Roles of lipids and fatty acids through the spermatogenesis of European eel (Anguilla anguilla)

Tesis por compendio[EN] During the last 25 years, eel populations have declined considerably, this species is considered outside safe biological limits. The life cycle of the eel is quite complex including transoceanic migration whose conditions are still so unknown that even the natural spawning process has never been observed. Eels cease feeding during migration and that is why their energy reserves such as lipids play a crucial role and thus their in deep study is essential to obtain gametes (eggs and sperm) quality as the first step for their reproduction in captivity. In the last years, many efforts have been directed at the study of the influence of fatty acids on the reproductive performance in females, but not in male eels. The present manuscript describes the results obtained in two experiments. The first experiment (which led to the three first articles included in the memory) focused on the effect of fatty acids along maturation, their relationship with steroid hormones and their effect on sperm quality parameters. The second experiment involved the comparison of different diets designed, considering the previously acquired insights into the most influential fatty acids on male eel reproduction, and the assessment of their influence on the reproductive performance of males. The first experiment was used to assess the changes in the fat content, as well as fatty acids, in different tissues of male eels hormonally induced to sexual maturation under different thermal regimes (two variables and one constant). Males finished spermatogenesis earlier with a constant temperature of 20 ºC, suggesting that eel spermatogenesis is tightly regulated by temperature. The fat content did not change significantly in the muscle, but was increased in the liver and decreased in testes during testicular development. With regard to fatty acids, during sperm maturation, the liver was highlighted as the main site of synthesis. Finally, EPA, ARA and DHA remained constant in testes, while the level of the rest of fatty acids decreased significantly. Using samples from the same experiment, the correlation between the main steroid hormones and fatty acids at different stages of testicular development were investigated. Similarly as occurs in mammals, EPA and DHA were highlighted as possible modulators of androgen synthesis. The set of the results obtained suggests new perspectives concerning the functions and interactions between fatty acids and steroids in fish spermatogenesis. Also, with samples from the first experiment, a study was carried out seeking to establish the possible correlations between different fatty acids and several sperm quality parameters. Correlations between several highly unsaturated fatty acids with parameters such as volume, the percentage of motile sperm and sperm velocity were found. Finally, with the acquired knowledge so far, a second experiment was carried out designing feeds with different fatty acid percentages in order to evaluate the influence of the diets on sperm quality. The results showed that high levels of DHA and EPA in the diets induce higher volumes and high sperm motility. This study allowed obtaining results applicable to the design of optimum broodstock diets for this species.[ES] Durante los últimos 25 años las poblaciones de anguila han disminuido considerablemente, de modo que la especie se considera fuera de los límites biológicos de seguridad. El ciclo vital de la anguila es bastante complejo incluyendo una migración transoceánica cuyas condiciones son todavía tan desconocidas que incluso el proceso natural de desove no se ha observado nunca. Las anguilas dejan de alimentarse durante la migración y es por ello que sus reservas energéticas en forma de lípidos tienen un papel crucial y su estudio en profundidad es importante para obtener gametos (huevos y esperma) de calidad como primer paso para su reproducción en cautividad. En los últimos años se han dirigido muchos esfuerzos al estudio de la influencia de los ácidos grasos en el rendimiento reproductivo de las hembras, pero no en el de los machos de anguila. El presente documento describe los resultados obtenidos en dos experimentos. El primer experimento (que dio lugar a los tres primeros artículos incluidos en la memoria) se centró en el efecto de los ácidos grasos durante la maduración, su relación con las hormonas esteroideas y su efecto sobre los parámetros de calidad espermática. El segundo experimento consistió en la comparación de diferentes dietas, diseñadas considerando los conocimientos adquiridos previamente sobre los ácidos grasos más influyentes en la reproducción de machos de anguila, y en la evaluación de su influencia en el rendimiento reproductivo de los machos. El primer experimento sirvió para evaluar los cambios en el contenido graso, así como en los ácidos grasos, en diferentes tejidos de machos de anguila hormonalmente inducidos a la maduración sexual bajo diferentes regímenes térmicos (dos variables y uno constante). Los machos concluyeron la espermatogénesis más temprano a una temperatura contante de 20 ºC, sugiriendo que la espermatogénesis de anguila está estrechamente regulada por la temperatura. El contenido en grasa no varió significativamente en el músculo, se incrementó en el hígado y bajó en el testículo a lo largo del desarrollo testicular. Con respecto a