Effects of the slow cooling during cryopreservation on the survival and morphology of Taiwan shoveljaw carp (Varicorhinus barbatulus) spermatozoa

Over the past decades, pollution, overfishing, and habitat degradation have driven the population size of Taiwan shoveljaw carp down markedly in Taiwan. Cryopreservation is a useful tool which could be used to maintain genetic resources to protect and preserve this endemic species. Four cryoprotectants [dimethyl sulphoxide (DMSO), dimethylacetamide (DMA), glycerol and methanol] and six freezing rates (0.5, 1, 2, 4, 8, 16 °C min-1) were tested in order to develop an optimal controlled slow-freezing protocol for Taiwan shoveljaw carp spermatozoa. Samples were subsequently examined under the scanning electron microscope to reveal whether cryopreservation had affected their ultrastructural morphology. The highest survival rate (50.1 ± 2.0%) was observed with a freezing rate of 8 °C min-1 in 1M DMSO, using SYBR-14 + PI staining. Fertility and hatching rate results using frozen-thawed spermatozoa (90.2 ± 2.2% and 22.3 ± 2.5%, respectively) were not significantly different from results with fresh spermatozoa. After cryopreservation, 21.0 ± 1.6% of frozen-thawed spermatozoa had mid-piece swelling and rupture of the head. Cryopreservation might, therefore, slightly affect Taiwan shoveljaw carp spermatozoa in terms of morphological change. However, these alterations could be compensated by using large enough numbers of normally functioning frozen-thawed spermatozoa to achieve a standard equal to fresh spermatozoa. This is the first report of successful cryopreservation of Taiwan shoveljaw carp spermatozoa using a controlled slow-cooling method

First successful production of adult corals derived from cryopreserved larvae

Coral reefs worldwide are declining due to increasing concentrations of greenhouse gases, which, combined with local anthropogenic pressure, are exacerbating unprecedented mass coral bleaching. For corals to survive, restoring coral reefs through cryopreservation is crucial. The aim of this study was to vitrify and laser-warm Stylophora pistillata planulae to allow for feasible settlement, post-settlement survival, and the production of adult corals. The no-observed-effect concentrations were used to determine the best cryoprotective agents for S. pistillata. The larvae were then subjected to cooling and nanolaser warming (300 V, 10 ms pulse width, 2 mm beam diameter) by using two vitrification solutions (VSs; VS1: 2 M dimethyl sulfoxide and 1 M ethylene glycol [EG]; VS2: 2 M propylene glycol and 1 M EG) and gold nanoparticles. The results revealed that VS1-treated larvae had a higher vitrification rate (65%), swimming rate (23.1%), settlement rate (11.54%), and post settlement survival rate (11.54%) than those treated with VS2. Seasonal variations also affected the cryopreservation of the planulae; VS1 was more favorable for the planulae in spring than in fall. Although laser-warmed larvae developed slower morphologically than their controlled counterparts, the production of cryopreserved adult S. pistillata corals was achieved. The proposed technique can improve the cryopreservation of corals and advance efforts to protect endangered coral species

Development of cryopreservation techniques for early stages zebrafish (Danio rerio) oocytes

A thesis submitted to the university of Bedfordshire in accordance with the requirements for the degree of Doctor of PhilosophyCryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, the sensitivity to chilling and toxicity of cryoprotectants of early stage zebrafish ovarian follicles were studied before designing protocols for their cryopreservation using controlled slow cooling. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. In vitro culture method for early stage zebrafish ovarian follicles were also developed. The studies showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles and methanol was the least toxic cryoprotectant. 4M methanol in potassium chloride (KCl) buffer was found to be the optimal cryoprotective solution and the optimum cooling rate was 4 °C/min for stage I and II follicles. Although the highest survivals after 2 h post-thawed incubation were 50.7 ± 4.0% for stage II ovarian follicles obtained with FDA+PI staining, ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell death. Furthermore, in vitro culture experiments showed that there was no growth for stage I and II ovarian follicles after cryopreservation, indicating that successful cryopreservation of early stage zebrafish ovarian follicles at liquid nitrogen still remains elusive. From in vitro culture study, 90% L-15 medium at pH 9.0 containing 10 IU/ml hCG was effective for in vitro culture of stage I and II ovarian follicles. Systematic study on cryopreservation of early stage fish ovarian follicles at liquid nitrogen temperature is reported ii here for the first time. The results will provide useful information on the future development of protocol design for successful cryopreservation of early stage fish ovarian follicles

The effect of cryopreservation on DNA damage, gene expression and protein abundance in vertebrate

Cryopreservation techniques allow the long-term storage of a wide variety of biological material without significant deterioration in quality. Immediate post-thaw survival is most often used to assess the effect of the freeze-thaw process on cells. However, this parameter provides no information on possible subtle effects of cryopreservation, including DNA damage, alteration of mRNA levels and protein function that may not be evident immediately post thaw. These potential adverse effects don’t necessarily result in cell death. While there are many comprehensive reviews of gamete and embryo cryopreservation in vertebrate species, we review here the publications relating to cryopreservation impact on the genome of sperm, embryos and oocytes

Study on the mitochondrial activity and membrane potential after exposing later stage oocytes of two gorgonian corals (Junceella juncea and Junceella fragilis) to cryoprotectants.

