INTRODUCTION

Coral reefs are the most complex and productive of all marine ecosystems (Grigg et al., 1984; Díaz et al., 1996). Their biodiversity has been compared to that found in tropical rain forests (^{Connell, 1978}), and have been called ‘oases of the sea’ due to their high productivity (^{Hoegh-Guldberg, 1999}). Unfortunately, during recent decades, coral reefs have undergone major changes worldwide. Several nature- and human-based threats such as global climate change, coral bleaching and diseases contribute to their degradation and destruction (^{Goldberg and Wilkinson, 2004}). Although reef vulnerability and resilience vary in relation to many factors including different species and population-specific responses (Rowan et al., 1977), most coral reefs show changes on community composition (Reaser et al., 2000), and even in some cases both reef structures and marine communities have collapsed (^{Graham et al., 2006}). Consequently, human communities living off these ecosystems have been affected (Wilkinson, 1999).

Different coral conservation strategies have been developed, including in situ conservation practices, which comprise the design and implementation of effective Marine Protected Areas (^{Carr, 2000}), governance, and regulatory and economic policies (^{White and Courtney, 2004}), as well as ecological restoration methods (^{Edwards, 2010}). However, the interaction of global and local threats calls for ex situ conservation practices to maintain and secure the future of coral reefs. Ex situ practices includes different approaches such as the use of on land nurseries for asexual rearing of corals, rearing coral larvae and recruits to juveniles (^{Rinkevich, 2005}; Edwards, 2010) and the preservation of biological samples in frozen banks by cryopreservation (^{Hagedorn et al., 2010}). The latter is an effective conservation tool where cells are frozen in sugar-like compounds called cryoprotectans (Hagedorn and Spindler, 2014). These substances preserve the cell structures intact during the freezing process (^{Pegg, 2007}), while the freezing temperatures stabilize conditions preserving living tissues for decades (Hagedorn and Spindler, 2014).

Gamete cryopreservation has already acted as an effective insurance policy to maintain the genetic diversity of many wildlife species including ferrets, antelopes (^{Wolf et al., 2001}; ^{Wildt et al., 2010}) and also marine invertebrates as oysters and sea urchins (^{Gakhova et al., 1988}; Paniagua-Chaves and Tiersch, 2001). Cells that are cryopreserved and banked properly can retain viability for years without DNA damage (^{Hagedorn et al., 2012}). However, the use of these frozen banks is new for coral species (Hagedorn et al., 2012), and is expected to secure existing species and genetic diversity as part of rehabilitation processes. Sperm cryopreservation has been successfully applied in some species of corals, including Acropora palmata (from the Caribbean), A. millepora, A. tenuis, A. loripes, Fungia scutaria, Platygyra lamolina, P. daedalea and Goniastrea aspera (Hagedorn et al., 2006; Hagedorn and Spindler, 2014). No coral larvae and eggs have yet been successfully cryopreserved (Hagedorn and Spindler, 2014).

As most Caribbean coral reef areas, Colombian coral reefs health and ecosystem services are decreasing. Between 2010 and 2013, three Colombian institutions (Universidad Jorge Tadeo Lozano, Calipso Dive Center and Tayrona National Natural Park Authority) joined their work towards coral reef restoration projects. This study was part of this effort, and aim to describe the spermatozoa morphology on the Caribbean reef building coral Orbicella faveolata and to assess gamete performance in terms of motility and vitality after two different cryopreservation treatments.

MATERIALS AND METHODS

RESULTS

Sperm morphology, motility and vitality

Orbicella faveolata sperm have spade shape heads and long tails (Figure 1). Mature spermatozoa had a mean flagellum length of 43.24 ± 7.99 µm (mean ± SD), and a mean head length of 4.10 ± 0.69 µm (N= 40 spermatozoa were measured). Sperm density was 8.58 x106 spermatozoa ml-1.

Figure 1 Sperm morphology of Orbicella faveolata: head (A) and flagellum (B). 

