (0.02 to 0.12) and VZ (0.04-0.33) were observed. The lowest RX activities were observed on the maize and soybean derived fibres, with similarly low VZ activity on MC fibre. Variation in the activity of faecal microbial enzymes was similarly indicated by the variable production of fermentation gas (51.8-299.4 mL g-1 DM) and short chain fatty acids (SCFA) (2.3-6.0 mMol g-1 DM). Soy hull, dSBH, MM and HC fibres were highly fermentable, with low fermentability of BG, MC and WB fibres. The fibres differed in the composition of fermentation SCFA, whereby SH, LH and MC shifted fermentation to Ace, and BG, dSBM, WB, MM, HC favoured Pro, while MM and HC favoured But production. The same nutritional properties were similarly evaluated in complete diets which were formulated from the ingredients for growth, and metabolic trials. For the growth trial, a standard (STD) (control), 141 g total dietary fibre (TDF) kg-1 dry matter (DM) maize-soybean growing pig diet, and five iso-nutritive, 246 g TDF kg-1 DM nutritionally balanced diets were formulated. The high DF was achieved by partial replacement of the MM and dSBM in the STD diet with MC, SH, BG, LH or WB. The differences in RX and VZ activities and in the fermentation characteristics which were observed on the fibre extracts from the high fibre ingredients were reflected in the DF from the respective complete diets in which they were included. However, the fibre from the basal dietary ingredients reduced the absolute values and the variation in the activities of RX (0.03-0.06) and VZ (0.16-0.22), and similarly reduced the variation in gas (126.6-187.6 mL g-1 DM) and SCFA (4.1-5.4 mMol g-1 DM) production of the DF from the fibrous diets. Enzyme activities on the STD DF were low for RX (0.03) and high for VZ (0.25). The STD DF produced 205.3 mL gas g-1 DM, which was similar to SH DF, and higher than all the other diets. The STD DF produced 5.0-mMol SCFA g-1 DM, which was quantitatively, and not statistically higher than the other fibres. The composition of SCFA was similar across all diets, except for the high percent Ace, with low Pro by the SH DF. Compared to the STD, the high DF diets increased percent Ace, with reduced Pro and But. The STD, MC, SH, BG, LH and WB diets were each prepared in duplicate mixes, one of which was fortified with 200 mg RX kg-1 feed (as fed). Seventy-two intact Large White X Landrace, male, 32.0 ± 5.6 kg live weight (LW) pigs were allocated to the diets in two completely randomised weight blocks in a 2 (fibre source) X 2 (enzyme) factorial arrangement. The pigs were fed ad libitum for 10 weeks. Cumulative LW gain and feed intake were measured at different stages of growth, and at slaughter. Apparent total tract digestibility (ATTD) of nutrients was estimated at 65-70 kg LW, using 0.2% (as fed) chromium oxide as the indigestible marker. Ileal tissue was sampled 50 cm above the ileo-caecal valve, on which villi height and area, and crypt depth were evaluated by computerised image analysis. Blood was sampled at slaughter from the severed vena jugularis, 16 hours after feeding. Serum urea, creatinine, triglycerides, glucose, and total cholesterol were analysed chemically. The serum metabolome was further explored using Proton Nuclear Magnetic Resonance Spectroscopy (1H -NMRS). There was fibre X RX interaction for villi height, whereby the enzyme reduced the villi height in pigs on the SH, STD and WB diets, with an opposite effect on pigs on the MC, BG, LH diets. The soluble fibre content was negatively correlated with crypt depth. Chemical analysis did not detect differences in metabolite concentration between the STD and the high fibre diets. However, more serum cholesterol was observed in pigs fed the WB compared to the LH and MC diets. 1H-NMRS indicated that feeding pigs the WB diet increased serum Cys and His, while supplementation of RX increased serum formate, glucose, and urea. There was diet X enzyme interaction for fructose, glucose, Arg, Cys, Ser, and Trp, whereby RX increased the levels in pigs on MC and WB, with an opposite effect in pigs on the other diets. There was large DF source-dependent variation among diets in ATTD of DM (0.80-0.85), organic matter (OM) (0.81-0.87), gross energy (GE) (079-0.85) and CP (0.81-0.85), whereby, relative to the STD diet, high DF reduced the ATTD of DM (all diets except SH), organic matter (OM) and energy and CP (all diets except the MC). Positive correlation was observed between fermentability and the ATTD digestibility of DM, OM, energy, ADF, NDF, and