Growth of shallot plants could be increased through good plant cultivation such as using organic materials that can improve physical, chemical and biological properties in the soil and contain macro and micronutrients so that organic matter is needed in the form of municipal waste compost. The research objective was to study the responsiveness of the growth of onion varieties due to the application of municipal waste compost. This research was carried out in the Bandar Senembah village Binjai district Barat in February-March 2019. The study used a randomized block design (RAK) with 2 factors and 3 blocks. The first factor is the variety (V) and the second factor is Municipal waste compost (K). The results showed that that the best varieties are varieties Bima Brebes. Where the variety showed the highest leaf length per sample and highest number of tillers per sample while the application of municipal waste compost does not show a significant effect on parameters of leaf length per sample but for the number of tillers per sample shows a significant effect where the best results in the application of 3 kg/m2 (plot) municipal waste compost. REFERENCES Ahmed, M. E., El-Kader, N. I. A. & Derbala, A.A.E. (2009). Effect of Irrigation Frequency and Potassium Source on the Productivity, Quality, and Storability of Garlic. Australian Journal Of Basic and Applied Sciences, 3(4), 4490–4497. Alfian, D. F., Nelvia & Yetti, H. (2015). The Effect of Potassium Fertilizer and Compost Mixture of Oil Palm Empty Bunches with Boiler Ash on Growth and Yield of Onion (Allium ascalonicum L.). Jurnal Agroekoteknologi, 5(2), 1-6. Amiroh, A. (2017). Pengaplikasian dosis pupuk bokashi dan KNO3 terhadap pertumbuhan dan hasil tanaman melon (Cucumis melo L.). Jurnal Saintis, 9(1), 25 - 36. Arisha, H. M. E.,. Ibraheim, S. K. A & El-Sarkassy, N. M. (2017). The response of garlic (Allium sativum L.) yield, volatile oil, and nitrate content to foliar and soil application of potassium fertilizer under sandy soil conditions. Middle East Journal of Applied Sciences, 7(1), 44-56. Aslamiah, I. D., dan Sularno. (2017). The response of growth and production of peanut plants of the addition of organic fertilizer concentration and reduction of an organic fertilizer dosage. Prosiding Seminas Nasional Fakultas Pertanian UMJ. BPS. (2018). Statistik Indonesia. Badan Pusat Statistik Republik Indonesia, Jakarta. Gunadi, N. (2009). Kalium sulfat dan kalium klorida sebagai sumber pupuk kalium pada tanaman bawang merah. Jurnal Hortikultura, 19(2),174-185. Hickey, M. (2012). Growing Garlic in NSW Second Edition. Primefact 259. Department of Primary Industries. NSW Government. Australia. Hilal, M.H., Selim, A.M. & El-Neklawy, A.S. (1992). Enhancing and retarding effect of combined sulfur and fertilizer applications on crop production in different soils. In Proceedings Middle East Sulphur Symposium 12-16 February, Cairo, Egypt. Marschner, P.( 2012). Mineral Nutrition of Higher Plants Third Edition. Elsevier Ltd. Oxford. Nainwal, R. C., Sigh, D., Katiyar, R. S., Sharma, I & Tewari, S. K. (2015). The response of garlic to integrated nutrient management practices in a sodic soil of Uttar Pradesh, India. Journal of Spices and Aromatic Crops, 24(1), 33-36. Putra, A. A. G. (2013). Kajian aplikasi dosis pupuk ZA dan kalium pada tanaman bawang putih (Allium sativum L.). Jurnal Ganec Swara, 7(2), 10–18. Setiawati, W., Murtiningsih, R., Sopha, G. A & Handayani, T. (2007). Petunjuk Teknis Budidaya Tanaman Sayuran. Balai Penelitian Tanaman Sayuran. Shafeek, M. R., Nagwa, M. H., Singer, S. M., & El-Greadly, N. H. (2013). Effect of potassium fertilizer and foliar spraying with Ethereal on plant development, yield, and bulb quality of onion plants (Allium cepa L). Journal of Applied Sciences Research, 9(2), 1140-1146. Sholihin, Y., Suminar, E., Rizky, W.H. & Pitaloka, G.G. (2016). Meristem explants growth of garlic (Allium sativum L.) Cv. tawangmangu on various compositions of kinetin and ga3 in vitro. Jurnal Kultivasi, 15(3), 172–179. Sulichantini, E. D. (2016). Effect of plant growth regulator Concentration Against Regeneration Garlic (Allium sativum L) In the Tissue Culture. Jurnal Agrifor, 15(1), 29–38. Suminarti, N.E. (2010). The Effects of N and K Fertilization on the Growth and Yield of Taro on Dry Land. Akta Agrosia, 13(1), 1–7. Uke, K. H. Y., Barus, H & Madauna, I. W. (2015). Effect of Tuber Sizes and Potassium Dosages on Growth and Production of Shallots var. Lembah Palu. Jurnal Agrotekbis, 3(6), 655 - 661. Utomo, P.S & Suprianto, A. (2019). Respon pertumbuhan dan produksi tanaman bawang merah (Allium ascalonicum L.) varietas thailand terhadap perlakuan dosis pupuk kusuma bioplus dan KNO3 putih. Jurnal Ilmiah Hijau Cendekia, 4(1), 28–34. Wu, C., Wang, M., Cheng, Z & Meng, H. (2016). The response of garlic (Allium sativum L.) bolting and bulbing to temperature and photoperiod treatments. Biol Open, 5(4), 507-518.
The purpose of this research was to assess the effect of different potassium doses and fertilizer sources on growth rate and time of bulb formation of garlic (Allium sativum L.). The research was conducted in April to Agustus 2019 in Sidomukti Village, Bandungan District, Semarang Regency and at Ecology and Crop Production Laboratory, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang. The research used a factorial randomized block design with three groups. The first factor was the dose of potassium fertilizer which consisted of a dose of 60 kg K2O/ha, 120 kg K2O/ha, 180 kg K2O/ha, and 240 kg K2O/ha. The second factor was the source of potassium fertilizer which consisted of KCl, ZK, and KNO3. Parameters that collect were time of bulb formation, growth rate, relative growth rate, and potassium absorption of bulb. The data obtained were analyzed by analysis of variance and obtained further by the Duncan test (Duncan's Multiple Range Test) at a significance level of 5%. The results showed that the application of ZK and KNO3 fertilizers at 240 kg K2O/ha had been able to increase the growth rate and the relative growth rate. The higher dose of fertilizer was increasing of potassium absorption of the bulb and made time of bulb formation getting slower. References Ahmed, M. E., El-Kader, N. I. A. & Derbala, A.A.E. (2009). Effect of Irrigation Frequency and Potassium Source on the Productivity, Quality, and Storability of Garlic. Australian Journal Of Basic and Applied Sciences, 3(4), 4490–4497. Alfian, D. F., Nelvia & Yetti, H. (2015). The Effect of Potassium Fertilizer and Compost Mixture of Oil Palm Empty Bunches with Boiler Ash on Growth and Yield of Onion (Allium ascalonicum L.). Jurnal Agroekoteknologi, 5(2), 1-6. Amiroh, A. (2017). Pengaplikasian dosis pupuk bokashi dan KNO3 terhadap pertumbuhan dan hasil tanaman melon (Cucumis melo L.). Jurnal Saintis, 9(1), 25 - 36. Arisha, H. M. E.,. Ibraheim, S. K. A & El-Sarkassy, N. M. (2017). Response of garlic (Allium sativum L.) yield, volatile oil, and nitrate content to foliar and soil application of potassium fertilizer under sandy soil conditions. Middle East Journal of Applied Sciences, 7(1), 44-56. Aslamiah, I. D., dan Sularno. (2017). Response of growth and production of peanut plants of the addition of organic fertilizer concentration and reduction of an organic fertilizer dosage. Prodising Seminas Nasional Fakultas Pertanian UMJ. BPS. (2018). Statistik Indonesia. Badan Pusat Statistik Republik Indonesia, Jakarta. Gunadi, N. (2009). Kalium sulfat dan kalium klorida sebagai sumber pupuk kalium pada tanaman bawang merah. Jurnal Hortikultura, 19(2),174-185. Hickey, M. (2012). Growing Garlic in NSW Second Edition. Primefact 259. Department of Primary Industries. NSW Government. Australia. Hilal, M.H., Selim, A.M. & El-Neklawy, A.S. (1992). Enhancing and retarding effect of combined sulfur and fertilizer applications on crop production in different soils. In Proceedings Middle East Sulphur Symposium 12-16 February, Cairo, Egypt. Marschner, P.