Article

How we can store sperm to prevent amphibians from going extinct


Amphibian sperm can be stored to conserve threatened species (credit: YULIA_988 at Shutterstock)
Amphibian sperm can be stored to conserve threatened species (credit: YULIA_988 at Shutterstock)

Even though this may not be obvious in your daily life, we're currently facing a mass extinction of animal and plant species. This is because issues caused by humans such as climate change, habitat loss, invasive species, diseases, and environmental pollution negatively affect many species. One of the animals facing extinction is monarch butterflies as I recently explained in my article "Protecting the Monarch: Strategies to Prevent Butterfly Extinction".
While butterflies are insects, also other types of animals are at risk. For example, amphibians are facing a similar faith: a shocking 41% are at risk of extinction! Although amphibians are also affected by all mentioned environmental issues, the most important cause is infectious diseases. One of these diseases is chytridiomycosis, caused by chytrid fungus (Batrachochytrium dendrobatidis). This fungus causes damage by invading the surface of amphibians' skin. While it is unclear why this disease has become so common and why it kills amphibians, it is possible that environmental stress, for example caused by climate change or increased exposure to ultraviolet radiation, reduces amphibians' resistance to infection. This reduced resistance allows the fungus to disrupt normal functioning of the skin, resulting in an internal imbalance.
Axolotl (Ambystoma mexicanum) are threatened with extinction (credit: axolotlowner on Shutterstock)
Axolotl (Ambystoma mexicanum) are threatened with extinction (credit: axolotlowner on Shutterstock)

As amphibians are facing so many environmental threats, conservation efforts in their natural environment may not be enough to save them from extinction. That is why also other efforts, away from their natural habitat, are needed. One of the possibilities is applying reproductive technologies. These technologies allow humans to increase the number of offspring, improve genetic diversity, and exchange genetic material between animals living in captivation and the wild. Genetic diversity can for example reduce when populations become isolated. The exchange of genetic material is even possible with previous generations!
An effective way to exchange genetic material in amphibians is using sperm. Sperm from living animals can be used but recent advances in reproductive technology allow us to use sperm from non-living animals. This is possible because amphibian sperm can now be stored by keeping the sperm cool for short-term storage or freezing it for long-term storage. Short-term is usually up to about a week and occasionally up to a month; long-term is up to several decades or even longer. This is how this works:

Obtaining amphibian sperm

The first step to ensure a species' future conservation by storing amphibian sperm is obtaining sperm from living animals. Obtaining sperm can be done during surgery by taking out the male animal's testes, which is of cause very harmful - unless it is done immediately after an animal died naturally for other reasons.
A more gentle way to obtain sperm is by applying hormone therapy, which causes sperm cells to develop and be released so that they can be captured - similar to when a fruit from a tree falls off when it is ripe so that it can be harvested. As the effects of hormone therapy are only temporary and it allows sperm to be obtained more often, this is the preferred method. To make hormone therapy successful, it is important to choose the optimal hormone type, dose, and frequency because males from different amphibian species react to hormones differently.
Hormone therapy is used to cause sperm cells to develop and be released (credit: Maria Sbytova on Shutterstock)
Hormone therapy is used to cause sperm cells to develop and be released (credit: Maria Sbytova on Shutterstock)


Storing sperm

The second step to ensure a species' future conservation by storing amphibian sperm is storing the sperm. Here, it is important that the conditions are right. This is important because whether fertilization is successful mostly depends on the number of healthy sperm cells and their speed. This means that storage conditions should ensure that these parameters are kept intact as much as possible.

Short-term storage

One example of a storage condition that influences whether sperm remains intact short-term is osmolality. Osmolality is the concentration of all dissolved particles in the sperm fluid. Before ejaculation, this concentration is the same as the concentration in the surrounding environment. This causes the sperm cells to stay where they are. After ejaculation, the difference in concentration between sperm and the surroundings suddenly increases. This causes chemical reactions in the sperm cells, which in turn causes sperm to move. But their movement also uses up their energy reserves quickly. This means that when storing sperm, it is helpful to keep high osmolality so that sperm remains inactive.
Another example of a storage condition that influences whether sperm remains intact short-term is temperature. Usually, temperatures between 0 to 5 °C (32 to 41 °F) are optimal for short-term storage of amphibian sperm, although this differs between species and can be higher as well. Low temperatures reduce the energy use of sperm cells and reduce bacterial growth.