los ácidos grasos, durante la maduración del esperma, el hígado destacó como el principal sitio de síntesis. Finalmente, EPA, ARA y DHA permanecieron constantes en el testículo, mientras que el nivel del resto de ácidos grasos bajó significativamente. Usando muestras del mismo experimento, se investigaron las correlaciones entre las principales hormonas esteroideas y los ácidos grasos en los diferentes estadios del desarrollo testicular. De forma similar a lo que ocurre en mamíferos, EPA y DHA destacaron como posibles moduladores de la síntesis de andrógenos. El conjunto de los resultados obtenidos sugiere nuevas perspectivas sobre las funciones y las interacciones entre los ácidos grasos y los esteroides en la espermatogénesis de peces. También con muestras del primer experimento, se llevó a cabo un estudio que buscó las posibles correlaciones de los diferentes ácidos grasos con varios parámetros de calidad espermática. Se encontraron correlaciones entre varios ácidos grasos altamente insaturados con parámetros como el volumen, el porcentaje de espermatozoides móviles y la velocidad del esperma. Finalmente, con los conocimientos adquiridos hasta el momento, se realizó un segundo experimento para el que se diseñaron piensos con diferentes porcentajes de ácidos grasos con el fin de evaluar la influencia de las dietas sobre la calidad del esperma. Los resultados mostraron que niveles altos de DHA y EPA en las dietas inducen mayores volúmenes y una alta motilidad del esperma. Este estudio permitió obtener resultados aplicables al diseño de dietas óptimas para reproductores de esta especie.[CA] Durant els últims 25 anys les poblacions d'anguila han disminuït considerablement, de manera que l'espècie es considera fora dels límits biològics de seguretat. El cicle vital de l'anguila és bastant complex incloint una migració transoceànica les condicions de la qual són encara tan desconegudes que fins i tot el procés natural de fresa mai ha estat observat. Les anguiles deixen d'alimentar-se durant la migració i és per això que les seves reserves energètiques en forma de lípids tenen un paper crucial i el seu estudi en profunditat és important per a obtenir gamets (ous i esperma) de qualitat com a primer pas per a la reproducció en captivitat. En els últims anys s'han dirigit molts esforços a l'estudi de la influència dels àcids grassos en el rendiment reproductiu de les femelles, però no en el dels mascles d'anguila. El present document descriu els resultats obtinguts en dos experiments. El primer (que dona lloc als tres primers articles inclosos en la memòria) es va centrar en l'efecte dels àcids grassos durant la maduració, la seva relació amb les hormones esteroides i el seu efecte sobre els paràmetres de qualitat espermàtica. El segon experiment va consistir en la comparació de diferents dietes, dissenyades considerant els coneixements adquirits prèviament sobre els àcids grasos més influents en la reproducció dels mascles d'anguila, i en l'avaluació de la seva influència en el rendiment reproductiu dels mascles. El primer experiment va servir per avaluar els canvis en el contingut de gras, així com en els àcids grassos, en els diferents teixits de mascles d'anguila hormonalment induïts a la maduració sexual baix diferents règims tèrmics (dos variables i un constant). Els mascles van concloure la espermatogènesis més prompte a una temperatura de 20 ºC, suggerint que l'espermatogènesis d'anguila està estretament regulada per la temperatura. El contingut en greix, no va variar significativament en el múscul, es va incrementar en el fetge i va baixar en els testicles al llarg del desenvolupament testicular. Pel que fa als àcids grassos, durant la maduració de l'esperma, el fetge va destacar com al principal lloc de síntesi. Finalment, EPA, ARA y DHA van romandre constants en el testicle, mentre que el nivell de la resta d'àcids grassos va baixar significativament. Emprant mostres del mateix experiment, es van investigar les correlacions entre les principals hormones esteroides i els àcids grassos en els diferents estadis de desenvolupament testicular. De manera semblant al que ocorre en mamífers, EPA i DHA van destacar com a possibles moduladors de la síntesi d'andrògens. El conjunt dels resultats obtinguts suggerixen noves prespectives sobre les funcions i les interaccions entre els àcids grassos i els esteroides en l'espermatogènesi dels peixos. També amb mostres del primer experiment, es va dur a terme un estudi que va buscar les possibles correlacions dels diferents àcids grassos amb diversos paràmetres de qualitat espermàtica. Es van trobar correlacions entre diversos àcids grassos altament insaturats amb paràmetres com el volum, el percentatge d'espermatozoides mòbils i la velocitat de l'esperma. Finalment, amb els coneixements adquirits fins al moment, es va realitzar un experiment per al qual es van dissenyar pinsos mb diferents percentatges d'àcids grassos per tal d'avaluar la influència de les dietes sobre la qualitat de l'esperma. Els resultats van mostrar que nivells alts de DHA i EPA en les dietes indueixen a majors volums i una alta motilitat de l'esperma. Aquest estudi va permetre obtenir resultats aplicables al disseny de dietes òptimes per a reproductors d'aquesta espècie.Baeza Ariño, R. (2015). Roles of lipids and fatty acids through the spermatogenesis of European eel (Anguilla anguilla) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/49988TESISCompendi