( 2012). Mineral Nutrition of Higher Plants Third Edition. Elsevier Ltd. Oxford. Nainwal, R. C., Sigh, D., Katiyar, R. S., Sharma, I & Tewari, S. K. (2015). The response of garlic to integrated nutrient management practices in a sodic soil of Uttar Pradesh, India. Journal of Spices and Aromatic Crops, 24(1), 33-36. Putra, A. A. G. (2013). Kajian aplikasi dosis pupuk ZA dan kalium pada tanaman bawang putih (Allium sativum L.). Jurnal Ganec Swara, 7(2), 10–18. Setiawati, W., Murtiningsih, R., Sopha, G. A & Handayani, T. (2007). Petunjuk Teknis Budidaya Tanaman Sayuran. Balai Penelitian Tanaman Sayuran. Shafeek, M. R., Nagwa, M. H., Singer, S. M., & El-Greadly, N. H. (2013). Effect of potassium fertilizer and foliar spraying with Ethereal on plant development, yield, and bulb quality of onion plants (Allium cepa L). Journal of Applied Sciences Research, 9(2), 1140-1146. Sholihin, Y., Suminar, E., Rizky, W.H. & Pitaloka, G.G. (2016). Meristem explants growth of garlic (Allium sativum L.) Cv. tawangmangu on various compositions of kinetin and ga3 in vitro. Jurnal Kultivasi, 15(3), 172–179. Sulichantini, E. D. (2016). Effect of plant growth regulator Concentration Against Regeneration Garlic (Allium sativum L) In the Tissue Culture. Jurnal Agrifor, 15(1), 29–38. Suminarti, N.E. (2010). The Effects of N and K Fertilization on the Growth and Yield of Taro on Dry Land. Akta Agrosia, 13(1), 1–7. Uke, K. H. Y., Barus, H & Madauna, I. W. (2015). Effect of Tuber Sizes and Potassium Dosages on Growth and Production of Shallots var. Lembah Palu. Jurnal Agrotekbis, 3(6), 655 - 661. Utomo, P.S & Suprianto, A. (2019). Respon pertumbuhan dan produksi tanaman bawang merah (Allium ascalonicum L.) varietas thailand terhadap perlakuan dosis pupuk kusuma bioplus dan KNO3 putih. Jurnal Ilmiah Hijau Cendekia, 4(1), 28–34. Wu, C., Wang, M., Cheng, Z & Meng, H. (2016). The response of garlic (Allium sativum L.) bolting and bulbing to temperature and photoperiod treatments. Biol Open, 5(4), 507-518.
The new legislative situation raises once again the question which alternatives exist in order to avoid boar taint exist, if piglet castration would be forbidden and fattening of boars would be a common practise. The aim this study was to contrast approaches from the large number of new and elder literatures with the subjects of reducing and eliminating boar taint and to assess the effectiveness of these methods to avoid boar taint. For the first time, these findings were presented in a summarised and concentrated form. After reviewing 857 titles out of the year 1911 up to 2015, 664 sources of literature were chosen from all fields of pig breeding, housing and feeding, application of drugs, meat processing and the answer of consumers give a comprehensive understanding on the term of boar taint and the alternatives of piglet castration. There is no single strategy against boar taint being exposed after this work. There are some approaches possible: the immunocastration that gained acceptance in Australia and Belgium is an animal welfare friendly and in practice feasibly method. But it needs an inspection for boar taint at the slaughter line. The fattening of young boars, supported by Switzerland, could be improved by an optimised housing and feeding strategy, as well as the use of breeding lines for low boar taint. The processing of boar meat to premium products is possible. However, the amount of tainted boar meat coming into processing is still not clarified. It remains to be seen, how Germany will solve the problem of piglet castration.:Table of contents II Abbreviations and Definitions V 1 Introduction 1 2 Alternatives for boar taint reduction or elimination 3 2.1 Breed and genetics 3 2.1.1 Literature review 3 2.1.2 Breeding efforts 6 2.1.2.1 Differences in breeds 6 Breeding differences with view to boar taint perception and androstenone 6 Breeding differences on chromosomal and enzymatic level 9 Breeding differences with view to skatole and indole 10 Breeding differences with view to boar taint compounds 11 2.1.2.2 Heritability 13 Heritability for boar taint in general 13 Heritability for androstenone 14 Heritability for skatole and indole 15 2.1.2.3 Breeding selection 16 Breeding selection for boar taint 16 Breeding selection for androstenone 18 Breeding selection for skatole and indole 19 Using selection against boar taint in breeding programs 19 2.1.3 Genetic selection 20 2.1.3.1 Genetics of enzymes 20 Enzymes and QTL for androstenone 21 Enzymes and QTL for skatole and indole 25 2.1.3.2 Genetics on the chromosomal level 27 Genetic features for lowering androstenone and boar taint levels 29 Genetic features for lowering skatole and indole levels 35 2.2 Sperm-sexing 37 2.3 Killing of newborn male piglets 39 2.4 Fattening of boars 39 2.4.1 Feeding of boars – literature review 39 2.4.2 Feeding strategies 42 2.4.2.1 Energy/nutrient-density/protein diet 42 2.4.2.2 Ad libitum/restrictive feeding/fasting 43 2.4.2.3 Wet feeding/whey/lactose 48 2.4.2.4 RPS - raw potato starch 51 2.4.2.5 FOS - fructooligosaccarides 56 2.4.2.6 NSP- non starch polysaccharides/readily digestible raw fibre/alternative energy – pectin 64 2.4.2.7 Raw protein 68 2.4.2.8 Corn/cereals/plants with cellulose, a NSP 72 2.4.2.9 Garlic 76 2.4.2.10 Bicarbonate/organic acids 76 2.4.2.11 Enzyme/vitamins/trace elements 76 2.4.2.12 Other feedstuffs 76 2.4.2.13 Activated carbon/Tween-60: polyoxyethylene sorbitan monostearate 76 2.4.3 Housing conditions of boars – literature review 80 2.4.4 Housing conditions to avoid boar taint 81 2.4.4.1 Hygiene/fully slatted floor 81 2.4.4.2 Space/employment 84 2.4.4.3 Photoperiod/season/temperature 87 2.4.4.4 Sex composition of pig groups/mixed or single sex group 94 2.4.4.5 Social hierarchy/split marketing/farrow-to-finish system 102 2.4.5 Age and weight of boars 111 2.5 Application of drugs 112 2.5.1 Literature review 112 2.5.2 Down–regulation of the hypothalamic-pituitary-gonadal axis by exogenous hormonal drugs 112 2.5.2.1 Foragynol 113 2.5.2.2 Stilbene/DES 113 2.5.2.3 Estrogens 114 2.5.2.4 