Cincy Science: Cincinnati Zoo CREW uses deep freeze to help animals breed, even after they're gone

CINCINNATI - When an Indian rhino gave birth to a calf at the Buffalo Zoo, it grabbed national attention. With only 60 of the species in captivity in North America, the birth seemed even more amazing considering the calf's father "Jimmy" died at the Cincinnati Zoo more than 10 years ago.

Terri Roth, Director of CREW (Conservation and Research of Endangered Wildlife) and vice president of conservation at the Cincinnati Zoo, explained when it comes to saving endangered species, the Cincinnati Zoo is banking on its cryopreservation research.

The CryoBioBanks at the Cincinnati Zoo contain preserved genetic material from endangered species. 

1. Tell me about Cincinnati Zoo’s CREW 

We’re kind of unique in Cincinnati actually having a zoo that supports a conservation research center like CREW. Most zoos don’t have an established research facility like ours.The focus is on propagation for both endangered animals and endangered plants – we work on both. Along those lines, we’re very interested in cryopreserving samples.

Primarily we’re looking at managing these populations long term and we want to make sure we maintain genetic diversity. We’re trying to facilitate natural breeding and enhanced reproduction anyway we can that way. But in addition to that we develop assisted reproduction and artificial insemination (AI) and in some cases, in vitro fertilization, and some of the higher tech methods as well to try and ensure these species reproduce and to enable us to move genetic material around the country and even internationally without having to necessarily move the animal for breeding purposes.

2. Could you elaborate on the process?

So along those lines we have to learn how to cryopreserve semen, we have to learn how to cryopreserve embryos from all these different species and if often takes different protocol. Different species react differently to different methodologies, so there’s a lot of research that goes into that for each species that we focus on.

And it’s parallel to that in the plant division. We work with a lot of endangered plants in North America, but our expertise is in tissue culture. So we work with the Botanical Gardens and they can tell us which species are difficult to propagate by traditional means. 

3. So you store the genetic material here?

We have here a large CryoBioBank and it’s basically a bunch of liquid nitrogen tanks that contain all this very valuable genetic material. And some of it is moved out quite frequently, for example our rhino program. If we’re doing artificial insemination for rhinos in different zoos across the nation, we’ll often take samples from our CryoBioBank to these other zoos to do the AI procedures. In other cases we have samples that may sit in the tanks and just be stored for the next 20 years. I just depends on what the needs are.

"We’re kind of unique in Cincinnati actually having a zoo that supports a conservation research center like CREW. Most zoos don’t have an established research facility like ours." Terri Roth, CREW director

4. You recently had success with an Indian rhino? 

Yes, the rhino up at the Buffalo Zoo recently gave birth. That was a classic case of exactly what we were trying to accomplish here because the male was a rhino we had maintained here at the Cincinnati Zoo for a long time, but has arthritis when we had him and he was incapable of breeding. So he never sired any offspring, but we were able to collect and bank semen from him on several occasions. We were able to use that semen to inseminate the female rhino at the Buffalo Zoo.

She’s now produced basically his offspring and he’s been dead for 10 years now. So we like to tell people if we save the genetic material now, we have more genetic diversity to fuse back into the populations in the future. So there are a lot of different ways we can benefit from incorporating this kind of technology into a breeding program.”

5. I saw on your Polar Bear Signature Project that polar bears are difficult to breed in captivity. Are you using this technology with them?

We’re just at the starting point. We’ve been working on rhinos since the 1990s, so we’ve had a little bit more time and a lot of experience under our belt. But the polar bear we just started working with in 2008, and then really became aware of how poorly they are reproducing. So, we’ve developed some methods to monitor their reproductive activity and we’ve been trying to determine where the reproduction is going wrong.

But at the same time we’ve had to act and jump into the artificial insemination earlier than we anticipated simply because some facilities are so desperate to try and get cubs - and right now there simply aren’t enough bears to match up so we have breeding pairs in every facility. So in the case of polar bears we have done a couple of AI attempts, and so far nothing has been successful. But again we’re just starting, so it’s a really steep learning curve.”

6. How crucial is this type of research to preserving species?

It depends. In some species, it could end up playing a pretty significant role and in other species it may not. What we try to do is prepare for the future and make sure we have as many options available to us as possible. So we’re not always sure. Typically with the rhinos in particular, we kind of focused on the Asian Rhinos because behaviorally they can be very incompatible. So if you think you have a great genetic match, but behaviorally they just fight, this kind of technology helps you overcome those behavioral problems.

But there are other situations too. We had an ocelot that lost her leg as a kitten and couldn’t be paired with a male for breeding because of the concerns that if the male started getting rough, she would be at a disadvantage. The only way she was going to give birth was through assisted reproduction. We did artificial insemination on her and she was able to produce a kitten twice now, otherwise she would have probably gone throughout her life without ever reproducing. So you never know, there are individual cases that become very important that we can contribute to and also the species at large.

7. So you’re increasing numbers both in captivity and in nature?

There are a couple of species that have truly benefited from this kind of work. The one that first comes to mind is the black footed ferret. And this is work we have not been involved with, but one of our collaborators at the National Zoo has been very involved with it. It’s through a combination of natural breeding and artificial insemination that they have built up the black footed ferret population and then those animals are the ones that have been reintroduced back to the wild. So it’s played a very important role in that particular species. Actually getting the species numbers up, getting them back out to the wild and making sure they’re genetically viable because there’s enough diversities there.

8. Are you trying to keep breed purity or can you mix different breeds of say for instance, rhinos?

We try to stay pretty pure with it. What you’re referring to is interspecies embryo transfer and there was a lot of interest in it back in the 1980s. The idea was that we could use these related surrogates that were maybe more abundant and we could put a lot of these endangered embryos into them and produce much faster. But it’s had very limited success and scientists have become a little more concerned about potential differences about those species in terms of the uterine environment, in terms of the milk produced, to the immunity it’s passed on.

I think we’ve kind of pulled back from that in the most part, although it’s still an idea that’s floated around especially for the species we’re in desperate shape with where it might be useful. And one example is a polar bear and a brown bear. We know the polar bears and brown bears actually hybridize on occasion in the wild. So our thought is their reproduction must be pretty similar and there is a possibility you could put embryos from one into the other. But we’re not at the point of marching down that path yet.

9. So you’re not trying to create new breeds like they do with dogs?

“When you get into dogs and cats and what we’ve done with breeding, those are the extremes. With wildlife you kind of don’t breed specific traits so much, but there is some effort sometimes to preserve even at a subspecies level. For example, the Florida panther, versus a western puma, there’s something that makes that Florida panther adapted to it’s environment. If we were to delude that out too much, would they still be as successful in Florida – we don’t know. But when the Florida panther became very, very inbred and it became apparent that if we didn’t get some genetic diversity somehow that they probably wouldn’t survive, we then did make the decision to bring in some Western pumas.

What they ended up doing was just introducing the western pumas until a few litters of cubs were produced and then they pulled the western pumas back out, so they didn’t dilute the Florida genes too much. Every situation is unique. We then have to look at the problems and if the inbreeding is more of concern than maintaining the pure subspecies, then sometimes you’ll just cross that line and say we need some new genetic diversity and let’s get it in here.

(Photos courtesy of Cincinnati Zoo)

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