Relationship between sperm quality parameters and fatty acid composition of the muscle, liver and testis of European eel

[EN] This study looks at the correlations that fatty acids have with different tissues in the European eel (Anguilla anguilla L.) during hormonally-induced sexual maturation, with different sperm quality parameters. In order to evaluate the different dynamics of the use of fatty acids, a categorization of the results from each sperm quality parameter (volume, concentration, motility and velocity) was performed. Low and moderate correlations were observed between muscle tissue and some sperm quality parameters but no high correlations were found. Eicosapentaenoic add (20:5n3, EPA) in the liver seems to have a role in determining the volume of sperm produced. This can be explained by the fact that EPA is a major requirement in the early phases of sperm production (probably as a component of the spermatozoal membrane). In addition, the levels of alpha-linolenic acid (18:3-n3, ALA) and linoleic acid (18:2-n6, LA) in the liver decreased when sperm motility increased. In all the tissues, a negative correlation was observed between arachidonic acid (20:4n-6, ARA) and the different sperm velocity parameters. The fact that an increase in the consumption of ARA coincides with an increase in the speed of spermatozoa, highlights the important role that this fatty acid plays not only in sperm production, but also in sperm velocity. All this information could prove useful in the development of suitable broodstock diets to improve sperm quality and subsequently, the larval development of this species. (C) 2014 Elsevier Inc All rights reserved.This work was funded by the European Community's Seventh Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL), and COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE). Victor Gallego, Ilaria Mazzeo and M. Carmen Vilchez had predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), respectively. David S. Penaranda was supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I). Rosa Baeza was supported by a contract funded by PRO-EEL. Authors want to thank Vicente Javier Moya Salvador for his technical assistance with gas chromatography analyses.Baeza Ariño, R.; Mazzeo, I.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Peñaranda, D.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2015). Relationship between sperm quality parameters and fatty acid composition of the muscle, liver and testis of European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 181:79-86. https://doi.org/10.1016/j.cbpa.2014.11.022S798618

Effect of thermal regime on fatty acid dynamics in male European eels (Anguilla anguilla) during hormonally-induced spermatogenesis

[EN] Little is known about the role of fat and fatty acids in European eel spermatogenesis. The aim of this research was to study the changes in fat content and to carry out a quantitative analysis of the fatty acid composition of the muscle, liver and gonad of European male eels during hormonally induced sexual maturation. Three different thermal regimes were used (two variable: T10 and T15; and one constant: T20) to replicate the changes in temperature that these fish experience during their transoceanic reproductive migration. Spermatogenesis was reached earlier in treatment T20, suggesting that spermatogenesis in the European eel is closely regulated by water temperature. Although eels lose body mass due to the period of fasting that accompanies gonadal growth, no significant changes were found in the fat and fatty acid content of the muscle during the experimental period. With regard to the liver, the levels of palmitic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids increased significantly at the start of the spermiation process in all the thermal treatments. In the testis, levels of EPA, arachidonic acid (ARA) and DHA remained constant during the maturation process whereas the levels of the rest of the fatty acids decreased significantly. The stability of the ARA and EPA levels in the testis may have a physiological significance, whereas the stability of the DHA levels may have a structural significance. The results suggest that the progression of spermiation is influenced by water temperature and demonstrate the importance of EPA, ARA and DHA in European eel reproduction. This study demonstrates that complementary research focusing on the lipid composition of commercial diets could improve the sperm quality of this species.Funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL). Victor Gallego, Ilaria Mazzeo and M. Carmen Vilchez had predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), respectively. David S. Penaranda was supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I). Rosa Baeza was supported by contract financed by PRO-EEL. The authors want to thank Vicente Javier Moya Salvador for his technical assistance with gas chromatography analyses.Baeza Ariño, R.; Mazzeo, I.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Peñaranda, D.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2014). Effect of thermal regime on fatty acid dynamics in male European eels (Anguilla anguilla) during hormonally-induced spermatogenesis. Aquaculture. 430:86-97. https://doi.org/10.1016/j.aquaculture.2014.03.045S869743

ESTUDIO DE LAS ALTERACIONES HEPÁTICAS E INTESTINALES DE DORADAS (SPARUS AURATA) ALIMENTADAS CON DIFERENTES NIVELES DE PROTEINAS VEGETALES