Progesterone 114 2.5.2.5 Testosterone and Trenbolone together with Estrogens 114 2.5.2.6 Testosterone 115 2.5.2.7 Gestagens/inhibitors: NTÖ, CMA, CPA, 19-NTA 115 2.5.2.8 GnRH 117 2.5.2.9 pST 118 2.5.3 Inhibitors of metabolism pathways 118 2.5.3.1 3β-HSD 118 2.5.3.2 Progesterone/5α-pregnane-3,20-dione 119 2.5.3.3 5α- reductase and CYP 17 119 2.5.4 Antibiotics 119 2.5.5 Immunocastration 121 2.6 Consumer evaluation 122 2.7 Processing of tainted boar meat 127 2.7.1 Literature review 127 2.7.2 Hanging/chilling 129 2.7.3 Mix/dilution 130 2.7.4 Heating/cold consumption 130 2.7.5 Masking 132 2.7.5.1 Masking (spices, starter cultures, liver tissue) 132 2.7.5.2 Smoking 133 2.7.6 Curing 134 2.7.7 Varieties of processed meat 135 2.7.7.1 Ham 135 2.7.7.2 Bacon 137 2.7.7.3 Salami sausages 137 2.7.7.4 Sausages 138 2.7.8 Other processing methods 139 3 Discussion 140 3.1 Breed and genetics 140 3.1.1 Breed difference 140 3.1.2 Heritability 141 3.1.3 Breeding selection 141 3.1.4 Genetics of enzymes 142 3.1.5 Genetics on chromosomal level 143 3.2 Fattening of boars 144 3.2.1 Feeding 144 3.2.2 Housing 147 3.3 Application of drugs 149 3.4 Processing of boar meat 151 4 Conclusion 154 4.1 Breed and genetics 154 4.2 Fattening of boars 155 4.2.1 Feeding 155 4.2.2 Housing 155 4.3 Application of drugs 156 4.4 Processing of boar meat 156 5 Summary 157 6 Zusammenfassung 159 7 Annexe 161 8 List of tables 225 9 List of figures 226 10 References 227 Acknowledgment 272
El ajo (Allium sativum L) es una planta monocotiledónea de la familia Alliaceae, su bulbo es ampliamente consumido por sus atributos culinarios y sus propiedades medicinales. Por su volumen de producción, el ajo, se ubica dentro de las 20 principales hortalizas a nivel mundial. Aunque la producción de ajo en México es baja; su calidad es muy apreciada en el mercado de exportación lo que permite su comercialización a Estados Unidos de América y La Unión Europea. Los factores de calidad del bulbo están relacionados con la adecuada secuencia de fotoperiodo y termoperiodo durante el ciclo de cultivo. No obstante, existen problemas de calidad cuando los factores ambientales no cumplen los requerimientos demandados por cada variedad. Se ha observado que un almacenamiento a 5 ºC durante cinco semanas previo a la siembra, adelanta la cosecha y disminuye la incidencia de bulbos con defectos. En este trabajo se comparó el efecto del almacenamiento de la semilla por 5 semanas a temperatura ambiente (TA) y a 5 ºC (5 ºC) antes de la siembra en el desarrollo de la planta, contenido de fenoles, flavonoides y fructanos totales, actividad enzimática de fenilalanina amonioliasa (PAL) y sacarosa-sacarosa 1-fructosiltransferasa (1-SST) y capacidad antioxidante de ajos de la variedad Coreano, los cuales fueron cultivados en la localidad de Cosío, Aguascalientes, Ags. En la temporada 2012-2013. El almacenamiento a 5 ºC incrementó el contenido de fenoles totales (39 %), flavonoides (24 %) y capacidad antioxidante (39 %) en de túnicas y el contenido de fructanos totales (17%) en bulbo durante el desarrollo en campo respecto al almacenamiento a TA. A su vez se encontró mayor actividad enzimática específica de PAL en túnicas (32 %) y de 1-SST (sacarosa-sacarosa-1-fructosiltransferasa) en bulbo (25 %) en la condición de 5°C al final del desarrollo. Después de las 5 semanas de almacenamiento a 5 °C, en los brotes de las semillas se incrementó la expresión en aproximadamente 9, 5, 24 y 18 veces los genes AsPAL, AsC4H (Cinnamato 4- hydroxilasa), As4CL (4Cumarato-CoA ligasa) y As1-SST respectivamente. A los 44 días de después de siembra (DDS) se observó una sobreexpresión en raíces de 5 °C de los genes AsPAL y AsC4H (33 y 15 veces respectivamente), y a los 106 DDS de los genes As4CL y As1-SST (6 y 7 veces respectivamente). Mientras que en disco basal proveniente de 5 °C se sobreexpresaron los genes AsPAL, AsC4H, As4CL y As1-SST (10, 21, 10, 25 veces respectivamente) hacia el final del desarrollo (106 DDS). Estos resultados indican que el almacenamiento a 5°C por 5 semanas induce la sobreexpresión de genes de la ruta de los fenilpropanoides (AsPAL, AsC4H y As4CL) y fructanos (As1-SST) en diferentes tejidos la cual se mantiene durante el desarrollo de la planta y se correlaciona con el aumento de la actividad enzimática de PAL y 1-SST, así como con la acumulación de metabolitos primarios (fructanos) y secundarios (fenoles y flavonoides) como una respuesta al estrés generado por el acondicionamiento en frío. El acondicionamiento al frío de la semilla fue suficiente para inducir una respuesta de defensa contra el estrés oxidativo en la nueva planta durante el desarrollo, lo cual podría llevar a una mayor tolerancia del ajo a otros tipos de estrés y/o enfermedades. ; "Garlic (Allium sativum L.) is a monocotyledonous plant of the Alliaceae family, its bulb is widely consumed for its culinary attributes and their medicinal properties. By production volume, garlic, ranks among the 20 major crops worldwide. Although garlic production in Mexico is low; its quality is highly appreciated in the export market allowing trading with the United States and the European Union. The bulb quality factors are related to the proper sequence of thermoperiod and - photoperiod during the growing season. However, there are quality problems when environmental factors do not meet the requirements demanded by each variety. It has been observed that storage at 5 ° C for five weeks prior planting, forward harvest and decreases the incidence of defective bulbs. In this study, the effect of seed storage for 5 weeks at room temperature (RT) and at 5 °C (5 °C) before planting on total phenolic, flavonoids and fructans contents, enzyme activity of phenylalanine ammonia-lyase (PAL), sucrose-sucrose 1- fructosyltransferase (1-SST) and antioxidant capacity of garlic of the Coreano cultivar grown at Cosío, Aguascalientes, Ags. at the 2012-2013 season was determined during crop development. Storage at 5 ° C increased the total phenolic content (39%), flavonoids (24%) and antioxidant capacity (39%) in cataphyll tissues and the content of total fructans (17%) in the bulb during development in comparison with storage at RT. In turn higher specific enzymatic activity of PAL in cataphylls (32%) and 1-SST (sucrose-sucrose-fructosyltransferase-1) activity in bulb (25%) for the condition 5 °C at the end of development was found. After 5 weeks of storage at 5 °C, expression of genes AsPAL, AsC4H (cinnamate 4-hydroxylase), As4CL (4Coumarate-CoA ligase) and As1-SST in sprouting seeds increased approximately 9, 5, 24 and 18 fold respectively. At 44 days after sowing (DAS) overexpression of As1-SST and As4CL genes was observed ( 33 and 15 fold respectively) in roots of 5 °C, and at 106 DAS, AsPAL and AsC4H were overexpressed (6 and 7 times respectively). While in basal plate of 5 °C, AsPAL, AsC4H, As4CL and As1-SST genes were overexpressed (10, 21, 10, 25 fold respectively) towards the end of development (DDS 106). These results indicate that storage at 5 °C for 5 weeks induces overexpression of genes of the phenylpropanoid pathway (AsPAL, AsC4H and As4CL) and fructans metabolism (As1-SST) in different tissues of garlic which is maintained during the development of the plant and correlates with increased enzymatic activity of PAL and 1-SST as well as with the accumulation of primary (fructans) and secondary metabolites (phenols and flavonoids) as a response to stress generated by cold conditioning. The cold conditioning of the seed was sufficient to induce a defense response against oxidative stress in the new plant during development, which could lead to greater tolerance of garlic to other type of stress and / or diseases."