fat. Negative correlation was observed between the swelling capacity and the ATTD of DM, OM, energy and protein, between DF solubility and DM, OM, protein, ADF and NDF, and between water binding capacity and ATTD of DM and OM, energy and NDF. At slaughter, there was similarly large, and DF source-dependent variation among the high fibre diets in feed intake (2.31-2.71 kg as fed day-1), live weight gain (0.75-0.86 kg day-1), and feed: gain ratio (2.73-3.00). Corresponding values for the STD diet were 2.44 kg day-1, 0.83 kg day-1and 2.86 kg day-1, respectively. Relative to the STD, LH reduced feed intake and live weight gain, and MC increased the feed: gain ratio. Predictions based on the in vitro fermentability of DF and feed intake suggested that due to poor fermentability, and or restriction of feed intake, relative to a standard fibre diet, high dietary levels of MC, WB and BG may reduce fermentation in the lower gut, while similar dietary levels of SH and LH may result in substantial increases in fermentation. At 50 kg LW, the fermentability of DF was positively correlated with feed intake and with weight gain, while water binding capacity and solubility of DF were negatively correlated with feed intake. At slaughter, the solubility of DF was negatively correlated with feed intake and feed: gain ratio. Large variation among the high fibre diets was also observed in the slaughter weight (89.2-96.8 kg), dressing % (68.6-76.4), meat colour (80.4-82.3), lean % (69.5-71.2), and fat % (10.1-12.6). In comparison, pigs on the STD diet scored 94.7 kg slaughter weight, 75.1% dressing, 81.6 cm carcass length, 82.5 meat colour, 68.4% lean, and 15.0% fat. Relative to the STD, LH reduced dressing and fat %. Lucerne hay and WB increased the lean%. For the metabolic trial, two iso-nutritive, mixed high fibre (319 g TDF kg-1 DM), nutritionally balanced diets were formulated to contain DF of high (HF) versus low (LF) fermentability. The diets had similar content of soluble DF and similar swelling and water binding capacities. Viscozyme was more active than RX on both the HF (0.20 versus 0.04) and the LF (0.17 versus 0.07) DF. The combination of RX and VZ statistically increased the enzyme activity on the HF (0.25) and quantitatively increased enzyme activity on the LF (0.18) DF, suggesting additive or synergistic effects. More gas was produced by the HF (159.5 mL g-1 DM) compared to the LF DF (96.6 mL g-1 DM). More SCFA were produced by HF (5.0 mMol g-1 DM), compared to the LF DF (3.6 mMol g-1 DM). Compared to the STD, HF DF increased percent Ace, with reduced Pro and But. The LF DF increased percent Ace, with quantitative, and not statistical reduction of Pro and But. In a metabolic trial, the HF and LF diets, and their duplicates containing 0.270 g RX kg-1 DM of feed (as fed) were fed ad libitum to eight ileum T-cannulised, intact Large White X Landrace male pigs weighing 65.0 ± 5.1 kg. The diets were allocated to the pigs in a duplicate 4 x 4 Latin Square design, in a 2 (enzyme) x 2 (fermentability) factorial arrangement. Each period consisted of two weeks of adaptation followed by five days of sampling. The ileal digesta was collected in each period and was similarly subjected to the fermentation test. Apparent ileal digestibility (AID) and ATTD were determined using 0.2% (as fed) chromium oxide as the indigestible marker. N excretion in faeces and urine were measured, and N retention was calculated. Blood was sampled by vena jugularis puncture on the last day of each period. Two blood samples were collected, the first 15 hours after removal from feed (15-hour serum), and the second 3 hours after re-introduction to feed (3-hour serum). Serum metabolites were evaluated by both chemical analyses and by 1H-NMRS, as described for the growth trial. Roxazyme did not affect the fermentation characteristics of the ileal digesta. In similar proportion to the fermentability of the PP digesta, the HF ileal digesta was more fermentable (65.4 mL gas g-1 DM and 6.1 mMol SCFA g-1 DM) than the LF ileal digesta (46.7 mL gas g-1 DM and 4.4 mMol SCFA g-1 DM SCFA). Prediction based on the in vitro fermentability of DF and feed intake suggested the HF diet could support one half times more fermentation in the lower gut compared to the LF diet. The HF diet had higher AID of DM (62.5 vs. 58.6), OM (65.6 vs. 62.1), energy (64.4 vs. 61.0), fat (85.8 vs. 81.7) and ash (41.8 vs. 32.7). The AID of HO-Pro, Met and Val were higher for the LF diet. There was diet X enzyme interaction