Applied Zoo-ology

Analysing black rhino faeces might not be every scientists’ idea of fun, but for Sue Walker, wildlife endocrinologist at Chester Zoo, UK, there is valid method behind apparent madness. 

There are less than 600 East African black rhinos left in Kenya, which make up 70 per cent of the global population. Therefore, maintining a genetically healthy and viable captive population is incredibly important; however,  managing and breeding black rhinos in captivity is not straightforward.

 “When working with wildlife, we try to do everything non-invasively. We don’t even want to touch the animals, so we use faeces, urine – anything we can easily get our hands on,” says Walker. But stepping away from traditional blood sample analysis causes problems, especially when it comes to studying the hormones that can help improve breeding program success. 

“If you analyse blood from the animal, the hormones are equivalent to human progesterone and oestrogen. In other words, native hormones that are quite easy to identify with enzyme immunoassay (EIA) tests,” Walker explains. “But in our complex samples, those hormones have been broken down, and we need to confirm that the metabolites measured with EIA are reflective of what’s going on in the animal’s endocrine system.”

Linking unknown metabolites to their parent hormones with EIA alone is difficult, because of antibody cross-reactivity. The complex relationship between hormones related to reproductive health (progesterone, oestrogen and testosterone) and those known to indicate the impact of potential stress (glucocorticoids) further clouds the issue. Confirmation of the results is essential.

How one company’s scientific know-how is helping a black rhino breeding program

Analysing black rhino faeces might not be every scientists’ idea of fun, but for Sue Walker, wildlife endocrinologist at Chester Zoo, UK, there is valid method behind apparent madness. 

There are less than 600 East African black rhinos left in Kenya, which make up 70 per cent of the global population. Therefore, maintining a genetically healthy and viable captive population is incredibly important; however,  managing and breeding black rhinos in captivity is not straightforward.

“When working with wildlife, we try to do everything non-invasively. We don’t even want to touch the animals, so we use faeces, urine – anything we can easily get our hands on,” says Walker. But stepping away from traditional blood sample analysis causes problems, especially when it comes to studying the hormones that can help improve breeding program success. 

“If you analyse blood from the animal, the hormones are equivalent to human progesterone and oestrogen. In other words, native hormones that are quite easy to identify with enzyme immunoassay (EIA) tests,” Walker explains. “But in our complex samples, those hormones have been broken down, and we need to confirm that the metabolites measured with EIA are reflective of what’s going on in the animal’s endocrine system.”

Linking unknown metabolites to their parent hormones with EIA alone is difficult, because of antibody cross-reactivity. The complex relationship between hormones related to reproductive health (progesterone, oestrogen and testosterone) and those known to indicate the impact of potential stress (glucocorticoids) further clouds the issue. Confirmation of the results is essential.

Improved analysis = better breeding

“Anytime something comes up that’s out of the ordinary, we jump at the chance to help,” says Tim Liddicoat, a Thermo Fisher Scientific Support  manager and key member of the collaboration.

In 2006, Harald Ritchie, Director, Business Development at Thermo Fisher Scientific, was touring the zoo’s vet facility and asked Walker if she would ever consider using HPLC.  “I pretty much jumped up and down and said ‘I would love to,’ but we didn’t have the money or the support,” says Walker. HPLC can fractionate the sample and allow different EIA tests to be run in parallel, which eliminates cross-reactivity and provides confirmation, despite an unknown target.

For the female black rhino, long and short reproductive cycles have been identified. Analysis using HPLC and EIA have confirmed that long cycles are linked to increases in adrenal function as reflected by higher concentrations of glucocorticoids. Research is now underway to understand why these long cycles occur. Despite this unknown, the HPLC and EIA analysis is currently being used to determine the most appropriate time to introduce males to the females without aggression. The accompanying photo illustrates the tangible benefits of getting this timing right.

Recently, the work has gone beyond HPLC and into mass spectrometry (MS). The flexibility of MS allows qualitative and quantitative analysis of extremely complex samples. “Initial results by quadrupole MS have been very promising. If we consider using an instrument capable of accurate mass scanning it will not be necessary to know which metabolites you are looking for because you can reexamine the data at a later date and identify compounds you did not initially target,” says Liddicoat. Walker’s dream, and the subject of continued collaboration between the Zoo and Thermo Fisher Scientific, is being able to measure multiple hormones from a single sample.

Sample stability

Another notable development from the collaboration is the eyebrow-raising use of consumables. “Our sample preparation is novel,” Liddicoat explains. “We store rhino faecal extracts on solid phase extraction (SPE) cartridges. It’s a really inventive thing to do”.

Walker’s team have shown in tests that faecal extract can be stored for about six months on SPE cartridges, and that has particular benefits in the field. For example, a PhD student from the University of Nottingham, UK who is tracking wild elephants in Malaysia is using the cartridges to collect and store faecal extract on the trail. Back in the lab, the sample can be pulled from the cartridges for further analysis. 

The cartridge loading procedure is fairly standard but with some important differences: it is performed in the field with no electricity or expensive equipment and the methanol concentration is higher than a typical reverse-phase extraction because of the need to extract compounds of interest from the sample. 

The complete process allows for easy storage and shipping. “I am aware that this type of protocol has been carried out previously on on-line SPE cartridges for storage of pharmaceutical library samples, but not routinely and not with biological samples. But it does make sense as chemists have known about the stability of solid-supported compounds for a while,” says Liddicoat.

An additional benefit? “It’s great when someone asks me what I do for a living and I’m able to describe the project at the zoo without sending them to sleep in 20 seconds flat,” he says. “Having something good to talk about at the pub? You can't buy that.”

Look at the Chester Zoo’s Website for more information about ongoing projects.