After being released from the gamete bundles, 90% of the sperm had motility; as expected, sperm motility decreased over time and the last movement was observed on less of 15% of sperm after 6 h and 14 min of breaking the gamete bundles. Fresh sperm motility varied from swimming (65.0 ± 1.26 %) to vibrating (whip-like motion; 23.5 ± 1.29 %) and, 12.3 ± 1.43 % was inactive (no motile). Three hours after breaking the gamete bundles 42.3 ± 1.01 % were swimming, 30.1 ± 0.91 % vibrating, and 25.1 ± 1.26 % inactive. After five hours 38.6 ± 1.29 % were vibrating, 11.8 ± 0.34 % swimming, and 47.9 ± 2.80 % inactive. Although sperm viability decreased with time as well, its reduction was less pronounced than its motility. Alive spermatozoa in fresh sperm were 75%, and after five hours around 60%, declining to less than 30% after eight hours (Figure 2).

Figure 2 Assessment of sperm motility, inactive and vitality over time. Motility includes swimming and vibrating. 

Cryopreservation assays

Although there was a slight decrease in the sperm swimming abilities, no statistical differences were found between fresh sperm and both treatments for cryopreserved sperm motility and vitality (X2=2.92, p=0.572; X2=1.69, p=0.428 respectively. Figure 3). After 24 h, the sperm from the slow cryopreservation treatment (4 °C) was 70% viable and moving, while the sperm exposed to the quick cryopreservation (liquid nitrogen) treatment had a vitality average of 55% and motility average of 75% after 24 h. No changes in the sperm morphology were observed within the treatments.

Figure 3 Motility percent (mean ± SD) of Orbicella faveolata on the fresh sample (2 hours after broken bundles) and after thawing in two frozen treatments. 

DISCUSSION

In this study, we describe the spermatozoa morphology of the coral Orbicella faveolata as well as two cryopreservation methods (at 4 °C and in liquid nitrogen) used to successfully preserve sperm for 24 h, maintaining at least 70% of sperm motility and vitality after thawing. This is the first study on O. faveolata sperm cryopreservation.

The observed triangular-like head shape sperm observed for O. faveolata is common in many marine invertebrates such as sea urchins (^{Landry et al., 2003}). Specifically for corals, two different spermatozoa head shapes have been previously described: pear-shaped and elongated conical head (^{Harrison and Wallace, 1990}). O. faveolata spermatozoa shape resembles the second one. As described by Harrison and Wallace (1990), the anterior process located on the spermatozoa head could have an acrosomal function; however, this has never been studied. Although differences in head length could be a species- specific taxonomic trait and be used to identify genetic clades (^{Briones et al., 2012}), there is no data for O. faveolata or other Orbicellids for comparisons. Available data from ^{Albright (2011}), who measured the O. faveolata head area using scanning electron micrographs, could not be used for comparison due to the difference on the resolution between images.

Sperm limitation, defined as the dilution of sperm in seawater (^{Yund, 2000}), is one of the key factors limiting reproductive success of sessile broadcast- spawning invertebrates (^{Levitan et al., 1992}; ^{Oliver and Babcock, 1992}). To maximize the success of fertilization and hence reproduction, multiple strategies have evolved (Yund, 2000). Some of these strategies in broadcasting corals are spawning synchrony (^{Harrison, 2011}), floating gametes (Oliver and Babcock, 1992), and specific sperm morphology and longevity (^{Benzie and Dixon, 1994}). O. faveolata colonies have simultaneous spawning events, and both egg and sperm bundles are positively buoyant. Gamete interactions are controlled by attributes like egg size, sperm swimming ability, and how likely a sperm-egg collision is to result in fertilization (Levitan and Petersen, 1995). Long spermatozoa flagella are associated to higher swimming speed and longer distances traveled, which is highly important for fertilization success (^{Fitzpatrick et al., 2010}). In other species of marine invertebrates as the black sea urchin, Diadema antillarum, has been measured that sperm availability and proximity between male and female during the spawn events affects the fertilization success (Levitan, 1991).

Sperm longevity in free-spawning marine invertebrates varies from few hours to days and, besides being species specific depends on concentration, temperature and egg contact (Jhonson and Yun, 2004). Our results shown than more than 50% of the spermatozoa can stay alive and mobile for six hours after being released. This factor could play an important part in the reproductive success of O. faveolata despite ocean dilution (Jhonson and Yun, 2004). It has been generally assumed that dilution of sperm in the water reduced sperm longevity in tens of minutes (^{Levitan, 1991}); however, our results show that it is possible that O. faveolata sperm remains viable for long times despite seawater dilution, increasing the chances of fertilizing eggs from isolated colonies.

