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- Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesisPublication . Diogo, Patricia; Martins, Gil; Nogueira, Rita; Marreiros, Ana; Gavaia, Paulo; Cabrita, ElsaThe 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.
- Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in Zebrafish (Danio rerio)Publication . Martins, Gil; Diogo, Patricia; Pinto, Wilson; Gavaia, PauloZebrafish is a model species with a high variability of feeding regimes among fish facilities. The use of live feeds for early life stages is a common practice, and few studies have focused early weaning into microdiets. The lack of standardized feeding protocols among research facilities promotes discrepancies in biological performances, and few studies relate dietary regimes to zebrafish development. The objective of this work was to assess the effect of an early transition into microdiets in zebrafish development by evaluating growth, survival, reproductive performance, and skeletal anomalies. These parameters were assessed in one group exclusively fed on Artemia nauplii and two groups fed on microdiets (commercial and experimental). Results showed that an early weaning with the two microdiets significantly improved zebrafish growth and reproductive performance, while a decrease in incidence of vertebral column anomalies was observed. A high survival was also maintained in fish fed microdiets at an early developmental stage when comparing to exclusive Artemia nauplii feeding. In conclusion, early weaning with high quality microdiets is beneficial for zebrafish growth, reproductive performance, and skeletal development, contributing to the standardization of zebrafish husbandry practices.
- Analysis of sperm quality in a type I diabetes zebrafish modelPublication . Diogo, Patricia; Eufrásio, Ana; Martins, Gil; Cardeira, João; Cancela, M. Leonor; Cabrita, Elsa; Gavaia, PauloDiabetes is a fast growing disease in human populaon and the study of its impact on mammalian reproducve traits has been con-troversial. Some authors showed a negave eect on sperm mol-ity and DNA fragmentaon in some species, while others failed to detect any eects. In the present study zebrash was used as a model to study the eect of diabetes in sperm traits such as mol-ity, viability and DNA fragmentaon
- Health behaviours as predictors of the Mediterranean diet adherence: a decision tree approachPublication . Bôto, Joana Margarida; Marreiros, Ana; Diogo, Patricia; Pinto, Ezequiel; Mateus, Maria PalmaObjective: This study aimed to identify health behaviours that determine adolescent's adherence to the Mediterranean diet (MD) through a decision tree statistical approach. Design: Cross-sectional study, with data collected through a self-fulfilment questionnaire with five sections: (1) eating habits; (2) adherence to the MD (KIDMED index); (3) physical activity; (4) health habits and (5) socio-demographic characteristics. Anthropometric and blood pressure data were collected by a trained research team. The Automatic Chi-square Interaction Detection (CHAID) method was used to identify health behaviours that contribute to a better adherence to the MD. Setting: Eight public secondary schools, in Algarve, Portugal. Participants: Adolescents with ages between 15 and 19 years (n 325). Results: According to the KIDMED index, we found a low adherence to MD in 9 center dot 0 % of the participants, an intermediate adherence in 45 center dot 5 % and a high adherence in 45 center dot 5 %. Participants that regularly have breakfast, eat vegetable soup, have a second piece of fruit/d, eat fresh or cooked vegetables 1 or more times a day, eat oleaginous fruits at least 2 to 3 times a week, and practice sports and leisure physical activities outside school show higher adherence to the MD (P < 0 center dot 001). Conclusions: The daily intake of two pieces of fruit and vegetables proved to be a determinant health behaviour for high adherence to MD. Strategies to promote the intake of these foods among adolescents must be developed and implemented.
- Type I Diabetes in Zebrafish reduces sperm quality and increases Insulin and Glucose transporter transcriptsPublication . Diogo, Patricia; Martins, Gil; F. Simao, Marcio; Marreiros, Ana; Eufrásio, Ana Catarina; Cabrita, Elsa; J. Gavaia, PauloType I diabetes is a prominent human pathology with increasing incidence in the population; however, its cause is still unknown. This disease promotes detrimental effects on reproduction, such as lower sperm motility and DNA integrity. Hence, the investigation of the underlying mechanisms of this metabolic disturbance in reproduction and its transgenerational consequences is of the utmost importance. The zebrafish is a useful model for this research considering its high homology with human genes as well as its fast generation and regeneration abilities. Therefore, we aimed to investigate sperm quality and genes relevant to diabetes in the spermatozoa of Tg(ins:nfsb-mCherry) zebrafish, a model for type I diabetes. Diabetic Tg(ins:nfsb-mCherry) males showed significantly higher expression of transcripts for insulin a (insa) and glucose transporter (slc2a2) compared to controls. Sperm obtained from the same treatment group showed significantly lower sperm motility, plasma membrane viability, and DNA integrity compared to that from the control group. Upon sperm cryopreservation, sperm freezability was reduced, which could be a consequence of poor initial sperm quality. Altogether, the data showed similar detrimental effects related to type I diabetes in zebrafish spermatozoa at the cellular and molecular levels. Therefore, our study validates the zebrafish model for type I diabetes research in germ cells.
- Selection criteria of Zebrafish male donors for sperm cryopreservationPublication . Diogo, Patricia; Martins, Gil; Eufrásio, Ana; Silva, Tomé; Cabrita, Elsa; Gavaia, PauloSelection 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.
- Reduction of skeletal anomalies in meagre (Argyrosomus regius, Asso, 1801) through early introduction of inert dietPublication . Martins, Gil; Ribeiro, Laura; Candeias-Mendes, Ana; Diogo, Patricia; Gamboa, Margarida; Barata, Marisa; Leonor Cancela, M.; Pousao-Ferreira, Pedro; J. Gavaia, PauloThe consolidation of meagre (Argyrosomus regius) in aquaculture requires an understanding and optimization of larval rearing and nutritional conditions. The aim of this study was to analyse the effects of an early introduction of inert diets during larval rearing, on growth performance, digestive enzymes activity and development of skeletal anomalies. This study evaluated the effects of three different timings for the introduction of inert diet during larval rearing: a control group (CTRL) where inert diet was initiated at 14 days after hatching (DAH) and two treatment groups that had an earlier introduction of inert diet at 8 DAH (T1) and 11 DAH (T2). Meagre larvae exhibited similar pancreatic and intestinal enzymatic activities among the different dietary treatments. No differences in the overall prevalence of anomalies were observed between treatments at 25 or 50 DAH, however, a significant reduction was observed in all groups with the transition from larval to juvenile stage. The precocious introduction of inert diet shifted the distribution of vertebral anomalies to a more anterior vertebral column region. Altogether, this study shows that earlier introduction of inert diets in meagre hatcheries can be beneficial for meagre production in aquaculture.
- Microdiet formulation with phospholipid modulate zebrafish skeletal development and reproductionPublication . Martins, Gil; Diogo, Patricia; Santos, Tamara; Cabrita, Elsa; Pinto, Wilson; Dias, Jorge; J. Gavaia, PauloDietary 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.