UNA SUSTITUCION CRECIENTE DE LA HARINA DE PESCADO POR UNA MEZCLA DE PROTEINAS VEGETALES (GUISANTE-ARROZ) FUE PROBADA EN DORADA(SPARUS AURATA) .SE REALIZO EL ESTUDIO DE LA POSIBLE INFLUENCIA DE LA DIETA EN LA INTEGRIDAD DEL INTESTINO Y LA ESTRUCTURA DEL HIGADO.Baeza Ariño, R. (2009). ESTUDIO DE LAS ALTERACIONES HEPÁTICAS E INTESTINALES DE DORADAS (SPARUS AURATA) ALIMENTADAS CON DIFERENTES NIVELES DE PROTEINAS VEGETALES. http://hdl.handle.net/10251/14048Archivo delegad

Carob seed germ meal as a partial substitute in gilthead sea bream (Sparus aurata) diets: Amino acid retention, digestibility, gut and liver histology

The use of carob seed germ meal (CG) as a substitute for fish meal was evaluated in fingerlings (average weight 10g) of gilthead sea bream fed isonitrogenous (46% Crude Protein, CP) and isolipidic (19.5% Crude Lipid, CL) diets containing four CG levels (0, 17, 34 and 52%). The duration of the trial was 83days. The diets were tested in triplicate, and the fish were fed to satiation twice daily. The apparent protein and energy digestibility coefficients decreased in response to the dietary inclusion of CG. The decreases in the values of the ADC ranged from 93% for the 0 diet to 80% for the 52 diet. The amount of digestible Arg in the diets increased with the inclusion of CG, from 3.2g Arg 100g -1 in the 0 diet to 4.81g Arg 100g -1 in the 52 diet. The digestible Arg content increased in the diets because the amounts of the remaining digestible amino acids decreased. The fish reached weights of 72, 72, 69 and 53g on the 0, 17, 34 and 52 diets, respectively. The 52 diet gave the lowest specific growth rate (SGR, 1.95%day -1) relative to those found for the other three diets (2.32%day -1). The 52 diet produced the least satisfactory results for feed intake (FI) and the food conversion ratio (FCR) (2.23g 100gfish -1day -1 and 1.38, respectively). The efficiency of retention of His, Phe and Leu in the fish fed the 52 diet showed the lowest values (17.3, 25.5 and 28.5%, respectively), but the efficiency of retention of Met in the fish fed the 52 diet showed the highest value (48.2%).In the distal intestine and liver, histological alterations were found in the fish fed the 34 and the 52 diets. In fish fed the 52 diet, the mucosal fold was significantly shorter and thinner and exhibited a smaller number of goblet cells. These changes could affect nutrient uptake by modifying the digestibility of the diet. The histology of the liver did not show severe degradation in any treatment. The results of the present experiment demonstrated that CG can be included at levels up to 34% in diets for short term feeding of gilthead sea bream without any adverse effects on fish growth and nutritive parameters.Present work was financed by AGROALIMED and GV/2010/046 (Conselleria de Agricultura, Pesca y Alimentacion, Generalitat Valenciana).Martínez-Llorens, S.; Baeza Ariño, R.; Nogales Mérida, S.; Jover Cerdá, M.; Tomás Vidal, A. (2012). Carob seed germ meal as a partial substitute in gilthead sea bream (Sparus aurata) diets: Amino acid retention, digestibility, gut and liver histology. Aquaculture. 338(341):124-133. https://doi.org/10.1016/j.aquaculture.2012.01.029S12413333834

Study of liver and gut alterations in sea bream, Sparus aurata L., fed a mixture of vegetable protein concentrates

Version of Record online (earlyview): 30 JUN 2014[EN] This study evaluated liver and gut alterations of gilthead sea bream, Sparus aurata L., fed diets in which 12 fish meal was substituted by a mixture (M) of pea protein concentrate (PPC) and rice protein concentrate 13 (RPC). Histological studies were conducted to evaluate the possible changes produced by four 14 experimental isolipidic (18%) and isoproteic (48%CP) diets with 30, 60 and 90% of fish meal substitution 15 by a pea and rice mixture and compared with a control diet (0%). 16 The results obtained in the present histological study showed significant changes especially in the case of 17 the 90% substitution in parameters such as thickness of the layers, goblet cells and villi measurements. 18 Structural integrity of the gut would not be significantly affected by a diet of up to 60% substitution. The 19 alterations observed in the liver could not only be attributed to diet but also to possible individual variations.This study was supported by the Universitat Politecnica de Valencia (UPV PAID-06-08).Baeza Ariño, R.; Martínez-Llorens, S.; Nogales Mérida, S.; Jover Cerda, M.; Tomas-Vidal, A. (2016). Study of liver and gut alterations in sea bream, Sparus aurata L., fed a mixture of vegetable protein concentrates. Aquaculture Research. 47(2):460-471. doi:10.1111/are.12507S46047147