Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

P. 122-130.e2eCryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage

Resultados preliminares de fertilizaciones artificiales realizadas con esperma crioconservado de rodaballo Scophthalmus maximus (Linnaeus, 1758)

Se crioconservó una mezcla de esperma procedente de cuatro machos sometidos a control de fotoperíodo. Después de valorar individualmente el esperma, la mezcla fue homogeneizada y preparada para la crioconservación. Se utilizaron dos diluyentes diferentes: la solución salina modificada de Ringer y la solución de Mounib, a los que se añadió como crioprotectores un 10% de yema de huevo y un 10% de dimetil sulfóxido (DMSO). Las relaciones esperma-diluyente utilizadas fueron 1:2 y 1:3. Una vez congelado en nitrógeno líquido a -196°C, se mantuvo durante 24 horas, período tras el cual fue descongelado y utilizado en la fecundación artificial. Las tasas de fecundación (85-92%) y de eclosión (23-35%) obtenidas con esperma crioconservado no difieren significativamente para los dos diluyentes ni para las dos diluciones (p < 0,001).Publicado

Natural feed after weaning improves the reproductive status of "Solea senegalensis" breeders

[EN] The aim of this study was to evaluate the effect of long term natural feeding in the subsequent reproductive status of 4 years old cultured Solea senegalensis, and to determine if the potential changes were structural or feeding dependent. To this aim, two different feeding regimes were used from one year after weaning and during the following 3 years; 1) a commercial dry food diet and 2) a natural feeding regime. After this period, the proportion of fluent males and the evolution of maturity stages of females over a breeding season were studied. A complete sperm quality analysis assessment was carried out, including individual volumes, motility, density and curvilinear, rectilinear and mean velocities of spermatozoa. Moreover, viability and apoptosis indexes were analyzed as indicator of molecular sperm membrane integrity. Additionally, a morphological characterization of the testes during the spawning season was conducted. Finally, both groups were fed with the same commercial pellets during one year to evaluate the effect of the diet of previous years on sperm quality. The results of this study showed how feeding can improve not only sperm quality and quantity, but also the proportion of fluent males and females in advanced maturity stages. All the sperm quality parameters resulted significantly higher in the group fed with a natural diet. Moreover, the number of apoptotic cells was significantly higher in the group fed with a commercial diet. According to the morphological features of the testes, the animals fed with a natural diet presented more basal position, less protuberances and irregular edges when compared with the animals fed with commercial diet. Interestingly, the progression of the spermatogenesis determined by the proportion of germ cells and the production of spermatozoa determined by the wider of the ducts system was also significantly larger in the natural diet group. After the standardization of the diets, mean volume per male and production of total motile cells were significantly higher in the group that was previously fed a natural diet, confirming structural improvements.S

Establishment and characterization of a sperm bank of Mediterranean seabass Dicentrarchus labrax (L., 1758) stock

Con el objetivo de conservar los recursos genéticos de la lubina Dicentrarchus labrax (L., 1758), se ha creado un banco de esperma con 42 machos de una población mediterránea. Se determinaron las características del esperma –concentración, motilidad y duración de la activación– que resultaron adecuadas para la fertilización artificial. La variabilidad genética de las lubinas procedentes del medio natural fue analizada mediante un conjunto de 10 loci microsatélites. A partir del genotipo de estos marcadores se obtuvo la huella de ADN de cada individuo con fines de identificación y estudios de paternidad. El análisis conjunto de todos los individuos permitió determinar los registros de las variables genéticas de este grupo. Los resultados muestran unos niveles altos de variabilidad genética representativos de una población natural y acentúan la importancia de este grupo como reservorio de esta especie en términos genéticosWith the aim of the conservation of the genetic resources of the European seabass Dicentrarchus labrax (L., 1758), a sperm bank of 42 males from a Mediterranean population has been established. The sperm characteristics –concentration, motility and duration of fertility– were assessed, and they proved suitable for artificial fertilization. The genetic structure of the original seabass broodstock from the wild was assessed by means of a set of 10 microsatellites. These genotype markers make it possible to obtain genetic fingerprinting from each individual for identification purposes. The population structure and genetic variability parameters found agree with those of natural populations from the same species, confirming its wild origin and enhancing its value as a genetic resource for the species in terms of genetic variabilityInstituto Español de Oceanografía. El proyecto RZ03-022 en el que se enmarca este trabajo ha sido financiado por el Programa Nacional de Recursos y Tecnologías Agroalimentarias, del Plan Nacional I+D+i del MCY

Effects of in vivo treatment with the dopamine antagonist pimozide and gonadotropin-releasing hormone agonist (GnRHa) on the reproductive axis of Senegalese sole (Solea senegalensis)

Publicado

Estudio preliminar sobre una posible inhibición dopaminérgia en la reproducción del lenguado senegalés (Solea senegalensis)