Introduction Aggression is a competition based survival strategy. The spiegeldanio (spd) strain of zebrafish (Danio rerio), which has a mutation in the fibroblast growth factor receptor 1a, is bolder and more aggressive than the wild type fish [1]. Usually a socially dominant fish has preferential access to food, mate and shelter, and shows very characteristic postures like erection of the fins. It is also aggressive frequently biting, striking and chasing the subordinate fish as well as threatening its own mirror image in mirror tests [2]. However, what happens when an already known bold and dominant fish like spiegeldanio loses a dyadic fight. Spd fish are more aggressive in mirror tests, attacking their mirror image more frequently than wild type conspecifics. However, are they more aggressive in dyadic fights? Do they show an inhibition of aggressive behaviour when losing fights, the typical loser effect? The behavioural inhibition observed in animals losing fights for dominance is at least in part believed to be mediated by an activation of the brain serotonin (5-hydroxytryptamine, 5-HT) system. Do spd fish show a typical increase in brain 5-HT activity in response to social subordination? Dopamine (DA), on the other hand, is associated with aggression and social dominance. What are the effects of winning and losing fights for social dominance in spd fish? In the present study these questions were addressed in an attempt to increase or understanding of the control of agonistic behaviour and social stress. Animals and Methods The Spd strain of zebrafish were raised and reared at 27°C in an Aquaneering Zebrafish system at Uppsala University Biomedical Center. The animals were kept at a 14:10 h of light-dark photoperiod. The water used in the fish tanks was Uppsala municipal tap water (pH 7.2-7.6) of which 10% was exchanged daily. Fish were fed twice daily with Tropical energy food (Aquatic Nature, Belgium) and Artemia (Platinum Grade 0, Argentemia, Argent, Aquaculture, Redmond, USA). The use of animals was approved by the Uppsala Animal Ethical Committee (permit Dnr 55/13) and followed the guidelines of the Swedish Legislation on Animal Experimentation (Animal Welfare Act SFS1998:56), and the European Union Directive on the Protection of Animals Used for Scientific Purposes (Directive 2010/63/EU). The fish were transferred to the individual compartments of dimension 29 x 7.5 x 20 cm (length x breadth x height) in experimental tanks used for dyadic interaction and allowed to recover in isolation overnight. These experimental tanks were made from poly methyl methacrylate plastic and each tank was equipped with a submerged pump with filter (Eheim, typ 2006020, pumping capacity 1/h180, made in China), a heater (Sera aquarium, 25W, made in EU) and an air stone, all of which were placed at the back of the tank separated from the fish by a white perforated PVC screen (Figure 1). The setup of the arena was such that the two fish (1 dyadic pair) had an olfactory but not any visual cue of each other before the dyadic interaction. In the mirror test the fish were made to fight against the mirror image that was displayed in the mirror which was pasted on the wall of the arena. Prior to the beginning of the dyadic contest the mirror was covered with a black plexiglas slide cover. The experiment was carried out in the following sequence: The fishes were netted out and placed in the arena in the compartments A and B (Figure 1) and separated from each other by a partition. The cover of the mirror (opaque black PVC partition, Figure 1) was then removed and fish were made to interact with their own mirror image for 10 minutes. Then the slide covering the mirror was pulled down and the middle separating partition was pulled out and the fish were given an opportunity to fight. Dyadic fight was recorded two times, morning and evening on day one with the help of a video filming camera. Then next day in the morning the dyadic fight was again recorded. During the dyadic interaction the two fishes indulged in mutual display of aggressive behaviour which was followed by chasing and biting attacks performed by the dominant fish over the subordinate fish. Then middle partition was introduced again. Fish were given 6 minutes to habituate and the cover from the mirror was removed and fishes were again allowed to interact with their mirror image. Again the mirror was covered and the fish was allowed to get involved in the dyadic fight. Then each fish was taken out from the compartment at the same time and sacrificed for sampling of brain tissue. The three dimensional model of tank used in the behavioural tests I) Tank used for mirror test and for dyadic fight later on. It consists of two compartments, A and B. The movable partition separating the two compartments would be removed during the dyadic fight test. Compartment C is located at the back and is separated from the compartment A and B with the help of white coloured opaque perforated partition. It contains an air stone (for diffusion of air bubbles), heater (27°C), water pump (for circulation of water) and a drainage tube to exchange the water. II) Diagram of the settings used for dyadic interactions. The mirrors are covered with the help of a black PVC slide and the middle partition is pulled out. This allows the fish to interact. Brain dissection and analysis of monaoamines and monoamine metabolites Brains were divided into forebrain (telencephalon and diencephalon), optic tectum and the rest (here denoted brain stem). The frozen brains were homogenised in 4% (w/v) ice-cold perchloric acid containing 100 ng/ml 3, 4-dihydroxybenzylamine (DHBA, the internal standard) using a Sonifier cell disruptor B-30 (Branson Ultrasonics, Danbury, CT, USA) and were immediately put on dry ice. Subsequently, the homogenised samples were thawed and centrifuged at 15,000 rpm for 10 min at 4o C. The supernatant was used for high performance liquid chromatography with electrochemical detection (HPLC-EC), analysing the monoamines dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) as well as the DA metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA), as described by Øverli et al. [3]. In short, the HPLC-EC system consisted of a solvent delivery system model 582 (ESA, Bedford, MA, USA), an autoinjector Midas type 830 (Spark Holland, Emmen, the Netherlands), a reverse phase column (Reprosil-Pur C18-AQ 3 µm, 100 mm × 4 mm column, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) kept at 40° C and an ESA 5200 Coulochem II EC detector (ESA, Bedford, MA, USA) with two electrodes at reducing and oxidizing potentials of -40 mV and +320 mV. A guarding electrode with a potential of +450 mV was employed before the analytical electrodes to oxidize any contaminants. The mobile phase consisted of 75 mM sodium phosphate, 1.4 mM sodium octyl sulphate and 10 µM EDTA in deionised water containing 7 % acetonitrile brought to pH 3.1 with phosphoric acid. The quantification of samples was done by comparing it with standard solutions of known concentrations. DHBA was used as an internal standard to correct for recovery with the help of HPLC software ClarityTM (Data Apex Ltd, Czech Republic). The serotonergic and dopaminergic activity was measured as the ratio of 5-HIAA/5-HT and DOPAC/DA respectively. The brain monoamines were normalized with respect to brain protein weights which were determined with Bicinchoninic acid protein determination kit (Sigma Aldrich, Sweden). The assay was read at a wavelength of 570 nm with the help of a plate reader (Labsystems multiskan 352, Labsystems Thermo Fisher Scientific). Results A clear dominant subordinate hierarchy was established within 30 minutes of dyadic interaction. The number of aggressive acts (bites, strikes and chases) performed by the looser fish decreased significantly from the first dyadic fight to the last (i.e. the fourth) dyadic fight. For the winner fish the number of aggressive acts performed against a mirror during the second mirror test increased or remained same as before after winning a dyadic fight, whereas for the looser fish it decreased significantly. The results from the present study indicate that subordinate fish have higher 5-HIAA/5-HT ratio in the optic tectum as compared to the dominants. More results from this study would be presented at the conference. References 1. Norton W, Bally-Cuif L (2010) Adult zebrafish as a model organism for behavioural genetics. BMC Neurosci. 11:90. 2. Rowland WJ (1999) Studying visual cues in fish behaviour: a review of ethological techniques. Env Biol Fishes. 56:285-305. 3. Øverli Ø, Harris CA, Winberg S (1999) Short-term effects of fights for social dominance and the establishment of dominant-subordinate relationships on brain monoamines and cortisol in rainbow trout. Brain Behav Evol. 54:263-275.
Esta tesis tiene como objetivo investigar los efectos del etanol y metales pesados (cadmio y mercurio) en los patrones diarios de dos especies acuáticas clave: pez cebra (Darío rerio) y anfípodo gammarus (G. aequicauda y G. chevreuxi). La investigación se realizó en dos niveles organizativos: comportamental y molecular. Con este fin, hemos establecido cuatro objetivos específicos: 1. Investigar los efectos al etanol en el pez cebra en función en su ritmo circadiano. 2. Determinar la existencia de un ritmo diario de la toxicidad del cadmio en el pez cebra. 3. Caracterizar en Gammarus chevreuxi los efectos en la actividad locomotora debido a sedimentos contaminados con mercurio. 4. Evaluar la calidad ambiental de los sedimentos de la cuenca sur del Mar Menor influenciados por históricas actividades mineras. Por lo tanto, estos nuevos datos tiene la intención de señalar la importancia de aplicar un enfoque multidisciplinario para estudios toxicológicos, así como elevar la importancia de la elección del momento adecuado para realizar el ensayo de toxicidad Metodología Los experimentos llevados a cabo en la presente investigación siguieron la legislación española sobre protección de animales y prácticas de laboratorio y fue aprobado tanto por el Comité Nacional de Protección de los Animales y el Comité de Bioética de la Universidad de Murcia - Los muestreo de crustáceos se realizaron en las zonas próximas a la costa mediante el uso de redes de 5 µ de luz de maya. Y su trasporte con aireación y refrigeración . Los muestreos de pez cebra se realizaron en las cámaras del departamento de Fisiología animal. - El etanol se adquirió a través de distribuidores comerciales y los sedimentos tóxicos directamente de las zonas de estudio mediante muestreos con barca. La extracción de los sedimentos se realizó utilizando cores y draga. Se almacenaran en refrigeradores a una temperatura de 4ºC. - Los test de mortalidad y comportamiento se realizaran en cámaras herméticas donde se permite el control de las condiciones de fotoperiodo y temperatura. - Las condiciones del agua antes y después de las exposiciones se medió con electrodos de pH y Oxígeno. - Lo análisis de la actividad ante exposiciones subletales se realizaron en las mismas cámaras y mediante la utilización de equipos de filmación sensibles a las luces infrarrojas, permitiendo así mantener las condiciones de oscuridad en los experimento que sea necesarios. - Determinación de metales pesados en tejidos, utilizamos La Espectroscopia de Emisión en Plasma Acoplado Inductivamente (ICP-AES) con Potencia RF: 1200W, Velocidad de bomba: 30r.p.m., Flujo de gas aux.:0.5l/min, Flujo de gas de nebulización: 0.5 l/min, Flujo de gas de refrigeración: 14 l/min, Flujo de gas de purga: Normal. - Para el estudio de expresión de diferentes genes realizamos la técnica de PCR cuantitativa utilizando SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) y un aparato ABI Prism 7500 (Applied Biosystems, Foster City, CA). Conclusiones 1. La exposición de pez cebra mediante baño a etanol a diferentes horas del día muestra un ritmo de toxicidad que refleja una mayor tasa de mortalidad en larvas durante el día comparado con los efectos durante la noche. Así mismo en adultos de pez cebra se observan efectos en su actividad natatoria y posición en la columna de agua siendo significativamente más severos durante la fase de luz. 2. La exposición de pez cebra mediante baño a cadmio durante diferentes horas del día reflejó un incremento de la mortalidad y acumulación del cadmio en tejidos durante la mitad de la fase de luz, así como una mayor reducción de la actividad locomotora cuando el pez es expuesto a concentraciones subletales, además la expresión de mt1 muestra un pico antes al final de la fase de luz. Todos estos resultados indican que el pez cebra es más vulnerable al cadmio durante la fase de luz que en la fase de oscuridad. 3. La exposición de Gammarus chevreuxi mediante sedimentos contaminados con mercurio mostró un efecto deletéreo en la actividad natatoria incluso en concentraciones consideradas no letales. Dichos efectos fueron más acusados durante el la fase de luz, que corresponde con la más activa del animal. 