on the AID of Met, whereby the RX reduced the AID of met in the LF diet, and not that of the HF diet. The ATTD was higher for the HF diet for DM (74.2 vs. 68.4), NDF (64.7 vs. 57.4), and ADF (35.1 vs. 21.0). There was positive correlation between the fermentability of DF and the AID DM, OM, ash, ash, fat and energy. The solubility of DF was negatively correlated with the AID of DM, OM, ash, fat, ADF and energy, and with the ATTD of DM, OM, ash, fat, energy, NDF, and ADF. Negative correlation was also observed between the swelling capacity of DF and the AID of protein, Trp and Lys. The solubility of DF was positively correlated with Ser, Ala, Val, Iso-Leu and His. There was diet X enzyme interaction for urea in the 15- hour serum, whereby RX tended to reduce the urea in the LF diet, while it increased that of the HF diet. Fermentability negatively correlated with urea in the 15- hour serum, and positively correlated with serum glucose in the 3-hour serum. In the 3-hour sample, 1H-NMRS indicated higher fucose, Pro and cholesterol in the LF diet. 1H-NMRS also indicated fermentability x RX interaction for Ser, Tyr, Lys, creatine, and possibly, glucose or fructose, glycerol or Gly and His or Arg, whereby RX increased the levels in the LF diets, with opposite effect in the HF diet. In conclusion, enzyme activities and fermentability were higly variable among different DF sources, and the effects were evident in the fibrous complete diets. The results of the in vitro studies supported the application of the methods to formulate fermentable insoluble fibre-rich, maize-soybean-mixed co-product diets. Correlation analyses suggested that DF fermentability, and solubility, swelling and water binding capacities explained significant proportions of the variances of the metabolic and physiological responses of the pigs to different feeds. Predictions based on the in vitro fermentability of DF and feed intake suggested that a strategy whereby pig diets are enriched in DF after the feedstuffs are screened on DF fermentability could substantially increase fermentation in the lower gut. Overall, the results suggested that productivity can be maintained in growing pigs fed diets containing up to twice the standard levels of DF, provided producers target co-product feeds that contain highly fermentable DF. The use of RX to improve nutrient digestion and to stimulate gut fermentation was not justified. / Environmental Sciences / Ph.D. (Environmental Sciences)

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2005. / The demand for protein for human and animal nutrition in South Africa is increasing and it will continuously increase. The effect of replacing soybean oilcake meal as protein source for broilers and full-fat soybean meal for weaner pigs, with different levels of sweet lupins (Lupinus angustifolius), canola oilcake and full-fat canola was examined. A basal diet with soybean oilcake as protein source was mixed with a diet using either sweet lupins or canola oilcake or full-fat canola in ratios of 100%, 67% and 33% respectively. In the broiler trial the test diets were fed for a period of six weeks with or without the enzyme Vegpro (Alltech). Pigs were fed the test diets, with and without Roxazyme® enzyme, ad libitum from 8.5 to 25 kg live weight. The fatty acid content of the fat pads of the broilers raised on the different diets was determined. The pigs were kept in the trial up to the grower- finisher phase. The fatty acid content of the carcass fat and muscle of pigs raised on the different diets was determined. The inclusion of enzymes had no effect on the growth, feed intake or feed conversion ratio of broilers fed the test diets. The provision of external dietary enzymes to the weaner pig diets failed to improve either dry matter intake or growth rate, but improved the feed conversion ratio. Broiler weights at six weeks of age were significantly higher for the control diet compared to the 20% lupin diet. There was no significant difference in the feed intake as the lupin content of the diets increased. The feed conversion ratio did not differ significantly between the control diet and the 6.6% lupin diet but became significantly poorer as the lupin content increased to 13.2% and 20% of the test diet. There were no significant differences in production performance of the control diet and the canola oilcake containing diet. The broiler weights at six weeks decreased significantly with each increase in the canola oilcake content of the diets. The