When sea urchins won’t spawn, cryobiologists step in.

Cryobiologists from Nature’s SAFE recently visited marine biologists at University of Southampton to help them preserve sea urchin sperm. But why does this help accelerate their research, and how did they do it?

At the University of Southampton, sea urchins are used as a model species to study processes of animal development such as skeletal development. Sea urchins are ideal for studying this because of their wide range of morphological diversity. As Jeff Thompson, Associate Professor at the University explains:

“There are about 1000 species of sea urchins which show a whole range of diverse shapes. They can be rounded or flat or they can be heart shaped, so they really show an array of anatomical diversity relative to other animal groups.”

This makes maintaining healthy, breeding urchin populations essential for their research. As sea urchins spawn eggs and sperm directly into the water, freshly spawned eggs can be fertilised easily when sperm is available. However, they have found that the process of inducing spawning in the male urchins is not always consistent, causing a pinch point in their captive breeding processes. To help address this breeding challenge, Nature’s SAFE was called in to attempt sperm cryopreservation:

“If we are able to cryopreserve sperm, which can be quite difficult [and inconsistent] to get from the animals… that will make this aspect of our research significantly more streamlined and reproducible”

Over the course of two days, Nature’s SAFE team members Tullis Matson and Alice Clark worked alongside Jeff Thompson, Robbie Robertson and Noé Wambreuse from the University of Southampton to cryopreserve sperm from two sea urchin species: the tuxedo urchin (Mespilia globulus) and the orange-spined ‘Halloween’ urchin (Tripneustes gratilla).

The process

The first step in the process is to induce spawning to collect sperm. This can be done in one of three ways: light shaking, thermal shock, or administering a low concentration potassium salt solution to cause muscle contraction and initiate spawning. As the success of these methods is inconsistent, the aquarium team attempted to initiate spawning from multiple males of each species using a combination of these methods. Luckily, the team were able to induce spawning in two male tuxedo urchins and two male Halloween urchins.

Semen was gently collected directly from the top surface of the urchins as they were spawning. Before freezing, the semen was analysed for sperm cell concentration and motility to make sure it was healthy and viable.

After confirming that the sperm samples were viable, they were cryopreserved by adding a cryoprotective solution to prevent cell damage during the freezing process and lowering the samples into the vapour of liquid nitrogen, using purpose-built cryo-racks designed by researchers from the Smithsonian Conservation Biology Institute. These racks allow them to freeze the samples at a rate of 50⁰C per minute until they dropped below -80⁰C. They were transferred directly into liquid nitrogen storage tanks to keep them frozen at -196⁰C for long-term storage.

The final step in this process was to test that the cryopreservation process had been effective in keeping the sperm cells alive. The team did this by thawing out one vial from each species and testing its ability to fertilise fresh eggs from females.

First up was the tuxedo urchin. Evidence of successful fertilisation of tuxedo urchin eggs was seen within an hour, indicated by the fertilisation envelope which appears as a halo around the eggs. Cell division then began and continued in multiple embryos into the next day, confirming that the eggs had been fertilised. These embryos will hopefully go on to form larvae and adult urchins. On day 2 of the visit, the team conducted a fertilisation test for the orange spined sea urchin, seeing initial evidence of fertilisation and cell division within a few hours which provides promising results that will require further follow up in a future visit.

How these samples will be used

So, what becomes of the sperm once it is frozen? Most of the samples preserved have been stored at the University so that researchers can use them to fertilise freshly spawned eggs with ease. Some of the samples were brought to the Nature’s SAFE laboratory in Shropshire, where they will remain in long-term storage for the university to access as needed.

What’s next?

The next step for Nature’s SAFE is to trial egg cryopreservation techniques to reduce the aquarium’s dependency on female spawning events. This is a much more challenging process due to the cell size of eggs in comparison to sperm, but one which they are hopeful to make progress on with the help of their expert network.  

Who made this possible

Nature’s SAFE is grateful to their collaborators and network of experts for making this work possible: the Smithsonian Conservation Biology Institute for providing their cryo-rack designs, Rob Lickley for providing 3D printing services for they cryo-racks, and the Cryo-CoRALS network (which includes the Zoological Society of London, the Natural History Museum in London, the Royal Zoological Society of Scotland, the Smithsonian Conservation Biology Institute, the Horniman Museum and Gardens, and Nature’s SAFE) for guiding the continued development of coral and sea urchin cryopreservation protocols.