Establishment of a genetic resource bank for restocking management in Portuguese oyster Crassostrea angulata and Striped-venus clam Chamelea gallina

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Effect of trehalose and sucrose in post-thaw quality of Crassostrea angulata sperm

Publication . Anjos, Catarina; Santos, Ana Luísa; Duarte, Daniel; Matias, Domitília; Cabrita, Elsa

Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4◦C for 10 min, frozen in a programmable biofreezer at −6 ◦C/min from 0 to −70◦C, and stored in liquid nitrogen. Samples were thawed in a 37◦C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.

2021-11-25Journal article

Open access

Assessment of larval quality of two bivalve species, Crassostrea angulata and Chamelea gallina, exposed and cryopreserved with different cryoprotectant solutions

Publication . Anjos, Catarina; Duarte, Daniel; Diogo, Patrícia; Matias, Domitília; Cabrita, Elsa

Marine bivalves are valuable resources, however, some shellfish populations are endangered due to factors such as anthropogenic pressure, pathologies or lack of reproduction synchrony. Portuguese oyster (Crassostrea angulata) and striped venus clam (Chamelea gallina) have high socio-economic value and their endangered natural populations require rehabilitation. Cryopreservation is a valuable method for the preservation and management of genetic resources for aquaculture and restocking. Larvae cryopreservation is particularly valuable since diploid organisms are obtained upon thawing. The objective of this work was the establishment of C. angulata and C. gallina D-larvae cryopreservation through the selection of permeant cryoprotectant in the freezing solution, namely ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). Cryoprotectants exposure showed that, in C. angulata, Me2SO promoted significantly higher incidence of abnormalities and enhanced glutathione reductase activity when compared to control (larvae without cryoprotectant exposure) or even to EG treatment. However, for both species, EG significantly reduced D-larvae average path velocity (VAP). In C. angulata post-thaw D-larvae, EG treatment promoted significantly lower motility and velocity when compared to control and Me2SO treatment. Superoxide dismutase (SOD) activity showed a reduction in C. angulata postthaw D-larvae when compared to control, which was compensated by the enhancement of glutathione peroxidase (GPX) activity. In C. gallina post-thaw D-larvae, only motility, velocity and SOD activity were significantly lower than control. Therefore, the best treatment to cryopreserve C. angulata D-larvae was EG while for C. gallina Me2SO produced better results. This work established for the first time D-larvae cryopreservation protocols for C. angulata and C. gallina.

2022Journal article

Restricted access

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure

Publication . Anjos, Catarina; Duarte, Daniel Filipe Correia; Fatsini Fernández, Elvira; Domitília Matias; Cabrita, Elsa

Background The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. Results Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. Conclusions The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

2024-06-12Journal article

Open access

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure

Publication . Anjos, Catarina; Duarte, Daniel Filipe Correia; Fatsini Fernández, Elvira; Matias, Domitília; Cabrita, Elsa

The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. Results Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. Conclusions The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

2024-06-12Journal article

Open access

Establishment of a genetic resource bank for restocking management in Portuguese oyster (Crassostrea angulata) and striped venus clam (Chamelea gallina)

Publication . Anjos, Catarina Miranda Castilho dos; Cabrita, Elsa; Matias, Domitília

Bivalves are essential for fisheries, aquaculture, and ecosystems, serving as nutrient-rich resources for human consumption. Despite their significance, many bivalve resources, including Crassostrea angulata (Portuguese oyster) and Chamelea gallina (striped venus clam) in Europe, evidence signs of depletion due to environmental change, anthropogenic impact, and overexploitation, requiring rehabilitation measures. One possible strategy involves establishing a genetic resource bank via cryopreservation. However, cryopreservation presents challenges, requiring optimization of freezing and thawing conditions, particularly the cryoprotectant solution, and understanding cryodamage. The present thesis aims to explore and establish conditions to store and preserve the genetic resources of C. angulata and C. gallina populations. Chapter 1 provides contextual background, on the current situation of bivalve production and the importance of these resources, with special attention on the endangered and valuable species for aquaculture/fisheries, C. angulata and C. gallina. The chapter addresses the fundamental principles of cryobiology and current knowledge on bivalve cryopreservation methodologies for sperm and larvae. This chapter discusses the value of cryodamage assessment tools, emphasizing “omics” molecular tools for high-potential analysis. Chapters 2 and 3 aim to optimize and develop new cryopreservation protocols for the target species. Chapter 2 investigates the effect of the cryoprotectant supplementation with sugars (trehalose and sucrose) on the post-thaw sperm quality of C. angulata. Several methodologies not commonly used in bivalve cryopreservation works were employed, including the determination of reactive oxygen species levels, acrosome integrity and the activity of antioxidant enzymes (superoxide dismutase - SOD, glutathione reductase - GR and glutathione peroxidase - GPX). Sugars supplementation, especially trehalose reduced lipid peroxidation and ROS levels having a positive effect in plasma membrane and acrosome integrity. Chapter 3 evaluates the larval quality of C. angulata and C. gallina exposed and cryopreserved with cryoprotectant solutions that differ in the permeant agent (dimethyl sulfoxide - DMSO and ethylene glycol - EG). The work aimed to understand the effects of cryoprotectant exposure and, cryopreservation on malformations, movement, and enzymatic activity compared with non-exposed larvae. The methodologies for cryopreserving D-larvae of both species were established for the first time. Chapter 4 investigates C. angulata D-larvae cryodamage during cryoprotectant exposure and cryopreservation, using RNA sequencing. This molecular approach was essential for providing evidence that the freezing process was the critical step rather exposure. Furthermore, identified 11 genes as relevant biomarkers of freezability for D-larvae quality assessment. This thesis presents strategies for cryopreserving the genetic material of C. angulata and C. gallina and for cryodamage evaluation.

2024-06-25Doctoral thesis

Open access