Coral reefs provide a valuable habitat for many economically valuable fish and invertebrates. However, they are in serious jeopardy, threatened by increasing over-exploitation, pollution, habitat destruction, disease and global climate change. Here, we examined the effect of cryoprotectant exposure on mitochondrial activity and membrane potential in coral oocytes in order to find suitable cryoprotectants towards their successful cryopreservation. According to the No Observed Effect Concentrations (NOECs), methanol was found to be the least toxic cryoprotectant whilst DMSO was the most toxic cryoprotectant. The results also demonstrated that there were no significant differences (p > 0.05) in ATP concentrations between Junceella juncea and Junceella fragilis after exposure to all concentrations of all cryoprotectants for 30 min. Using confocal microscopy, JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide) staining indicated that the mitochondrial membrane potential of Junceella fragilis oocytes reduced after 1 M and 2 M methanol treatment and a loss of the mitochondrial distribution pattern and poor green fluorescence after 3M methanol treatment. Therefore, even oocytes that show no adverse effect of cryoprotectants on survival might suffer some more subtle impacts. The results obtained from this study will provide a basis for development of protocols to cryopreserve the oocytes of gorgonian corals

Cryopreservation and Cryobanking of Cells from 100 Coral Species

When coral species become extinct, their genetic resources cannot be recovered. Coral cryobanks can be employed to preserve coral samples and thereby maintain the availability of the samples and increase their potential to be restocked. In this study, we developed a procedure to determine coral species-specific requirements for cryobank freezing through determining suitable cryoprotective agents (CPAs), CPA concentrations, equilibration times, holding durations, viability rates, and cell amounts for banked coral cells, and we established the first ever coral cell cryobank. Coral cells, including supporting and gland cells, epidermal nematocysts, Symbiodiniaceae and symbiotic endoderm cells (SEC) were found from the extracted protocol. Approximately half of the corals from the experimental corals consisted of spindle and cluster cells. Gastrodermal nematocysts were the least common. The overall concentration of Symbiodiniaceae in the coral cells was 8.6%. Freezing using DMSO as a CPA was suitable for approximately half of the corals, and for the other half of species, successful cell cryopreservation was achieved using MeOH and EG. EG and DMSO had similar suitabilities for Acanthastrea, Euphyllia, Favites, Lobophyllia, Pavona, Seriatopora, and Turbinaria, as did EG and MeOH for Acropora, Echinopyllia, and Sinularia and MeOH and DMSO for Platygyra after freezing. At least 14 straws from each species of coral were cryobanked in this study, totaling more than 1884 straws (0.5 mL) with an average concentration of 6.4 × 106 per mL. The results of this study may serve as a framework for cryobanks worldwide and contribute to the long-term conservation of coral reefs

Effect of Cryopreservation on Proteins from the Ubiquitous Marine Dinoflagellate Breviolum sp. (Family Symbiodiniaceae)

Coral reefs around the world are exposed to thermal stress from climate change, disrupting the delicate symbiosis between the coral host and its symbionts. Cryopreservation is an indispensable tool for the preservation of species, as well as the establishment of a gene bank. However, the development of cryopreservation techniques for application to symbiotic algae is limited, in addition to the scarceness of related studies on the molecular level impacts post-thawing. Hence, it is essential to set up a suitable freezing protocol for coral symbionts, as well as to analyze its cryo-injury at the molecular level. The objective of this study was to develop a suitable protocol for the coral symbiont Breviolum subjected to two-step freezing. The thawed Breviolum were then cultured for 3, 7, 14, and 28 days before they were analyzed by Western blot for protein expression, light-harvesting protein (LHP), and red fluorescent protein (RFP) and tested by adenosine triphosphate bioassay for cell viability. The results showed the highest cell viability for thawed Breviolum that was treated with 2 M propylene glycol (PG) and 2 M methanol (MeOH) and equilibrated with both cryoprotectants for 30 min and 20 min. Both treatment groups demonstrated a significant increase in cell population after 28 days of culture post-thawing, especially for the MeOH treatment group, whose growth rate was twice of the PG treatment group. Regarding protein expression, the total amounts of each type of protein were significantly affected by cryopreservation. After 28 days of culture, the protein expression for the MeOH treatment group showed no significant difference to that of the control group, whereas the protein expression for the PG treatment group showed a significant difference. Breviolum that were frozen with MeOH recovered faster upon thawing than those frozen with PG. LHP was positively and RFP was negatively correlated with Symbiodiniaceae viability and so could serve as health-informing biomarkers. This work represents the first time to document it in Symbiodiniaceae, and this study established a suitable protocol for the cryopreservation of Breviolum and further refined the current understanding of the impact of low temperature on its protein expression. By gaining further understanding of the use of cryopreservation as a way to conserve Symbiodiniaceae, we hope to make an effort in the remediation and conservation of the coral reef ecosystem and provide additional methods to rescue coral reefs

Correction: Lin et al. Study on the Development and Growth of Coral Larvae. <i>Appl. Sci.</i> 2022, <i>12</i>, 5255

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