El objetivo del presente estudio fue evaluar el efecto del sistema dopaminérgico sobre la reproducción del lenguado senegalés (Solea senegalensis). Para ello, reproductores de lenguado senegalés nacidos y criados en cautividad (generación F1) fueron tratados con 1) GnRHa, 2) inhibidor dopaminérgico (pimozide) o 3) un tratamiento combinado (GnRHa + pimozide). El efecto de los tratamientos se evaluó a nivel de producción de huevos y esperma (cantidad y calidad); además se estudió el efecto sobre niveles plasmáticos de esteroides sexuales y desarrollo gonadal (histología). Las hembras no mostraron una estimulación en la ocurrencia de puestas por el tratamiento combinado GnRHa + pimozide respecto a aquellas tratadas solo con GnRHa. Sin embargo, los machos que recibieron el tratamiento combinado mostraron una mayor estimulación en la producción de esperma y en el grado de maduración testicular respecto a aquellos que recibieron solo GnRHa o pimozide. Los resultados indican que el sistema dopaminérgico no parece ejercer una acción fuerte sobre la ocurrencia de puestas en hembras, aunque podría estimular la espermiación en machos de lenguado senegalés.Preliminary study on dopaminergic inhibition in Senegalese sole reproduction (Solea senegalensis) The aim of the present study was to evaluate the effect of the dopaminergic system on Senegalese sole (Solea senegalensis) reproduction. For this purpose, Senegalese sole breeders hatched and raised in captivity (F1 generation) were treated with 1) GnRHa, 2) a well-known dopaminergic inhibitor (pimozide) or 3) a combined treatment (GnRHa + pimozide). The effect of each treatment was determined by studying spawning occurrence and sperm (quantity and quality); in addition, effects on sex steroids levels and on gonadal development (histology) were also considered. Females did not show any improvement of spawning occurrence by combining GnRHa and pimozide respect to those females treated only with GnRHa. However, males which received the combined treatment showed a greater stimulation of sperm production and testicular maturity than those males treated with a single GnRHa or pimozide treatment. Our results indicate that dopaminergic system do not seem to play a strong action on eggs spawning in females, but may stimulate spermiation in Senegalese sole males

Estudio del ciclo del nitrógeno en la bahía de Santander

Publicado

Contribution to the management and conservation of gametes of the turbot Scophthalmus maximus (Linnaeus, 1758)

El propósito de esta memoria es contribuir a la mejora en la gestión de la reproducción y el perfeccionamiento en la conservación de gametos de rodaballo Scophthalmus maximus (L, 1758). La demanda por parte del sector productor de rodaballo de un control más riguroso de la reproducción y una gestión genética más conveniente de cara al incremento de la producción es manifiesta. En la gestión de reproductores se aprecia una variabilidad considerable en las tasas de fecundación, que puede ser atribuible a la calidad de los gametos o a la eficacia del método de fecundación. Por otro lado, la evolución del cultivo de esta especie requiere métodos que prolonguen la viabilidad de los gametos de alta calidad. Estas circunstancias han motivado que el Consejo Internacional para la Exploración del Mar (CIEM), en el ámbito de la acuicultura y siguiendo las líneas prioritarias del proyecto Programa Marco, recomiende el desarrollo de la investigación sobre la calidad de gametos, la optimización de métodos y el desarrollo de técnicas que permitan prolongar en el tiempo la viabilidad de gametos de alta calidad. A tenor de estas prioridades, el objetivo de esta tesis ha sido orientar en la optimización de la gestión de los stocks de reproductores en el cultivo de rodaballo para facilitar la determinación del método idóneo de fecundación artificial y el logro de la conservación de los gametos a corto y largo plazo, todo ello con el propósito de ir aproximando al rendimiento máximo los resultados de las explotaciones industriales. Adicionalmente, se ha realizado la evaluación del crecimiento de larvas y alevines producidos con gametos conservados para así poder garantizar al sector la fiabilidad de las técnicas desarrolladas.The turbot Scophthalmus maximus (Linnaeus, 1758) farming industry demands greater control over fish reproduction and genetic management in order to raise production levels. In reproductive management, there is a huge variation in fertility rates, which could be due to the quality of the gametes or the different fertilization methods currently in use. Moreover, the improvement of the species requires methods able to prolong the viability of high-quality gametes. Given this scenario, in the area of aquaculture, the International Council for the Exploration of the Sea (ICES), and the priority guidelines of the Framework Programme recommend studies on gamete quality, optimization of methods, and the development of techniques which would make it possible to prolong the viability of high-quality gametes over time. Following these priorities, the aim of this thesis has been to optimise reproductive stock management in turbot farming, in order to determine the most appropriate method for artificial fertilization and short- and long-term conservation of gametes, with the aim of obtaining maximum productivity in industrial settings. Furthermore, an assessment was made of the growth of larvae and fry produced with conserved gametes, in order to confirm the feasibility of these techniques in the turbot farming industry.Instituto Español de Oceanografí