martes, 22 de octubre de 2019

Patricia Diogo defiende su Tesis Doctoral en Faro

El pasado viernes 18, nuestra colega defendió su Tesis Doctoral, titulada: Cryopreservation of zebrafish germ cells: technological improvements and methodological standardization for gene banking and management en la Universidad del Algarve (Portugal). Durante la sesión pública Patricia llevó a cabo una presentación de 30 minutos en la que resumió su trabajo, que consta de 6 artículos, 4 de ellos ya publicados. Seguidamente, se estableció una discusión con los distintos miembros del tribunal en la que la candidata demostró su dominio del tema. Enhorabuena Dra. Diogo!

El tribunal estuvo presidido por el Dr. Sergio Manuel Machado Jesus, Presidente del Consejo Científico de la Facultad de Ciencias y Tecnología de la Universidad del Algarve. El Dr. Ákos Horváth (Szent István University, Hungría) y yo mismo (desde la UPV), actuamos como principales oponentes, realizando la mayor parte de las preguntas que se formularon durante la discusión que siguió a la presentación de Patricia. Los otros dos ponentes fueron el Dr. Paulo Gavaia (co-supervisor de la Tesis junto a Elsa Cabrita) y la Dra. Catarina Oliveira, ambos investigadores del Centro de Ciencias del Mar (CCMAR) de la Universidad del Algarve.

El resumen de la tesis es el siguiente:

Due to the increasing number of zebrafish (Danio rerio) mutant and transgenic lines, there is a high demand for assisted reproductive techniques to support facility management. Efficient zebrafish sperm cryopreservation is a pressing necessity to manage and preserve the valuable zebrafish genetic resources.
Although zebrafish sperm cryopreservation was first attempted more than 30 years ago, protocols still lack standardization, which translates into high variability in post-thaw sperm quality and in vitro fertilization success. Therefore, the present thesis aims to improve the current methodologies used for zebrafish sperm cryopreservation and broodstock management towards the standardization of procedures in this species.
The introductory context of the present thesis is approached in chapter 1. In this chapter the relevance of zebrafish model is discussed as well as this species sperm cryopreservation usefulness. The main factors affecting sperm quality and the application of reliable quality analysis are discussed in this chapter. The final objective of sperm cryopreservation is to obtain high quality offspring and therefore in vitro fertilization, early development and offspring quality analysis are important tools for the optimization of sperm cryopreservation methodologies. The current knowledge in sperm cryopreservation fundamentals is approached in this chapter, as well as the main advances and bottlenecks in zebrafish sperm cryopreservation.
In chapter 2, the zebrafish sperm motility activation was assessed under different conditions of water temperature and conductivity. The environmental conditions present in the fertilization microenvironment are responsible for the mechanism of spermatozoa motility activation and metabolic modulation that influence the probability of fertilization success. Zebrafish is commonly reared at 28°C, but with variable water conductivity conditions among facilities. However, sperm motility analysis is routinely performed with distilled water at room temperature. We aimed to understand the effect of water temperature and conductivity on sperm motility and fertilization ability. Water at 28°C with lower water conductivity (0 and 700 μS/cm) improve sperm motility parameters.
Standardization of the water conditions (of system water and activation medium used for motility analysis) among facilities is highly relevant to improve the reproducibility of sperm quality analysis and thus, to predict with higher accuracy fertilization ability.
Successful cryopreservation depends on high quality sperm, which depends on the quality of breeders. Consequently, broodstock selection and management is a priority to improve sperm cryopreservation. The broodstock diet has a preponderant effect on gamete quality, particularly in phospholipids and antioxidants content which are known to promote spermatogenesis. Therefore, in chapter 3 we aimed to determine the effects of a tailor-made purified diet supplemented with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the zebrafish reproductive performance, gamete quality and larval skeletal malformations. Both dietary supplementations with phospholipids improved sperm motility and eggs quality, however PC increased the incidence of skeletal malformations on the offspring, as previously observed in other teleosts. Although dietary phospholipids classes have a role in the ossification process of the vertebral column in teleosts, its mechanisms are still to be understood. Therefore, the development and use of a standardized diet for zebrafish broodstock is essential to reduce the variability of the reproductive performance among facilities. In chapter 4, the selection of optimal age and minimum sperm collection frequency was evaluated, since these factors are essential to obtain high quality samples. Our results indicate that young males (6-8 months) showed higher sperm quality and require a minimum of 14 days between sperm collections to recover sperm plasma membrane viability.
An important bottleneck in cryopreservation is the liquid nitrogen requirement for storage. Therefore, it was established in chapter 5 a new cryopreservation method using an electric ultrafreezer (-150°C) as an alternative to liquid nitrogen, for the first time in a teleost species. This protocol reaches a fast cooling rate (-66°C/min) in one single step and yields higher sperm viability and DNA integrity in comparison to the traditional methods (-20°C/min in liquid nitrogen). The synergy obtained by the combination of cryoprotectants is a successful cryopreservation strategy that can be beneficial in the optimization of zebrafish sperm cryopreservation. Therefore, it was selected the most adequate cryoprotectant combination that generates offspring with normal skeletogenesis. Data show that 15% of DMF with 50 mM of bicine or 10% of egg yolk is beneficial for the quality of zebrafish offspring sired by cryopreserved sperm. To the best of our knowledge, this is the first report on skeletal development of zebrafish offspring sired by cryopreserved sperm performed with different extender compositions.
Zebrafish is especially useful to investigate some of the most prominent human diseases such as diabetes. Among other consequences, diabetes (type I and II) causes disturbances in the male reproductive system, since glucose metabolism is an important event not only in spermatogenesis but also in mature spermatozoa metabolism. In chapter 6 we aimed to validate zebrafish as a useful model organism to investigate male reproductive dysfunctions mechanisms caused by type I diabetes. In this chapter, sperm cryopreservation was applied to a relevant zebrafish model of type I diabetes. The transgenic zebrafish under diabetic conditions shows higher levels of insulin a (insa), insulin receptor a (inra) and glucose carrier 2 (slc2a2) transcripts in spermatozoa when compared to the controls. This is because gametogenesis occurred under diabetic conditions, changing transcription in the germline. Consequently, spermatozoa carry the imprinted transcripts that will be transmitted during fertilization. Sperm quality (motility, viability and DNA integrity) was lower in the transgenic fish under (transient) diabetic state as observed in human and mouse model. Sperm cryopreservation affects sperm quality of fish both under diabetic and non-diabetic conditions. However, diabetic conditions were detrimental in sperm freezability, which can be explained by the lower initial sperm quality. In this chapter zebrafish was validated as a useful model organism to investigate male reproductive dysfunctions mechanisms caused by type I diabetes.
Relevant differences between different zebrafish lines are evidenced in terms of sperm quality and susceptibility to damage, which suggests that it is an important factor to consider while establishing sperm cryopreservation protocols. This thesis offers new insights and a set of guidelines on breeder’s management and sperm cryopreservation to improve zebrafish husbandry practices.