For several marine invertebrates, including some coral species of the genera Acropora, sperm motility initiates in response to specific chemical cues secreted by the eggs (^{Morisawa, 1994}; ^{Yoshida et al., 2002}; ^{Morita et al., 2006}). Specifically, for three species of Acropora from the Pacific, sperm movement was initiated in the presence of eggs at 150-300 mm distance when pH was increased by adding choline chloride (Na-free artificial seawater containing NH4Cl) to the artificial seawater (Morita et al., 2006). These authors argue that sperm regulatory mechanisms are modulated by egg secretions which play an important role in the fertilization success. As Acropora spp., the Orbicella annularis species complex has similar spawning times (^{Fukami et al., 2004}): nevertheless, in this study sperm motility in O. faveolata was observed from the beginning when gametes were separated and eggs were absent in the samples. Thus, as sperm regulatory mechanisms are modulated by egg secretions, it is possible that some of the egg secretions were washed with the sperm during separation or that the chemical inducing sperm movement for this species is present in the seawater. This aspect must be further studied.

Orbicella faveolata sperm cryopreservation assays were successful; the sperm exposed to the slow cryopreservation treatment had a post thaw average motility of 70%, and the one exposed to quick cryopreservation treatment had a motility of 75%. In both treatments, sperm morphology did not chanced. In comparison, ^{Hagedorn et al. (2006}) obtained a post-thaw sperm motility of >95% after storage but noticed a change in the morphology of the sperm in both the head diameter and tail length. It seems that these changes were a result of the cryoprotectant used. All cryoprotectants are toxic or lethal to sperm cells, especially at high concentrations. In our study, the absence of sperm morphology changes could be the result of combining intracellular (1,2-Propadiol) and extracellular (milk) cryoprotectant. The extracellular could provide an initial protection against the formation of intracellular ice (^{Gaitán-Espitia et al., 2013}) and the milk’s lipid content may stabilize cell membranes during freeze-thaw processes (^{Odintsova and Boroda, 2012}).

Although we assessed sperm performance in terms of motility and vitality, the next challenge is determining fertilization success of the sperm after cryogenic preservation. Frozen-thawed sperm from different species of corals have been used to fertilize conspecific egg released in the same spawn and from successive spawns, reaching fertilization success of 60%, and resulting in functioning larvae (^{Hagedorn et al., 2012}). Moreover, in preliminary experiments on A. clathrata, larvae produced from frozen-thawed sperm developed, settled and assimilated symbionts creating new corals in vitro (Hagedorn and Spindler, 2014).

All cryopreservation methods in coral have been successfully applied to sperm, however coral oocytes have not been successfully cryopreserved yet. One of the main reasons is eggs sensitivity to chilling (^{Hagedorn and Spindler, 2014}) which results on affectations on the cell membrane properties, function and integrity (^{Lin and Tsai, 2012}). We obtained similar results when tested cryopreservation of O. faveolata oocytes, after the freeze-thaw processes all oocytes disintegrated.

This work supports ^{Hagedorn et al. (2006}; 2012) calls for the development of cryopreservation protocols that can be used to create a cryobiology bank to secure existing coral species and their genetic diversity. Today, there are three long-term cryobanks in the world where coral cells are stored, two of them located in the U.S. at the Smithsonian Institution at the Hawaii Institute of Marine Biology and the U.S. department of Agriculture´s Animal Germplasm Program, and one in Australia at the Taronga Western Plains Zoo (Hagedorn and Spindler, 2014). Cryopreservation of coral gametes could play an important role in near-future coral restoration and may also contribute to expand coral nurseries projects by including sexually reproductive corals (Hagedorn and Spindler, 2014), that like O. faveolata, reproduces sexually only once a year.

More studies on coral cryopreservation techniques, fertilization success and development of larvae and recruits after cryopreservation are in need. The use of sperm cryopreservation techniques, such as the one tested in this study, can potentially help to preserve gene diversity, prevent coral extinctions, and create opportunities for diversifying shrinking populations by avoiding natural loss in heterozygosity due to genetic drift (^{Hagedorn and Spindler, 2014}). In addition, it can allows us to advance in research on coral developmental biology, genetics, systematics and molecular biology.