4. Nuestros resultados sugieren que la actividad locomotora y el número de surfacings se presentan cono una señal temprana de la interacción de los factores co-estresantes temperatura y sedimentos contaminados. Los parámetros comportamentales estudiados en Gammarus. aequicauda y Gammarus. chevreuxi tienen una conexión clara con la supervivencia de los organismos y pueden tener una relevancia ecológica. This thesis aims at investigating the effects of Ethanol and heavy metals (cadmium and mercury) on the daily patterns of two key aquatic species: zebrafish (Dario rerio) and amphipod gammarus (G. aequicauda and G. chevreuxi). The research was conducted in two organizative levels: Physiology behavior and molecular. With this purpose we have established four particular objectives: 1. To investigate time-dependent effects of ethanol exposure in zebrafish. 2. To determine the existence of a daily rhythm of cadmium toxicity in zebrafish. 3. To characterise the effects of locomotor activity to mercury-contaminated sediments in Gammarus chevreuxi. 4. To evaluate the environmental quality of the soft-bottoms from the southern basin of the Mar Menor lagoon influenced by historical mining activities. Hence, this new data intends to point out the importance of applying a multidisciplinary approach to toxicology studies, and also raise the importance of the time experimental setting of toxicity test. Methodology The experiments performed in the present research followed Spanish legislation on Animal Welfare and Laboratory Practices and was approved by both the National Committee on Animal Welfare and the Bioethics Committee of the University of Murcia. - The sampling of gammarids were conducted in the nearshore areas by using mesh of 5 μ. Their transportation was with aeration and cooling. The zebrafish samplings were performed in Animal Physiology's chambers of the department. - Ethanol was purchased by authorized distributors, and toxic sediments were extracted directly from the study areas by boat sampling. Sediments extraction was with cores and dredge. They were stored in the refrigerator at 4 ° C. - The test of mortality and behavior was conducted in chambers where control of the photoperiod and temperature conditions allowed. - Water conditions before and after exposure was measured with pH electrodes and oxygen. - The analysis of activity at sublethal exposures were conducted in the same chamber and using equipment sensitive to infrared light filming, thereby maintaining the conditions of darkness in the experiment is necessary. - Determination of heavy metals in tissues, we used emission spectroscopy Inductively Coupled Plasma (ICP-AES) RF Power: 1200W, pump speed: 30r.pm, aux gas flow: 0.5l / min gas flow. Misting: 0.5 l / min, the cooling gas flow: 14 l / min, purge gas flow: Normal. - Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and an ABI Prism 7500 apparatus (Applied Biosystems, Foster City, CA). Conclusions 1. Exposure of zebrafish using ethanol bath at different times of day shows a rate of toxicity reflecting a higher rate of mortality in larvae during the day compared to the effects overnight. Also effects in adult zebrafish are observed in their swim and activity position in the water column to be significantly more severe during the light phase. 2. Exposure zebrafish using cadmium bath during different times of day reflected an increase in mortality and cadmium accumulation in tissues during half of the light phase and a further reduction of locomotor activity when the fish is exposed to sublethal concentrations. Expression of mt1 shows a peak before the end of the light phase. All these results indicate that zebrafish are more vulnerable to cadmium during the light in the dark phase. 3. The exposure of Gammarus chevreuxi by mercury-contaminated sediments showed a deleterious effect on the swimming activity even considered non-lethal concentrations. These effects were most pronounced during the light phase, which corresponds to the most active animal. 4. Our results suggest that the locomotor activity and the number of surfacings an early sign of the interaction of co-temperature stressors and contaminated sediments occur cone. The behavioral parameters studied in Gammarus. aequicauda and Gammarus. chevreuxi have a clear connection to the survival of organisms and can have an environmental relevance.
Sturgeons (Family Acipenseridae) are one of the largest and most primitive fish families on Earth. Although they have always been typically distributed throughout the Northern Hemisphere, during the last decades wild populations have declined due to anthropogenic factors such as overfishing, poaching, pollution, and habitat loss. The situation is particularly dramatic for Atlantic sturgeon, one of the most ancient species among the family, which is currently extinct in Europe. In order to reintroduce the Atlantic sturgeon in Europe, several Baltic countries have been working together for more than two decades to build an ex-situ broodstock locally with fish derived from Canada, where the populations are not threatened, and releasing juveniles into the rivers flowing to the Baltic Sea.However, rearing fish aimed for restocking in the same manner as aquacultured fish has been shown to impact the post-release survival of juveniles in the long term. For example, in aquaculture conditions, fish are often maintained in high densities, at constant photoperiod and temperature conditions, and feeding on commercial pellets, leaving them cognitively naïve when released into natural environments. Additionally, increased water temperatures due to global warming have a strong influence on the geographic distribution of the species, resulting in local extinctions and population shifts. Although the effect of heat and cold stress on the juvenile and embryonic development have been assessed for several sturgeon species, most studies on gene expression have looked at a very limited number of genes due to the lack of sequence information and genomic resources. Also, most studies use other sturgeon species such as Siberian, Japanese, and white sturgeons, which are widespread aquacultured species. Exposing fish to temperatures higher than the optimal can trigger phenotypic adaptations leading to increase thermotolerance and potentially improve postrelease survival, however the impact of temperature-training protocols on the response to a subsequent heat shock has not yet been assessed in sturgeons. Therefore, the main aim of this thesis was to generate genomic and transcriptomic resources for Atlantic sturgeon, which are essential to improve and promote research in many fields, such as ecology, physiology and evolutionary studies. Moreover, it provides a reference for RNAseq-mediated transcriptome mapping. Additionally, we have used these resources to develop and evaluate the impact of novel rearing techniques toward improving restoration success, focusing on temperature training. First, we have assembled a high-quality de novo transcriptome, made an inventory of all the heat shock protein (HSP) gene family members and exposed a cell line derived from Atlantic sturgeon larvae to a moderate and severe heat shock in order to identify all heat-responsive genes using an RNAseq approach (Paper I). We found 76 HSP genes