lunes, 21 de octubre de 2019

Nuestro último artículo, aceptado en Aquaculture

Dietary amino acids impact sperm performance traits for a catadromous fish, Anguilla anguilla reared in captivity

I.A.E. Butts, G.S. Hilmarsdóttir, V. Zadmajid, V. Gallego, L.K. Holst, J.G. Støttrup, C. Jacobsen, M. Krüger-Johnsen, S.N. Politis, J.F. Asturiano, J. Tomkiewicz

Little is known about the role of dietary amino acids on male reproductive performance and gamete quality in fishes. Thus, the objective of this study was to investigate how “enhanced” feeds (EH- 4, EH-5, EH-6), with modified amino acid composition, and the standard on-growing diet (DAN-EX) impact body composition, milt biochemistry, and sperm performance in male European eel, Anguilla anguilla. The fatty acid composition of EH-4, EH-5, and EH-6 was similar but differed to that in DAN-EX, while amino acid composition varied between all four diets. Diet did not influence organ-somatic indices (e.g. HSI, GSI), while males fed EH-4 were heavier than other groups. Arginine, alanine, and lysine were the most abundant amino acids in milt (>11%), followed by glycine, aspartic acid, valine, glutamic acid, and leucine (>5.66%). Diet mpacted milt arginine, serine, proline, methionine, and histidine levels. Specifically, males fed DAN-EX, EH-4, and EH-5 had the highest percentages of arginine, while males fed EH-4 to EH-6 had higher percentages of serine. Proline was most abundant in males fed DAN-EX, EH-5, and EH-6. Both methionine and histidine were detected at low percentages (<2%), and were impacted by diet, where males fed EH-4 and EH-5 had higher percentages of methionine, and males fed DAN-EX, EH-4, and EH-6 had the highest percentage of histidine. Milt production increased over time, where eels fed EH-4 and EH-6 showed the highest probability of producing suited milt volumes (>0.5 mL) for fertilization procedures. Spermatocrit (43.1 ± 1.80%) did not differ between the diets (ranged from 37.57 to 47.21%). Dietary regime had an impact on sperm motility, such that eels fed EH-5 and EH-6 had the greatest percentage of motile cells. In addition, fish fed EH-5 and EH-6 (or DAN-EX) had the fastest swimming sperm. Spermatogenic maturity index of hormonally treated eels varied within groups but did not differ between dietary treatment groups after 9 weeks of injections (ranged from 0.54 to 0.80). The most interesting amino acids to scrutinize from PCA plots were proline, histidine, and valine as well as lysine and arginine. Here, eels with highly motile sperm had milt with high relative proportions of proline, histidine, and valine, but were particularly low in lysine and arginine. Together, our findings add evidence that certain amino acids regulate milt biochemistry, and that male ejaculate traits may be promoted by amino acid intake. Further studies to evaluate effects of supplemented amino acid diets on fertilization ability and inter-linked early developmental stages are required.

sábado, 12 de octubre de 2019

Workshops de EELRIC y del EAS Eel Thematic Group en Berlín

Coincidiendo con el inicio del Aquaculture Europe 2019 (Berlín, Alemania), el día 7 de octubre se celebraron dos reuniones de expertos en anguilas, a las que acudimos Luz Pérez y yo mismo.
Por la mañana, se llevó a cabo el 4th Workshop de EELRIC, la plataforma de investigadores sobre anguila, coordinado por Arjan Palstra. Durante la reunión, los diferentes grupos participantes realizamos presentaciones cortas para comunicar nuestros últimos resultados y publicaciones, informamos de los proyectos vigentes y discutimos sobre las opciones para lograr financiación para actividades comunes de investigación. Participamos grupos de España, Holanda, Italia, Dinamarca, Francia, Alemania, Japón y Nueva Zelanda.  
Por la tarde, se desarrolló un Workshop del European Aquaculture Society (EAS) Thematic Group on European eel on the development of breeding and hatchery Technology (EEL-DHBT), organizado por Jonna Tomkiewicz, y que se tituló: Impacts and Insights - progress and persistent challenges in develop­ing eel hatchery technology en la que algunos colegas presentaron el estado de sus investigaciones en varios aspectos relacionados con la reproducción de anguilas en varios puntos del mundo.