Sea urchin cryopreservation – why and how?

We recently visited the University of Southampton to help them cryopreserve sea urchin sperm. But why did they seek out our support to do this in the first place?

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.”

The anotomical diveristy of sea urchins can be seen here when comparing the short spines of the Halloween urchin (left) to the Black long spine urchin (right). This anatomical diversity makes urchins a great model organism for research on animal skeletal development.

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”

Researchers Jeff Thompson, Robbie Robertson and Noé Wambreuse from the University of Southampton welcomed Nature’s SAFE team members Tullis Matson and Alice Clark to their research aquarium with the hopes of cryopreserving sperm from their breeding population of sea urchins.

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, we 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.

*Robbie Robertson and Alice Clark extract semen from the opening of a spawning orange-spined ‘Halloween’ urchin, which will then be cryopreserved.*

After confirming that the sperm samples were viable, we cryopreserved them 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 allowed us 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 Nature’s SAFE team used portable freezing devices designed by researchers at the Smithsonian Conservation Biology Institute for on-site cryopreservation of sea urchin sperm. These devices use liquid nitrogen to freeze sperm at a rate of approximately 50⁰C per minute.

The final step in our process was to test that the cryopreservation process had been effective in keeping the sperm cells alive. We 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. We added the cryopreserved sperm to the fresh eggs at two concentrations and compared this to a batch of eggs with no sperm added. We saw evidence of successful fertilisation of tuxedo urchin eggs 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 our visit, we conducted a fertilisation test for the orange spined sea urchin. We saw initial evidence of successful fertilisation and cell division within a few hours, providing promising results which will require further follow up in a future visit.

The success of the cryopreservation process was tested by thawing out one vial of preserved sperm from each species and testing its ability to fertilise freshly spawned eggs. Pictured here is evidence of the successful fertilisation of tuxedo urchin eggs from cryopreserved sperm. A) The fertilisation envelope, which appears as a halo around the eggs, indicates successful fertilisation. B) Cell division begins, confirming that the egg has been fertilised. C) Cell division continues in multiple embryos, which will hopefully go on to form larvae and adult urchins.

How these sample 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 that we are hopeful to make progress on with the help of our expert network.

Who made this possible

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