in the Atlantic sturgeon transcriptome, only 16 of which were responsive to at least one of the applied heat shock protocols, and only 5 of which were consistently upregulated after both moderate and severe heat shock at all the tested timepoints. After building the reference transcriptome and annotating all the HSP genes, we have evaluated the differences in liver transcriptome between temperature-trained and nontrained juveniles upon exposure to a new heat shock (Paper II). After four weeks of treatment, fish exposed to temperature training showed between 2 to 4 fold less dysregulated genes in response to a new heat shock than the non-trained group, indicating their improved ability to maintain transcriptomic homeostasis during a new heat shock. Again, like in the in vitro experiment, very few of the annotated HSP genes were dysregulated in response to heat shock in the liver transcriptome, namely hspa1, hspc1 and dnajb4. Overall, the response to heat shock in the liver transcriptome was milder than the in vitro response, which is likely a consequence of the activation of compensatory mechanisms. These mechanisms include the neuroendocrine system and result in increased tissue protection and thermogenic capacities, especially in the trained fish. We therefore propose that temperature-training protocols like the one tested in this thesis should be included in the set of new rearing techniques for fish used for restocking; however, other protocols should be investigated. Since the main bottleneck in the evaluation of the effect of such training is the lack of sequence information and a reference genome for RNAseq experiments, we have additionally assembled a reference genome for Atlantic sturgeon using a combination of short and long-read sequencing technologies (Paper III). The assembled genome provides for the first time clear evidence of a sturgeon-specific whole-genome duplication event (SR), independent from the American paddlefish (Polyodon spathula), which is the main representative of the sister Family (Polyodontidae) within the same Order (Acipensiformes). The presence of duplicated Hox clusters, together with synteny and phylogenetic studies of these developmental genes, and the results of microsatellite loci analysis, suggests that sturgeons have a paleotetraploid origin, and that a rediploidization process is still ongoing. In summary, the results presented in this thesis advance the field of sturgeon research. We hypothesized that temperature training has a positive effect during the exposure to a subsequent heat shock, but its potential to improve post-release survival in the long term should still be assessed. We therefore suggest that future work should be aimed at the optimization of rearing methods for stocking programs and that a reference genome should be used. ; Stør (Familie Acipenseridae) er blant de største og mest primitive familier av benfisk. Mens de opprinnelig var utbredt over hele den nordlige halvkule, er mange populasjoner nå kritisk truet på grunn av menneskeskapte faktorer som overfiske, forurensning og tap av habitat. Situasjonen er spesielt dramatisk for atlantisk stør, en av de eldste artene i familien, som er utdødd i Europa. For å gjeninnføre atlantisk stør, har flere baltiske land arbeidet for å etablere en ex situ stamfiskpopulasjon basert på fisk fra Canada (hvor bestanden ikke er truet), for produksjon av yngel til utsetting i baltiske vassdrag. Imidlertid gir produksjon av settefisk etter samme prinsipper som for oppdrettsfisk svært lav overlevelse i naturen. Eksempelvis vil høy tetthet, konstant fotoperiode og vanntemperatur, og fôring med pellets til faste tider gi en kognitivt naiv fisk som ikke klarer seg i det fri. I tillegg kommer økte vanntemperaturer som resultat av global oppvarming, og som har stor innvirkning på den geografiske fordelingen av arter, som igjen resulterer i lokal utryddelse og forflytning av populasjoner. Selv om effekter av vanntemperatur har blitt undersøkt på embryonal- og yngelutviklingen i flere størarter, har de fleste studier fokusert på et lite sett av gener fordi genomiske ressurser har manglet. I tillegg er de fleste studiene utført på andre arter som er vanlig i akvakultur, slik som sibirsk, japansk og hvit stør. Eksponering til vanntemperaturer som er høyere enn artens optimum, kan utløse fenotypiske tilpasninger som fører til økt termotoleranse og potensielt forbedre overlevelse i naturen. Men effekten av slike temperaturtreningsprotokoller på responsen på et påfølgende varmesjokk er ikke studert i stør. Hovedmålet med denne avhandlingen var å generere genomiske og transkriptomiske ressurser for atlantisk stør, som en viktig faktor for å forbedre og fremme forskning innen økologi, fysiologi og evolusjon. Videre gir avhandlingen en referanse for RNAseq-mediert transkriptomkartlegging. Disse ressursene er så benyttet til å utvikle og evaluere virkningen av nye oppdrettsteknikker for settefisk, med fokus på temperaturtrening. Først har vi satt sammen et høykvalitets de novo transkriptom, deretter karakterisert genfamilien av varmesjokkproteiner (HSP), og så eksponert en cellelinje avledet fra atlantiske størlarver for et moderat og et kraftig varmesjokk for å identifisere alle varmeresponsive gener ved bruk av RNAseq (Artikkel I). Vi fant 76 HSP-gener i transkriptomene fra atlantisk stør, hvorav 16 responderte på minst en av de testede varmesjokkprotokollene, og bare 5 av disse ble konsekvent oppregulert etter både moderat og kraftig varmesjokk ved alle testede tidspunkt. Etter å ha bygget referansetranskriptomet og karakterisert alle HSP-gener, evaluerte vi forskjellene i levertranskriptom mellom temperaturtrent og ikke-trent yngel ved eksponering for et nytt varmesjokk (Artikkel II). Etter fire ukers behandling viste fisk som ble utsatt for temperaturtrening mellom 2-4 ganger færre dysregulerte gener som svar på et nytt varmesjokk sammenlignet med den ikke-trente gruppen. Dette antyder en forbedret evne til å opprettholde transkriptomisk homeostase under et nytt varmesjokk. I likhet med in vitro eksperimentet var svært få HSP-gener dysregulert som respons på varmesjokk i levertranskriptomet, nemlig hspa1, hspc1 og dnajb4. Totalt sett var responsen på varmesjokk i levertranskriptomet mildere enn in vitro responsen, noe som sannsynligvis skyldes ulike kompensatoriske mekanismer. Disse inkluderer det nevroendokrine systemet og resulterer i økt vevsbeskyttelse og termogen kapasitet, spesielt i trent fisk. Selv om andre protokoller bør undersøkes nærmere, foreslår vi at protokoller for temperaturtrening lik den som ble testet i denne avhandlingen bør vurderes i nye oppdrettsprotokoller for settefiskproduksjon av stør. Siden den viktigste flaskehalsen i evalueringen av effekten av slik trening er mangelen på sekvensinformasjon og et referansegenom for RNAseq-eksperimenter, har vi i tillegg produsert et referansegenom for atlantisk stør ved bruk av ulike sekvenseringsteknologier for korte og lange reads (Artikkel III). Genomet