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Microdiet formulation with phospholipid modulate zebrafish skeletal development and reproduction

Publication . Martins, Gil; Diogo, Patricia; Santos, Tamara; Cabrita, Elsa; Pinto, Wilson; Dias, Jorge; J. Gavaia, Paulo

Dietary phospholipids' (PLs) content, origin, and profile are known to affect fish development and reproductive performance, but their effects in zebrafish (Danio rerio) nutrition are still poorly investigated. Therefore, this study aimed to assess the effect of practical microdiets containing plant-based and marine PL sources in zebrafish growth, survival, skeletal development, and reproductive performance. Reproductive performance was evaluated according to sperm motility, number of eggs, egg morphometry, hatching rate, and offspring standard length at 5 days postfertilization (dpf). For this purpose, seven microdiets were used, where two control diets were tested along with a supplementation with soybean lecithin (SL) as a plant-based PL source, and krill oil (KO) and copepod oil (CO) as marine PL sources, or in combinations (SLCO and SLKO). KO supplementation decreased larval growth performance and induced severe skeletal anomalies. SL supplementation reduced sperm total motility but improved offspring length at 5 dpf. CO supplementation increased sperm motility and the number of spawned eggs. Our results showed that a careful selection of the origin of dietary PL sources for microdiet formulation is critical to ensure adequate skeletal development and reproductive success. This study contributes to the improvement of zebrafish microdiet formulation and optimization of zebrafish husbandry practices.

2020-02Journal article

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Analysis of sperm quality in a type I diabetes zebrafish model

Publication . Diogo, Patricia; Eufrásio, Ana; Martins, Gil; Cardeira, João; Cancela, M. Leonor; Cabrita, Elsa; Gavaia, Paulo

Diabetes is a fast growing disease in human populaon and the study of its impact on mammalian reproducve traits has been con-troversial. Some authors showed a negave eect on sperm mol-ity and DNA fragmentaon in some species, while others failed to detect any eects. In the present study zebrash was used as a model to study the eect of diabetes in sperm traits such as mol-ity, viability and DNA fragmentaon

2016-10Journal article

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Selection criteria of Zebrafish male donors for sperm cryopreservation

Publication . Diogo, Patricia; Martins, Gil; Eufrásio, Ana; Silva, Tomé; Cabrita, Elsa; Gavaia, Paulo

Selection criteria for sperm cryopreservation are highly relevant in zebrafish since sperm quality is particularly variable in this species. Successful cryopreservation depends on high-quality sperm, which can only be ensured by the selection of breeders. Consequently, male selection and management are a priority to improve cryopreservation, and therefore, this study aimed to characterize optimal age and sperm collection frequency in zebrafish. For this purpose, males from wild type (AB) and from a transgenic line [Tg(runx2:eGFP)] were sampled at 6, 8, 12, and 14 months. For each age, sperm were collected at time 0 followed by samplings at 2, 7, and 14 days of rest. Sperm quality was assessed according to motility and membrane viability parameters. Quality assessment showed that Tg(runx2:eGFP) displayed significantly higher motility than AB and younger males showed higher motility in both lines. Sperm collection frequency affected membrane viability. While AB fish recovered sperm viability after 14 days of rest, Tg(runx2:eGFP) could not recover. Consequently, it may be important to study the sperm quality of each zebrafish line before sperm cryopreservation. Taking into consideration the results achieved in both lines, sperm collection should be performed between 6 and 8 months of age with a minimum collection interval of 14 days.

2019Journal article

Open Access

Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis

Publication . Diogo, Patricia; Martins, Gil; Nogueira, Rita; Marreiros, Ana; Gavaia, Paulo; Cabrita, Elsa

The synergy obtained by the combination of cryoprotectants is a successful strategy that can be beneficial on the optimization of zebrafish sperm cryopreservation. Recently, a protocol was established for this species using an electric ultrafreezer (-150 degrees C) performing cooling rate (-66 degrees C/min) and storage within one step. The ultimate objective of sperm cryopreservation is to generate healthy offspring. Therefore, the objective of this study was to select the most adequate cryoprotectant combination, for the previously established protocol, that generate high quality offspring with normal skeletogenesis. Among the permeating cryoprotectant concentrations studied 12.5% and 15% of N,N-dimethylformamide (DMF) yielded high post-thaw sperm quality and hatching rates. For these two concentrations, the presence of bovine serum albumin (10 mg/mL), egg yolk (10%), glycine (30 mM) and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro fertilization success and offspring skeletal development (30 days post fertilization). Higher concentration of permeating cryoprotectant (15%) decreased the incidence of deformed arches and severe skeletal malformations, which suggests higher capacity to protect the cell against cold stress and DNA damage. Extender containing 15% DMF with Ctrl, Bicine and egg yolk were the non-permeating cryoprotectants with higher post-thaw quality. The use of these compounds results in a reduction in vertebral fusions, compressions and severity of skeletal malformations in the offspring. Therefore, these extender compositions are beneficial for the quality of zebrafish offspring sired by cryopreserved sperm with 66 degrees C/min freezing rate. To the best of our knowledge, this is the first report on skeletal development of the offspring sired by cryopreserved sperm performed with different freezing media compositions in zebrafish.

2019-12Journal article

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