Long-term storage

One example of a storage condition that influences whether sperm remains intact long-term is the preservation medium. The preservation medium is the substance surrounding the sperm during storage. This medium needs to have several characteristics that protect the sperm from freezing cold. These characteristics include preventing ice formation within the sperm cells and preventing dehydration. Also, the medium needs to contain substances such as antioxidants and antibiotics. The presence and concentration of components differ per species, as they need to be optimized for the physiological characteristics of the sperm cells.
Another example of a storage condition that influences whether sperm remains intact long-term is the freezing method. Freezing puts a lot of stress on sperm cells. Stress factors include ice that can form inside the cells, a sudden change in the medium's concentration, and toxicity. These stress factors can damage sperm cells and make them unable to fertilize an egg. To limit the damage as much as possible, optimal rates and conditions need to be determined. For example, whether a slow-cooling rate of -200 °C (360 °F) per minute or an ultra-rapid cooling rate of -1000 °C (1800 °F) per minute is better. With an ultra-rapid cooling rate, the formation of ice crystals is prevented by converting the sperm and medium to a glassy state immediately but this method results in a lot less usable sperm cells after thawing. Cooling can be done using programmable freezers, by holding sperm samples above the surface of liquid nitrogen, or a specially designed device for this purpose called dry shipper.

A tube above liquid nitrogen (credit: Elena Pavlovich on Shutterstock)
A tube above liquid nitrogen (credit: Elena Pavlovich on Shutterstock)


Using sperm

The third step to ensure a species' future conservation by storing amphibian sperm is using the sperm to produce offspring. Preferably offspring that can't be distinguished from natural offspring in terms of fitness, development, and survival, and without abnormalities.
Artificial fertilization involves several steps:
  1. An egg cell needs to be obtained from a female amphibian, by massaging the abdominal after providing hormones, by obtaining the egg from a mating female, or by obtaining it from the oviduct - the tube that leads away from the ovary - under anesthasia.
  2. The egg cell is placed onto a Petri dish.
  3. The reactivated thawed sperm and storage medium are poured onto fresh egg cells, into the Petri dish, for artificial fertilization.
Thawed sperm under a microscope, in this case of a bull (credit: "Bull Semen Motility" by Ghadeer Bustani is licensed under CC BY-SA 4.0)
Thawed sperm under a microscope, in this case of a bull (credit: "Bull Semen Motility" by Ghadeer Bustani is licensed under CC BY-SA 4.0)

This artificial fertilization process is in some species equally effective compared to natural fertilization when looking at hatching success and larval survival while in other species, it is less effective. Also, amphibians' body weight at metamorphosis and other characteristics such as length can be the same or differ, depending on the species. Metamorphosis involves in amphibians the changes that are needed for life on land as opposed to life in water. Their weight at this stage is important, as it impacts for example fertility and their ability to find food. And important for the conservation of species: whenever offspring from stored sperm reaches sexual maturity, they can produce a new generation.

Conclusion and how we can take action

So, sperm storage can be used to conserve threatened amphibian species by storing amphibian sperm, either short-term or long-term. This sperm can then be used to artificially fertilize an egg, which can produce healthy offspring. This healthy offspring can in turn grow the species' population by natural fertilization processes.
Here are practical ideas of what you and I can do to save amphibians:
  • Refraining from chemical fertilizers and pesticides to prevent water pollution
  • Properly disposing of waste to prevent pollution
  • Protecting natural habitats in which amphibians can thrive
  • Preventing fragmentation of habitats when building infrastructure
  • Building ecoducts (animal bridges) so that amphibians can safely cross busy roads
  • Supporting organizations that specialize in protecting amphibians
Ecoduct (credit: Maarten Zeehandelaar on Shutterstock)
Ecoduct (credit: Maarten Zeehandelaar on Shutterstock)

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Did you enjoy this article? Then I'm sure you'll love my book "A Guide to A Healthier Planet" as well. Have a peek inside and find out more at:


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About the author

Dr. Erlijn van Genuchten is a an internationally recognized environmental sustainability expert. She is a science communicator, helping scientists in the fields of nature and sustainability increase the outreach of their results and allowing us all to put scientific insights into practice and contribute to a sustainable future. Erlijn has inspired thousands of people around the world - for example - by supporting the United Nations with her expertise, her book "A Guide to A Healthier Planet" published by Springer Nature, her YouTube channel Xplore Nature, and her posts on social media.

Credit

This article is based on:
Image: "Bull Semen Motility" by Ghadeer Bustani is licensed under CC BY-SA 4.0
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