Development of Cryopreservation Techniques for Gorgonian (Junceella juncea) Oocytes through Vitrification.

Sujune Tsai,Suchana Chavanich,Chiahsin Lin,Wish Yen,Voranop Viyakarn,Kevin Mccluskey

doi:10.1371/journal.pone.0123409

Sujune Tsai, Suchana Chavanich + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0123409

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Abstract

Gorgonian corals are slowly declining due to human interaction and environmental impacts. Cryopreservation of gorgonian corals is an ex-situ method of conservation, ensuring future reproduction. The present study assessed the vitrification properties of cryoprotectant (CPT) mixtures using the cryotop, cryoloop and open pulled straw (OPS) cryopereservation methods prior to experimentation on gorgonian (Junceella juncea) oocytes. Investigations of the equilibration and vitrification solutions’ (ES and VS) effect on oocytes throughout different incubation periods were conducted. The cryotop method was found to be the most successful in ensuring vitrification. The most favourable VS was composed of propylene glycol (PG), ethylene glycol (EG) and methanol with concentrations of 3.5M, 1.5M and 2M respectively. Experiments were performed using the cryotop method to cryopreserve Junceella juncea oocytes using VS2, the solution had the least impact on oocytes at 5°C rather than at 26°C. The success of the vitrification procedures was determined by adenosine triphosphate (ATP) levels in cooled-thaw oocytes and the highest viability obtained from the present study was 76.6 ± 6.2%. This study provides information regarding gorgonian corals’ tolerance and viability throughout vitrification to further advance the vitrification protocol on whip corals.

Highlights

Gorgonian corals provide important habitats for many ecologically significant fish, as well as vital reef habitats for numerous invertebrates, including seahorse (Hippocampus) [1], oyster (Pteria colymbus) [2] and shrimp (Neopontonides beaufortensi) [3]
The oocytes were loaded with a micropipette in the cryotop, cryolop and open pulled straw (OPS) and subsequently submerged in liquid nitrogen
The lowest concentration of all the CPTs to be vitrified was found with the use of the cryotop device

Summary

Introduction

Gorgonian corals provide important habitats for many ecologically significant fish, as well as vital reef habitats for numerous invertebrates, including seahorse (Hippocampus) [1], oyster (Pteria colymbus) [2] and shrimp (Neopontonides beaufortensi) [3]. With regards to the cryopreservation of invertebrates, freezing techniques have been developed to successfully cryopreserve the eggs and embryos of oysters (Crassostrea virginica) [6], sea urchins (Evechinus chloroticus) [7], abalone (Haliotis diversicolor supertexa) [8], and polychaete worms (Nereis virens) [9]. As it stands there is no information available on the cryopreservation of coral oocytes and embryos. Factors limiting oocyte cryopreservation include their high chilling sensitivity, and low membrane permeability. Another factor is the large amount of yolk within, and their large size, resulting in a low surface area to volume ratio [10]

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