Elsevier

Cryobiology

Volume 80, February 2018, Pages 130-138
Cryobiology

Cryopreservation of sperm from the coral Acropora humilis

https://doi.org/10.1016/j.cryobiol.2017.10.007Get rights and content

Abstract

Corals are sensitive to minute changes in their environments, and their continued existence is substantially threatened by the increasing number of destructive anthropogenic activities and unprecedented rates of climate change. Although cryopreservation has been successfully to preserve mammalian gametes for decades, coral cryopreservation was attempted for the first time less than 15 years ago, and freezing protocols exist for only a handful of coral species. The present study developed a cryopreservation protocol for the sperm of the common Indo-Pacific reef-builder Acropora humilis. Colonies of reefs of Sattahip Bay, Chonburi Province, Thailand were collected from 3 m depth with a mesh net during a spawning event. Immediately after collection, the sperm were isolated and subjected to a two-step freezing method featuring DMSO, polyethylene glycol, or methanol as the cryoprotectant. Viability and motility were assessed via a bioluminescence technique and a “computer-assisted semen analysis, and it was found that a 15-min equilibration with 2 M DMSO followed by cooling at 41.7 °C was the optimum cryopreservation protocol for A. humilis sperm. The post-thaw sperm achieved 45% fertilization success, and 35% of the fertilized eggs developed into blastopore larvae. The present optimized protocol can therefore facilitate the preservation of sperm for future propagation efforts of this species and provide an experimental platform for optimizing cryopreservation protocols for gametes of other scleractinian coral species.

Introduction

Increasing unrestricted and destructive anthropogenic activities have not only significantly damaged the natural environment but also have played a leading role in causing harmful climatic alterations. In the marine ecosystem, increased carbon emission levels have led to global warming and major environmental changes such as increased seawater temperatures, seawater levels, and pH values, which are detrimental to aquatic organisms, particularly coral populations [25]. In addition, excessive urbanization and high tourist influx in coastal ranges have caused damages to reef and coral areas because of their direct and close contact with these habitats [15]. Consequently, 19% of the global reef cover has been lost, and such losses are predicted to increase in the next 40 years if effective remediation strategies are not adopted [64]. In addition, reef inhabitants were reported to be significantly affected by these unsolicited events [29].

Although many conservation strategies have been adopted for reef protection, bleaching and reef erosions are frequently observed due to increasing pollution and water quality degradation. In situ conservation may be effective and provide long-lasting solutions; however, a longer period of rehabilitation and recovery is required. Alternatively, ex situ conservation is adopted for the preservation of coral populations in a controlled artificial environment, and they are subsequently reintroduced to the wild. However, currently, all organisms cannot be cultured and reared in artificial environments, and reefs are continually plagued with various destructive activities. In the context of ex situ conservation, cryopreservation is an excellent tool for safeguarding coral populations by preserving their cellular function [35], [60]. Cryopreservation allows the temporary preservation of the gametes of extinct and endangered coral species. Therefore, by using their viable gene pool and DNA, such species can be rehabilitated using advanced technology in the future.

Below subphysiological temperatures, cellular metabolic activities can be arrested for an indefinite period to preserve cell viability. However, during this process, cells are susceptible to damage, such as intracellular ice crystal formation, freezing injuries, and osmotic stress. Therefore, chemicals referred to as cryoprotectants (CPAs) are essential for cell treatment before cryopreservation. CPAs, cooling rates, and freezing temperatures are tailored to each species and cells due to different tolerances to low-temperatures and CPA toxicity. Therefore, to identify optimum freezing conditions, toxicity tests and freezing rate experiments, which may include the determination of the type, concentration, and duration of CPA equilibration at varying freezing temperatures and cooling rates, have been conducted.

Most developments in coral cryopreservation are in early stages, with current studies concentrating on reproductive cells such as oocytes, sperm, and embryos, for future population recovery and increasing genomic data in gene banks. A current review revealed that protocols for the chilling and cryopreservation of oocytes [35], [36], [61], [62] larvae, and embryonic cells [23], [24] are being developed with promising results. The sperm of three coral species, Fungia scutaria, Acropora palmate, and A. digitifera, have been cryopreserved with favorable outcomes [23], [47]. Successful sperm cryopreservation in these species is crucial because hard corals are fundamental for reef building and sustaining important marine organisms.

The present study established an optimum cryopreservation protocol for A. humilis sperm. A. humilis is a hermatypic coral that is commonly found in shallow waters of up to 5-m depth. The spawning events of A. humilis gametes are highly synchronized events that occur annually or biannually depending on colony location [17]. The egg–sperm bundles disperse shortly after their release, and fertilization occurs externally in the water column. Fertilized oocytes develop into larvae and attach to the substrate for growth. A. humilis is a reef-building coral that plays a significant role in reef ecology and acts as a natural breakwater to reduce wave energy. Despite their large distribution across tropical waters, particularly in the reefs of the Pacific and Indian Oceans, this species is categorized as near threatened in the International Union for Conservation of Nature Red List [56]. Global climate change, anthropogenic activities, and natural predation are among the major causes of coral population decline. In addition, higher water temperatures affect coral fecundity and reproductive timing, further reducing their population [54]. Their threatened status suggests that coral reef areas are critically damaged and could jeopardize the entire marine food web and ecology. The present study is the first to perform cryopreservation of A. humulis sperm; therefore, we intended to identify optimum freezing conditions that may facilitate the restoration of endangered coral species and the maintenance of coral genetic diversity.

Section snippets

Evaluation of coral maturity and spawning periods

The A. humilis sperm were collected from the reefs in Sattahip Bay, Chonburi Province, Thailand (Fig. 1). The spawning periods of A. humilis in this region are usually during January to March. The spawning period was ascertained by examining coral reproductive organs: a small coral piece was fragmented by hand or chisel and observed under a light microscope (CCX31 Olympus, Japan). A mature coral with swollen polyps indicated that the gamete bundles were closer to the oral cavity for release.

Sperm motility and velocity

Post-thaw sperm motility and viability are critical for successful fertilization. Overall, the sperm cryopreserved in DMSO had the highest individual velocity and motility rates. The sperm that were equilibrated with 2 M DMSO for 15 and 20 min had the fastest individual velocity rates of 75 and 68 μm s−1, respectively (Fig. 3a), followed by those equilibrated with both 1 and 2 M PG (approximately 70 μm s−1; Fig. 3b). In the present study, the sperm with an individual velocity rate of less than

Discussion

CPAs play a critical role in preventing freezing injuries by reducing ice crystal formation through appropriate cell dehydration. However, the type, concentration, and incubation period of CPAs should be compatible with corresponding cells to prevent CPA toxicity [14]. In the present study, three CPAs (DMSO, MeOH, and PG) were used because they have been reported to be effective or used previously in coral sperm cryopreservation [23], [24], [47]. Our results revealed that DMSO (2 M) was the

Conclusion

A cryopreservation protocol for A. humilis sperm was successfully established. To ensure high-quality sperm and experimental reproducibility, sperm viability assessment prior to cryopreservation and the selection of high-motility sperm are highly recommended. Furthermore, gamete bundles should be collected and washed immediately after release to reduce contamination. In the present study, the fertilization success of post-thaw sperm was approximately 50% of the control group. The optimum

Acknowledgements

We would like to thank the Plant Genetic Conservation Project under the Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn, the Naval Special Warfare Command, Royal Thai Navy, Department of Marine and Coastal Resources Thailand, NRCT-JSPS Core to Core, and Thailand Research Fund (RSA 6080087) for their assistance during the study period.

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