Step by step optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772)

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the industry. Thus, the development of protocols for sperm storage are crucial. Four sequential experiments were conducted to optimize a sperm cryopreservation protocol for this species. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. No significant differences (p > 0.05) were detected between the control samples and cryoprotectants at concentration up to 10% DMSO, 10% propanediol, and 20% methanol in terms of motility parameters. Second, using the highest non-toxic concentrations of cryoprotectants, sperm was cryopreserved in 0.5 mL straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5 cm) that correspond to different freezing rates. Motility parameters after freezing/thawing decreased for all the cryoprotectants (p  0.05) between the two thawing rates. The best results were obtained using 10% DMSO. Finally, the fertilization capacity of cryopreserved sperm (10% DMSO and thawed at 5 °C for 1 min) was tested against fresh sperm using two spermatozoa:egg ratios and 4 h gametes contact time. The ratio of eggs with normal cell cleavage, abnormal cleavage or undeveloped were counted at the 2-4 cell stage. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5 × 104 spermatozoa:egg compared with fresh sperm (p  0.05). To cryopreserve spotted wolffish sperm it is recommended to use 10% DMSO, loaded in 0.5 mL straws, freeze at a height between 4.5 (-14.05 °C/min) and 7.5 cm (-5.9 °C/min) from liquid nitrogen for 10 min and thaw for 1 min at 5 °C (177.9 °C/min). In vitro fertilization with cryopreserved sperm should be performed with a concentration of at least 5 × 105 spermatozoa per egg.WOLFSTORE project (AF0078) supported by the MABIT program from Norway. JS was supported by a Cost action FA1205 AQUAGAMETE and an ERASMUS grant.info:eu-repo/semantics/publishedVersio

Antioxidants in fish sperm and the potential role of melatonin

In recent years, the effects of novel antioxidants have played an important role in the research focusing on fish cell protection. As food demand grows, aquaculture production becomes more intensive, and fish are more exposed to oxidative stress conditions, like high densities, temperature shifting, frequent fish handling and samplings, and prophylactic or disease treatments, which expose fish to a different environment. Particularly in reproduction, germ cells lose antioxidant capacity with spermatogenesis, as spermatozoa are more prone to oxidative stress. Antioxidants have been used in a variety of fish physiological problems including in reproduction and in the establishment of cryopreservation protocols. From the most used antioxidants to natural plant food and herbs, and endogenously produced antioxidants, like melatonin, a review of the literature available in terms of their effects on the protection of fish spermatozoa is presented here in a classified structure. Several direct and indirect approaches to improve gamete quality using antioxidants administration are mentioned (through feed supplementation or by adding in cryopreservation media), as well as factors affecting the efficiency of these molecules and their mechanisms of action. Special attention is given to the unclear melatonin pathway and its potential scavenger activity to prevent and counteract oxidative stress damage on fish spermatozoa.This work was part of a PhD programme funded by the Portuguese FCT—Foundation for Science and Technology, through the grant SFRH/BD/148280/2019 to F.F, and by “Fundo Social Europeu”. It had also the contribution of projects ReproF1 and Serinova (Programa Operacional Mar2020, MAR-16-02-01-FMP-0059 and MAR-16-02-01-FMP-0064), Portuguese national funds from FCT (Foundation for Science and Technology) through projects UIDB/04326/2020 and PTDC/CVTCVT/4109/2020 (SpermAntiOx) and contract DL 57/2016/CP1361/CT0007 to C.C.V.O.info:eu-repo/semantics/publishedVersio

Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review

Protocols for the cryopreservation of fish gametes have been developed for many different fish species, in special, freshwater salmonids and cyprinids. Methods for sperm freezing have progressed during the last decades due to the increasing number of potential applications: aquaculture (genetic improvement programs, broodstock management, helping with species having reproductive problems), biotechnology studies using model fish species (preservation of transgenic or mutant lines), cryobanking of genetic resources from endangered species, etc. This mini-review tries to give an overview of the present situation of this area of research, identifying the main challenges and perspectives, redirecting the reader to more in-depth reviews and papers. (C) 2016 Elsevier Inc. All rights reserved.European Training Network IMPRESS (Marie Sklodowska-Curie Actions) from PROMAR programme [642893]COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) from PROMAR programmeResearch Centre of Excellence from PROMAR programme [9878-3/2016/FEKUT, KMR_12-1-2012-0436]NKFIH (OTKA) from PROMAR programme [109847]KLING from PROMAR programme [31-03-05FEP-73]CRIOBIV from PROMAR programme [31-03-05-FEP-59]REPLING from PROMAR programme [31-03-05-FEP-69]info:eu-repo/semantics/publishedVersio

The use of sand substrate modulates dominance behaviour and brain gene expression in a Flatfish Species

Physical complexity adds physical enrichment to rearing conditions. This enrichment promotes fish welfare and reduces detrimental characteristics that fish develop in captivity. Senegalese sole (Solea senegalensis) is an important species for European aquaculture, where it is reared in intensive conditions using fibreglass tanks. However, reproductive dysfunctions present in this species do not allow it to complete its life cycle in captivity. Recently, dominance behaviour has been studied to try to solve this problem. The present study aimed to assess the effect of sand as environmental enrichment in the dominance behaviour and brain mRNA abundance of Senegalese sole juveniles. Four tanks of sole (n = 48 fish in total) were established in two different environments (with and without sand). Juveniles were subjected to dominance tests of feeding and territoriality. Behaviours analysed by video recordings related to the distance from the food delivered and harassment behaviour towards other individuals (e.g., resting of the head on another individual). In both environments, dominant sole were the first to feed, displayed more head-resting behaviour and dominated the area close to the feeding point, where the events were reduced in fish maintained in the sand. mRNA expression related to differentiation of dopamine neurons (nr4a2) and regulation of maturation (fshra) were significantly upregulated in dominant fish in the sand environment compared to dominants maintained without sand. The use of an enriched environment may affect Senegalese sole dominance, enhance welfare and possibly advance future maturation.Programa Operacional Mar2020, MAR-16-02-01-FMP-0059; d CONDISOLE (CeiMar funds, CEIJ-005); LA/P0101/2020;info:eu-repo/semantics/publishedVersio

O empreendedorismo no feminino na região do Algarve

Partindo do cruzamento da Sociologia do Trabalho com os Estudos de Género, este trabalho pretende contribuir para o estudo do empreendedorismo em Portugal. O Empreendedorismo é aqui compreendido como um modo-de-estar na vida que é também fruto de uma escolha racional do e da agente. O estudo da mulher no papel de empreendedora vem reforçar o seu empoderamento, ou seja, a afirmação do seu papel na sociedade e nos variados contextos da sua vida, abordagem com a qual assumidamente nos identificamos. Este trabalho procura assim investigar o empreendedorismo de mulheres no território do Algarve de forma a compreender os eventuais padrões que o caracterizam, as diversidades, dificuldades e conquistas destes percursos e o contributo de cada ação individual para o desenvolvimento local. Utilizando uma abordagem qualitativa, mas com recurso pontual a diferentes técnicas de recolha para potenciar a triangulação de dados, a investigação logrou encontrar perfis fortemente marcados pela diversidade onde são irrelevantes para além do género, outras variáveis como a idade e a nacionalidade. Os resultados sugerem uma primeira tipologia de empreendedorismo no feminino que, sendo indissociável do contexto específico desta pesquisa, pretende contribuir para estudos futuros sobre a temática a nível global.Based in the fields of Sociology of Work and Gender Studies, this work aims to contribute to the study of entrepreneurship in Portugal. Entrepreneurship is understood as an attitude towards life that is also the fruit of a rational choice of the agent. The study of women in the role of entrepreneurial can reinforce their empowerment, that is, the affirmation of their role in society and in the diverse dimensions contexts of their lives, an approach with which we clearly identify. Therefore, this work seeks to investigate the entrepreneurship of women in the Algarve, to understand the patterns, diversities, difficulties and achievements of these professional paths and the contribution of each individual action to local development. Using a qualitative approach, but with a point resource to different data collection techniques, looking forward the triangulation of this issue, the research was able to find profiles strongly marked by the diversity, where, besides gender other variables revealed irrelevant like as age and nationality. The results propose a first typology of entrepreneurship in the feminine that being inseparable from the specific context of this research intends to contribute to future studies on the theme at the global level