gir for første gang klare bevis for en størspesifikk helgenomdupliseringshendelse (SR), uavhengig av spadestør (Polyodon spathula), som er hovedrepresentanten for søsterfamilien (Polyodontidae) innenfor samme Orden (Acipensiformes). Tilstedeværelsen av dupliserte klynger av hox-gener som er sentrale i tidlig utvikling, i tillegg til hox-gen synteni og fylogeni, og mikrosatellitt loci-analyser, antyder at stør har en paleotetraploid opprinnelse, og at en rediploidiseringsprosess fortsatt pågår. Oppsummert vil de forbedrete genomiske og transkriptomiske verktøy presentert i denne avhandlingen åpne for nye muligheter innen størforskning. Videre har temperaturtrening en positiv effekt ved eksponeringen til varmesjokk, men potensialet for temperaturtrening og dermed økt overlevelse ved utsett bør undersøkes videre. ; Os esturións (Familia Acipenseridae) pertencen a unha das familias de peixes mais grandes e primitivas da Terra. Aínda que sempre estiveron distribuídos no hemisferio Norte, nas últimas décadas as poboacións salvaxes teñen diminuido debido a factores antropoxénicos como a sobrepesca, a caza furtiva, a contaminación e a perda do hábitat. A situación é especialmente dramática para o esturión Atlántico, unha das especies máis antigas da familia que se atopa extinta en Europa na actualidade. Co fin de reintroducir o esturión Atlántico en Europa, varios países bálticos levan traballando xuntos durante máis de dúas décadas para construír un núcleo reprodutor con peixes derivados de Canadá, onde a poboación non esta ameazada, e liberar xuvenís nos ríos que flúen ao Mar Báltico. Non obstante, cultivar peixe destinado ao repoboamento utilizando as mesmas técnicas típicamente utilizadas en acuicultura afecta negativamente á supervivencia a longo prazo. Por exemplo, en condicións de acuicultura os peixes adoitan producirse en altas densidades, baixo condicións abióticas constantes (fotoperíodo e temperatura), e aliméntanse de pellets comerciais, producindo animais congitivamente inxenuos cuando son libertados no ambiente natural. Ademais, o aumento das temperaturas da auga debido ao quentamento global ten unha forte influencia na distribución xeográfica das especies, dando lugar a extincións locais. Aínda que os efectos do estrés térmico no desenvolvemento embrionario e etapas xuvenís xa teñen sido avaliados en varias especies de esturións, a maioría dos estudos de expresión xénica céntranse en un número moi limitado de xenes debido á falta de recursos xenómicos. Ademáis, a maioría dos estudos utilizan outras especies de esturións como o branco, siberiano ou xaponés, mais comúns en acuicultura. A exposición dos peixes a temperaturas superiores ás óptimas pode desecandear adaptacións fenotípicas resultando nun incremento da tolerancia térmica e potencialmente unha mellora da supervivencia no hábitat onde son libertados. Sen embargo, os efectos do réxime de temperatura utilizado durante a cría na resposta a un choque de calor posterior non teñen sido avaliados. Por todo isto, o obxectivo principal desta tese foi xerar recursos xenómicos e transcriptómicos para o esturión Atlántico, esenciais para mellorar e promover a investigación en moitos campos da ciencia como a ecoloxía, fisioloxía e a xenómica evolutiva, ademais de fornecer unha referencia para o mapeado do transcriptoma. Ademais, empregamos estes recursos para desenvolver e avaliar o impacto de novas técnicas de cultivo para mellorar o proceso de repoboación, centrándose na xeración de individuos termotolerantes. En primeiro lugar, temos ensamblado un transcriptoma de alta calidade, fixemos un inventario de todos os membros da familia das proteínas de choque térmico (HSP) e expuxemos unha liña celular isolada a partir de larvas disgregadas de esturión Atlántico a un choque de calor moderado e severo para identificar xenes sensibles ó calor (Artigo I). Atopamos 76 HSP no transcriptoma de esturión Atlántico, dos cuáis só 16 foron sensibles a polo menos un dos protocolos de choque de calor avalidado, e 5 foron sensibles a ambos choques térmicos idenpendentemene do momento da amostraxe. Utilizando as secuencias dos xens HSP e o trancriptoma de esturión ensamblado no Artigo I como referencia, temos avaliado as diferenzas no transcriptoma hepático en resposta a un choque térmico entre xuvenís criados baixo un réxime de temperatura constante e en réxime fluctuante (Artigo II). Despois de catro semanas de tratamento, os peixes criados en réxime de temperatura fluctuante mostraron entre 2 e 4 veces menos xens diferencialmente expresados en resposta a un novo choque térmico que os peixes criados en réxime de temperatura constante, indicando a súa mellor capacidade para manter a homeostase transcriptómica durante un novo choque térmico. Como xa indicaron os resultados in vitro, moi poucos HSP foron diferencialmente expresados en resposta ó choque de calor no transcriptoma hepático, concretamente hspa1, hspc1 e dnajb4. En xeral, a resposta ao choque térmico no transcriptoma hepático foi máis leve que a resposta in vitro, o que é probablemente consecuencia da activación de mecanismos compensatorios. Estes mecanismos inclúen o sistema neuroendocrino e teñen como consecuencia un aumento da protección dos tecidos e das capacidades termoxénicas, especialmente no peixe criado a temperaturas fluctuantes. Polo tanto, propoñemos que a cría de peixes a temperaturas fluctuantes debe ser incluida no conxunto de novas técnicas empegadas en peixes criados con fins de repoboamento, non obstante, outros protocolos de temperatura deben ser investigados. Dado que unha importante limitación para a avaliación de novas técnicas de cría é a falta dun xenoma de referencia para experimentos de mapeado do transcriptoma, no Artigo III temos ensamblado un xenoma de referencia para o esturión Atlántico, combinando tecnoloxías de secuenciación de curta e longa lectura. O xenoma do esturión Atlántico evidencia por primeira vez a presenza dun evento de duplicación específico de esturión (SR) e independente do peixe-espátula (Polyodon spathula). A presenza de xenes Hox duplicados, xunto con estudos filoxenéticos e de sintenia e os resultados da análise de loci microsatélite suxire que os esturións teñen unha orixe paleotetraploide e que a rediploidización é aínda un proceso activo. En resumo, os resultados presentados nesta tese avanzan no campo da investigación con esturións. Os nosos resultados suxiren que a cría de peixes baixo un réxime de temperatura fluctuante ten un efecto positivo durante un choque térmico subsecuente, pero a influencia de esta nova técnica de cría na supervivencia dos xuvenís tras a súa liberación no habitat a repoboar aínda debe ser avaliada. Contudo, suxerimos que o traballo no futuro ten que estar centrando na optimización dos métodos de cría en programas de repoboación e que o xenoma de referencia debe ser usado. ; The European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 642893: Improved Production Strategies for Endangered Freshwater Species, "IMPRESS".