feed intake of the 20% canola oilcake diet at week six was significantly less than the intake of the control diet, but not significantly less than the 6.6% and 13.2% canola oilcake diets. The feed conversion ratio of the control diet was significantly better than the 13.2% and 20% canola oilcake diets. No significant differences were found in week six between the 6.6% full-fat canola diet and the control diet for broiler weights and feed intake. The feed conversion ratio of the broilers fed the 13.2% and 20% full-fat canola diets was significantly poorer than the control diet. The final body weights of the weaner piglets fed the control diet were significantly higher than the final body weights of weaner piglets fed the lupin containing diets. The average daily gain of the weaner piglets fed the control diet was significantly higher than the gain of the weaner piglets fed the lupin containing diets. No significant differences in the feed intake and feed conversion ratio between the different lupin inclusion levels were detected. There were no significant differences in body weight, feed intake, average daily gain and the feed conversion ratio, between the various full-fat canola containing diets and the control diet. A significant difference in body weight was found between the weaner piglets fed the 20% canola oilcake diet and the weaner piglets fed the control diet. There were no significant differences in feed intake between the various inclusion levels of canola oilcake. The control and 6.6% canola oilcake containing diets had significantly higher average daily gains than the 20% canola oilcake containing diet. In the final trial the finisher pigs fed the test diet containing 25.00% lupins, had a final body weight significantly lighter than the final body weight of the finisher pigs fed the other test diets. The finisher pigs fed the test diet containing 25.00% lupins, also had a significantly reduced average daily gain and feed intake. The feed intake of the 25.00% canola oilcake diet was significantly lees than the feed intake of the 16.75% full-fat canola containing diet. The feed conversion ratio of the 25.00% lupin containing diet was significantly poorer than the feed conversion ratio of the 16.75% lupin containing diet, 8.25% lupin containing diet, 16.75% full-fat canola containing diet, 25.00% canola oilcake containing diet and the control diet. In a choice feeding trial growing pigs were offered four diets with four different protein sources: sweet lupins (25% inclusion level), canola oilcake (25% inclusion level), full-fat canola (25% inclusion level) and soybean oilcake (25% inclusion level), while their daily intakes were recorded. In a second choice feeding trial pigs were offered ten different diets with increasing levels (6.6%, 13.2%, 20%) of either sweet lupins, canola oilcake meal or full-fat canola meal. The pigs consumed significantly more of the soybean oilcake containing diet compared to diets containing the alternative protein sources. Pigs consumed significantly less of the full-fat canola diet compared to the sweet lupin and canola oilcake diets. Ten different canola cultivars were collected from two different locations in the Mediterranean rainfall area of South Africa namely the Western Cape (Swartland) and Southern Cape (Rûens) grain producing areas. The sinapine and glucosinolate content of various canola cultivars was compared and the influence of locality on the sinapine and glucosinolate content of the canola cultivars was determined. There were no significant differences (P < 0.05) in sinapine content when the canola produced in the Western and Southern Cape were compared. Varola 54 and Rainbow cultivars had significantly higher (P < 0.05) sinapine contents compared to the Varola 50 cultivar. Samples of lupins, field peas, faba beans and narbon beans were collected and analysed for amino acids, alkaloids, non-starch polysaccharides, tannin and starch. The digestible energy value of these alternative protein sources for pigs was determined. Significant differences were found in the amino acid content of the various crops. The alkaloid content of the lupins varied significantly between the sweet and bitter lupin varieties. Sweet L. angustifolius cultivars contained ca 50mg/kg and the bitter L. angustifolius cultivars ca 15000mg/kg alkaloids. The mean alkaloid content of L. albus cultivars was ca 1300mg/kg. The faba beans, narbon beans and peas had significantly higher values for tannins and starch, compared to lupins.