O papel político-social do Diretor Escolar : perceções de uma Directora de Agrupamento de Escolas

Dissertação de mestrado, Educação (Área de especialidade em Administração Educacional), Universidade de Lisboa, Instituto de Educação, 2017Este trabalho reportou um estudo centrado na figura do diretor de um Agrupamento de Escolas, localizado na periferia de Lisboa, tendo como objetivo identificar, descrever e analisar as lógicas e racionalidades que conduzem ao desempenho e construção do papel político-social, junto dos atores educativos de um Agrupamento de Escolas. O contexto teórico sobre a problemática em análise recaiu sobre os conceitos de regulação, cultura e organização. Centrando-se o estudo no diretor escolar teve como conceitos centrais para a análise da ação deste ator educativo, os papéis, as conceções e as lógicas da ação. Trata-se de um estudo qualitativo e interpretativo, estruturado num estudo de caso. Como instrumentos de recolha de dados recorremos a duas entrevistas semiestruturadas ao sujeito em estudo, complementadas com a análise documental. O procedimento de análise dos dados da investigação realizou-se com base em dimensões temáticas, que reportaram as perspetivas e as ações desenvolvidas pelo diretor em estudo na promoção do papel político-social. O estudo permitiu retratar uma diretora que agiu no sentido de implementar consensos, num esforço de aliar o empenho interno de melhoria (da imagem e desempenho do agrupamento), às contribuições externas da comunidade educativa. Neste sentido, estruturou uma rede de compromissos entre os diferentes atores educativos valorizando o seu papel de amenizadora e articuladora das diferentes conveniências educativas. Indiciamos, assim, a promoção e valorização da ação colaborativa, o recurso à visibilidade e à regulação autónoma como estratégias utilizadas pela diretora em prol da melhoria do Agrupamento de Escolas.This work reports a study centered on the figure of the director of a Grouping of Schools, located in the periphery of Lisbon, aiming to identify, describe and analyze the logic and rationalities that lead to the performance and construction of the political-social role, together with the educational actors of a School Grouping. The theoretical context on the problematic in analysis fell on the concepts of regulation, culture and organization. Focusing the study on the school director, the study focused as central concepts for an analysis of the action of this educational actor, the roles, the conceptions and the logic of action. It is a qualitative and interpretative study, structured in a case study. As data collection instruments, we used two semi-structured interviews with the subject under study, complemented by the documentary analysis. The procedure for analyzing research data was based on thematic dimensions, which reported the perspectives and actions developed by the director under study in promoting the political-social role. The study allowed to portray a director who acted to implement consensus, in an effort to combine the internal commitment to improve (the image and performance of the grouping), the external contributions of the educational community. In this sense, it has structured a network of commitments among the different educational actors, valuing its role as facilitator and articulator of the different educational conveniences. Thus, we highlight the promotion and appreciation of collaborative action, the use of visibility and autonomous regulation as strategies used by the director to improve the School Grouping

Semen Cryopreservation in Brazilian Freshwater Fish: Advances and Main Challenges

Studies on semen cryopreservation in Brazilian freshwater fish have been growing in number of publications and investigated species. Despite this apparent increase in research, standardization of cryoprotocols is still missing, making it clear that the grounds on the quality of cryopreserved semen has not yet reached a level that guarantee satisfactory results for its replication. This chapter aims to make a critical and reflective analysis on the ways cryopreservation of freshwater fish semen has been conducted in Brazil. The difficulties in standardizing protocols, broodstock, and selection of genetically superior animals; the barriers in transferring technology from laboratory benches to the field and make feasible the use of cryopreserved semen on a commercial scale; the formation of germplasm banks and the responsible use of cryopreserved material are also discussed. We have no intention to point out the successes and mistakes that may have been committed in pursuing development of cryopreservation protocols, but a reflection on the future directions considering what should be pondered on this subject with objectivity and scientific consolidation

Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensis

Cryopreservation of germ cells would facilitate the availability of cells at any time allow ing the selection of donors and maintaining quality control for further applications such as transplan tation and germline recovery. In the present study, we analyzed the efciency of four cryopreservation protocols applied either to isolated cell suspensions or to testes fragments from Senegalese sole. In tes tes fragments, the quality of cryopreserved germ cells was analyzed in vitro in terms of cell recovery, integrity and viability, DNA integrity (fragmentation and apoptosis), and lipid peroxidation (malondialde hyde levels). Transplantation of cryopreserved germ cells was performed to check the capacity of cells to in vivo incorporate into the gonadal primordium of Senegalese sole early larval stages (6 days after hatch ing (dah), pelagic live), during metamorphosis (10 dah) and at post-metamorphic stages (16 dah and 20 dah, benthonic life). Protocols incorporating dimethyl sulfoxide (DMSO) as a cryoprotectant showed higher number of recovered spermatogonia, especially in samples cryopreserved with L-15 + DMSO (0.39 ± 0.18 × 106 cells). Lipid peroxidation and DNA frag mentation were also signifcantly lower in this treat ment compared with other treatments. An important increase in oxidation (MDA levels) was detected in samples containing glycerol as a cryoprotectant, refected also in terms of DNA damage. Transplan tation of L-15 + DMSO cryopreserved germ cells into larvae during early metamorphosis (10 dah, 5.2 mm) showed higher incorporation of cells (27.30 ± 5.27%) than other larval stages (lower than 11%). Cryopreservation of germ cells using testes fragments frozen with L-15 + DMSO was demonstrated to be a useful technique to store Senegalese sole germline.LA/P/0101/2020info:eu-repo/semantics/publishedVersio

The protective effect of endogenous melatonin on gilthead seabream sperm during cryopreservation

Cryopreservation is widely used for artificial reproduction techniques (ART) but requires good gamete quality to succeed. Considering the endogenously produced melatonin by the fish and its protective role in the organism, the objective of this experiment was to search for the best moment of the day to collect gilthead seabream (Sparus aurata) sperm with better quality and, at the same time, to test the potential protective effect of the exogenous melatonin during sperm freezing and thawing processes. Fish were first sampled every 6 h, corresponding to the beginning of the light period (6 h), mid-light (12h), beginning of the dark period (18 h), and mid-dark (24 h) to determine melatonin concentration in blood plasma by radioimmunoassay (RIA). The analysis showed higher values at 24 h (244 pg/mL) compared with 12 h (99 pg/mL), which allowed the selection of those moments for the next experiment. After, fish were sampled for sperm collection at mid-light (ML, 12 h) and mid-dark (MD, 24 h), and fresh sperm was used to assess gamete quality and for the cryopreservation assay. Sperm samples were cryopreserved to test supplementation of different melatonin concentrations (0.001 mM, 0.01 mM and 0.1 mM) together with a control group without added melatonin. Gamete quality was assessed through spermatozoa concentration and motility (CASA system), cell viability (PI/SYBR-green) and DNA fragmentation (Comet assay). Despite cell viability that was higher at ML, most of the fresh sperm motility parameters did not differ between ML and MD, only linearity (LIN) was enhanced at MD. Nevertheless, in cryopreserved samples, total motility (TM) was significantly higher at MD in all melatonin treatments, control, and fresh samples, revealing an endogenous night-effect. Moreover, spermatozoa concentration was also higher at MD (28.9 x 109/mL) than at ML (20.7 x 109/mL). Supplemented melatonin did not confer extra protection to gilthead seabream sperm during cryopreservation since the tested concentrations did not differ between the control in any sperm quality test. It is here suggested that endogenously produced melatonin may contribute to the improvement of some gamete quality parameters at mid-dark, allowing the aquaculture sector to select better sperm quality in a noninvasive way by choosing it as the best moment of the day for sperm collection.LA/P/0101/2020info:eu-repo/semantics/publishedVersio