Long-term study on survival and development of successive generations of Mytilus galloprovincialis cryopreserved larvae

Abstract

Shellfish aquaculture needs the development of new tools for the improvement of good practices avoiding the reliance on natural spat collection to increase production efficiently. The aim of this work was to improve the cryopreservation protocol for Mytilus galloprovincialis larvae described in Paredes et al. (in: Wolkers, Oldenhof (eds) Cryopreservation and freeze-drying protocol, methods in molecular biology, Humana Press, 2021, pp 2180, https://doi.org/10.1007/978-1-0716-0783-1_18). Moreover, the capability of producing adult mussels from cryopreserved 72 h-old D-larvae and potential long-term effects of cryopreservation through progenies were evaluated. The selection of 72-h old D-larvae for cryopreservation yielded 75% of recovery, higher than 50% from trochophores. The best combination was 10% Ethylene–Glycol + 0.4 M Trehalose in Filtered Sea Water (FSW) with cooling at − 1 °C/min and a water bath at 35 °C for thawing. Sucrose (SUC) solutions did not improve larval recovery (p > 0.05). At settlement, 5.26% of cryopreserved F1 larvae survived and over 70% settled. F2 cryopreservation produced 0.15% survival of spat and settlement varied from 35 to 50%. The delay of shell size showed on cryopreserved larvae declined throughout larval rearing without significant differences with controls from settlement point (p > 0.05). Long-term experiments showed that it is possible to obtain adult mussels from cryopreserved larvae and this tool does not compromise the quality of following progenies, neither for cryopreservation nor post-thawing development of them.

Handling, Reproducing and Cryopreserving Five European Sea Urchins (Echinodermata, Klein, 1778) for Biodiversity Conservation Purposes

“…Treatments were then fixed with formalin and examined under a microscope in order to determine the fertilization of the eggs (when studying Sperm after 15 min contact time) or the percentage of normal 4-arm pluteus larvae (n = 100), as well as the average larval growth (n = 35), when working with embryos (after 48 h of incubation at 18 °C). Whenever possible, the NOEC (No Observed Effect Concentration) and LOEC (Lowest Observed Effect Concentration) were determined by calculating the significant differences in larvae size between treatments and control after 48 h, and taking into account larval normal development, following [ 26 , 29 , 30 ]. Quality control of the gametes has been conducted according to [ 31 ].…”

Cryopreservation Enables Long-Term Studies ofOctopus vulgarisHemocyte Immune Functions

“…In addition to the CPAs, cryopreservation also requires optimal cooling and warming rates to control cellular dehydration during freezing, the intracellular ice formation, and also the possible deleterious effect of CPAs during the thawing process (Wolkers and Oldenhonf;. For the last point, according to previous data obtained for marine molluscs, we have used a cooling rate of -1°C/min (Heres et al, 2022) and it is also very important a progressive dilution of the freezing medium to dilute as much as possible the CPA and prevent the cells to damage. Different researches have shown that progressive dilution of CPA during the thawing process is superior to rapid thawing for maintaining cell viability allowing a gradual equilibration of osmolarity, reducing the risk of osmotic shock, which is detrimental to cell membranes and overall cell survival (Baust et al, 2017;Agarwal and Singh;.…”

“…Specifically, in the field of aquaculture, cryopreservation has already contributed to the implementation of selective breeding programs, to the preservation of biodiversity through biobanks, and to improve the hatchery spat overcoming the limitations of this important industry (Paredes et al, 2022;Heres et al, 2022). Despite the wide range of applications offered by this discipline, to our knowledge, the cryopreservation protocol of any immune cell in marine invertebrates species has not yet been optimized, making it difficult to study their identification, both molecular and functional, and keeping closed a door that would allow many studies that, to date, have not been possible.…”

Development of a non-programmable sperm cryopreservation technique in dwarf surfclams Mulinia lateralis–a potential model species for bivalve research

“…These concerns have been further stressed recently as negative effects of sperm cryopreservation on progeny fitness have been reported in fish and mammalian species, such as brown trout Salmo trutta (Nusbaumer et al, 2019), Japanese eels Anguilla japonica (Müller et al, 2018), horses (Ortiz-Rodriguez et al, 2019) and mice (Jia et al, 2015), and a urgent study to address these knowledge gaps was recommended at the development of Atlantic salmon gene banking (Bøe et al, 2021). However, investigations on performance of economic important traits and generations are time-consuming and challenging in farmed marine bivalve species because of (1) their long generation interval, from one year in Pacific oysters Crassostrea gigas (Yang et al, 2021) to multiple years in scallop Patinopecten yessoensis (Silina, 2018); and/ or (2) high costs and challenges to manage the experiments in the open marine environments (Heres et al, 2022). All these obstacles could be addressed if a model species could be applied.…”