Introduction
E lasmobranchs are an ecologically diverse vertebrate group that plays a key role in the regulation of the ecosystems they inhabit (Stevens, 2000). But the life histories of sharks and rays make this group extremely sensitive to elevated mortality from fishing habitat destruction (Dulvy et al., 2014). Given this situation, the use of ex situ conservation breeding techniques , including artificial insemination, could be a strategy worthy of consideration in elasmobranch conservation plans. However, to perform this technique a reliable sperm supply is needed, and despite cryobanking is the most common way to guarantee the availability of viable sperm (Asturiano et al., 2017 ), there are not widespread protocols for elasmobranch sperm cryopreservation. In fact, information on elasmobranch sperm cryopreservation is limited to just two scientific publications, with information regarding two ray species and one shark (Daly et al., 2011 ; Daly and Jones, 2017). While the authors reported successful results in the cryopreservation of both rays no conclusive information was given about the shark besides the toxic effect of the cryoprotectant used on the sperm. To date there is no reported information on successful cryopreservation of shark sperm.
Material and Methods
Sperm from two species, the small-spotted catshark (Scyliorhinus canicula) and the rough skate (Raja radula ) was obtained from alive animals kept in a public aquaria and from animals obtained from commercial fisheries.
An artificial elasmobranch seminal plasma extender (EE) was formulated to be similar in composition (solutes, pH, and osmolality) to the inner fluids of marine elasmobranchs : in mM; 433 Urea, 376 NaCl, 120 Trimethylamine N-oxide (TMAO), 8.4 KCl, 50 Glucose, 7 CaCl2-2H2O, 3.5 NaHCO3, 0.08 Na2SO4, 1.4 MgSO4 ; pH 6.5; Osmolality 1000 mOsm/kg). Short-term preservation of the sperm was tested by diluting ( dilution ratio 1:9; sperm:EE ) the small-spotted catshark sperm in EE and using sea water as a control. Samples were kept at 4 ºC. Sperm motility was assessed for a period 36 days. Only sperm samples with an initial motility higher than 60% were considered for the trials.
The cryopreservation protocols were performed by adding to the previous mixture of sperm and extender (EE) a series of different cryoprotectants: 10% methanol (MET) or 10 % dimethyl sulfoxide (DMSO) or 20% fresh egg yolk. The combination of the cryoprotectants was also tested: 10% MET plus 10% fresh egg yolk, or 10% DMSO plus fresh egg yolk, or 5% MET plus 5% DMSO plus 10% fresh egg yolk. The cryopreservation mixture was used to fill 1.5 ml cryotubes and left for an equilibration period of 15 min at 4 °C to ensure the correct functioning of the cryoprotectant s. Samples were frozen inside a styrofoam box partially filled with liquid nitrogen (LN). After the equilibration process, cryotubes were placed over a net metal platform floating 1 cm over the LN, for a period of 15 min. Cryotubes were completely submerged into the LN after that time, remain ing there for 5-60 min. For the thawing process, cryotubes were extracted from the LN and submerged for 75 s in a water bath at 70 °C. Samples post -thawing motility was assessed using a microscope and video recording, a nd cells membrane integrity was checked using a fluorescence LIVE/DEAD Sperm Viability Kit with SYBR-14, which stains intact cells green, and propidium iodide (PI) that stains damaged cells red.
Results
Short-term storage trial. Sperm mot ility in seawater showed a significant decrease after 5 days, reaching values close to 30%, and close to 0 on day 19. In contrast, diluted samples in EE showed a significant decrease in motility values with respect to day 0 (85-90%) after the first days of storage (days 5 and 12) , showing approximately 60-65% of motile cells, followed by a second significant motility decline on day 19, reaching values of 45-50%, and a progressive reduction until day 36 (<10% motility).
Cryopreservation trials. In the rough skate the use of 10% DMSO or 10% MET rendered post-thawing motility values higher than 40%. However, t he combination of 5% DMSO plus 5% MET caused a significantly lower result than the other protocols. The small volume of rays sperm samples avoided the test of egg yolk.
In the case of the shark sperm samples the best post-thawing motility values were obtained with a combination of 5% DMSO, 5% MET and 10% egg yolk, which induced mean values close to 35%. Overall, the addition of egg yolk increased the post-thawing motility values, by up to 42.1% in samples with initial motility values of 70%.
Discussion
We have formulated a specific sperm extender capable of maintaining spermatozoa motility capacity for several weeks in different elasmobranch species. Moreover, we achieved the cryopreservation of sperm supplementing the extender (EE) with different combinations of cryoprotectants. Best results were obtained with a combination of DMSO , methanol and egg yolk. Despite the setting up process was developed using sperm samples from small-spotted catshark (Scyliorhinus canicula) and rough skate (Raja radula ), additional cryopreservation attempts have been carried out using other 8 species, including sharks and rays classified as Critically Endangered, such as the blue shark (Prionace glauca) and the bull ray (Aetomylaeus bovinus ). In the end, the sperm of a total of 10 species of elasmobranchs have been cryopreserved for the first time, including sharks, whose cryopreservation had not previously been achieved.
These results can allow the creation of cryobanks for elasmobranch sperm , becoming new tools for their conservation, complementing ex situ conservation efforts developed by public aquaria worldwide. Besides gene conservation and reproductive research projects, a regular supply of frozen sperm will reduce the problems that result from the long-distance transport of specimens, inbreeding or the lack of synchronized reproductive cycles in captivity.
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Daly, J., Holland, M., Galloway, D. (2011) Preliminary investigations on sperm cryopreservation of a stingray, the sparsely spotted stingaree, in: Cryopreservation in Aquatic Species, 2nd Edition, eds. T. R. Tiersch and C. C. Green (World Aquaculture Society) 337–344.
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