Toxoplasma gondii ist weltweit einer der häufigsten zoonotischen Parasiten. Der obligat in-trazelluläre Gewebeparasit hat ein breites Wirtsspektrum als Zwischenwirte. Der Mensch infiziert sich häufig durch orale Aufnahme von Gewebezysten aus rohem oder unzureichend erhitztem Fleisch. Schweine und Hühner als fleischliefernde Tiere stellen eine wichtige Infek-tionsquelle für den Menschen dar. Ziel der Arbeit war die Verteilung und Parasitenbelastung von T. gondii in verschiedenen Geweben von infizierten Schweinen und Hühnern mit Hilfe quantitativer real-time PCR auf Basis des 529-bp-Fragmentes zu bestimmen. Experimentell wurden 10 Schweine und 12 Hühner mit unterschiedlicher Infektionsdosen von Toxoplasma-Oozysten infiziert. Anhand der 529-bp-PCR waren 90% der untersuchten Schweine und >90% der untersuchten Hühner Toxoplasma-DNA-positiv. In Schweinen gelang der Nach-weis von Toxoplasma-DNA in der Oberschenkelmuskulatur mit 70% und Bauchmuskulatur mit 60% am häufigsten. Gehirn und Vorderbeinmuskulatur waren mit jeweils 40%, Herz mit 30% und Zunge mit 10% Toxoplasma-DNA-positiv. In experimentell infizierten Hühnern wur-de T. gondii-DNA am häufigsten in Oberschenkelmuskulatur, Brustmuskulatur und Gehirn mit jeweils 50% und im Herz mit ungefähr 20% nachgewiesen. Die Quantifizierung des Erre-gers in den T. gondii-positiven Gewebeproben ergab eine Parasitenanzahl von 0,1 bis 4,1 in 25 mg Schweinegewebe und von 0,1 bis 4,9 in 25 mg Hühnergewebe, die unabhängig von der Gewebeart war. Es wurde in dieser Arbeit auch eine Reverse Transkriptase real-time PCR zur Bestimmung der Viabilität von Parasiten in den Gewebe infizierter Schweine und Hühner durch Nachweis von T. gondii-mRNA etabliert werden. Die Sensitivität dieser Metho-de war geringer als die der real-time PCR für T. gondii-DNA und konnte in experimentell infi-zierten Schweinen und Hühnern keine lebenden Parasiten detektieren. Die Ergebnisse die-ser Arbeit zeigen, dass Muskulatur von Schweinen und Hühnern bevorzugte Orte für die Persistenz von T. gondii in diesen fleischliefernden Tieren darstellen. Das ist ein wichtiger Hinweis, dass bei Verzehr von rohem oder nicht ausreichend erhitztem Fleisch oder Fleisch-produkten dieser Tiere ein potenzielles Infektionsrisiko für den Menschen besteht.