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For anyone who works with clouded leopards, Neofelis nebulosa,
it becomes evident very quickly that they are many things. They
are beautiful, secretive, often reclusive, amazing, and everything
about managing them is a challenge. To work with them is both
a privilege and at the same time frustrating.
The problems in managing clouded leopards are many. First and
foremost is the high incidence of males killing or injuring prospective
mates either on introduction or at some time during their time
together. (Clouded leopards have the largest canines relative
to body size of any cat species). Cats successfully paired often
spend their entire life with only one mate since most zoos won't
risk re-pairing. Some success in pairing has also come by introducing
cats before a year of age. The risk of injury at introduction
is less with animals that are not yet sexually mature. There have
been mortalities however when these young cats reach sexual maturity
and the relationship changes. When one of the animals behaves
in a "different manner", either male or female, become
vulnerable to the other animal misreading their body language
and acting in a defensive or aggressive way.
To successfully manage a captive population, it is important
to have as many "founder" animals as possible, (animals
unrelated to the rest of the population). Next, it is important
to equally represent all of the founder animals in the population
to maintain genetic diversity. To do this in most other species,
breeding recommendations would be made based on pairing animals
with the lowest mean kinship, (least amount of relatedness to
each other). With the problems of pairing and re-pairing of clouded
leopards, this has not been successful. If that wasn't bad enough
the captive population is dealing with effectively only three
founders, making the population fairly inbred. This is compounded
every year that we are unable to effectively breed the most genetically
desirable animals. Most of these animals haven't bred so far because
of unsuccessful pairing attempts, singly housed individuals that
are too risky to pair due to their age, or animals with very low
fertility. Bringing in wild clouded leopards has not been an option
for a very long time. The few wild animals brought in years ago
never reproduced, probably due in part to the fact that they did
not adapt well to the change to captivity.
Assisted reproduction is often a wonderful management tool to
use for these difficult situations. Remember earlier I mentioned
how difficult and challenging clouded leopards can be? Well, what
is a typical practice in farm animals is of course anything but
typical in clouded leopards! I think most people don't realize
how many years goes into research into the specific characteristics
of each species reproductive systems, before any thought of attempting
assisted reproduction. I know I had no idea about 15 years ago.
Since that time I have learned a lot! First, many questions have
to be answered, such as how long is the female's estrus cycle,
is she a spontaneous ovulator or an induced ovulator, or a combination
of both? What is the male's reproductive cycle like? Are they
seasonal breeders? Can this be changed by artificial light cycles,
and if so what is the optimum light cycle? Then there is the question
of sperm quality. Developing appropriate medias for holding semen,
as well as cryopreservation methods to successfully freeze it
for future use have to be researched. In most species this process
takes a minimum of two years.
Most of this groundwork was done for clouded leopards early on
due to the difficulties in successful pairing, and the problem
we were facing with inbreeding. Like I said earlier, everything
with clouded leopards is difficult. Nothing comes easily. We have
had a dedicated team of reproductive specialists from the National
Zoo working on the "clouded leopard problem" for about
15 years. The first obstacle was the overall poor semen quality
in most of the population. Compared to all other cat species,
they have the highest percentage of abnormal sperm, they have
a low sperm count, and the sperm are hard to keep alive long enough
to get through the freezing process. The females can be both induced
and spontaneous ovulators. These factors greatly complicated assisted
reproduction attempts.
Ten years ago artificial insemination was attempted in a clouded
leopard. It was a success! It must however, have been a perfect
situation and luck because there have been no further successes
since then. Hopes were maintained that "we" (a dedicated
group of clouded leopard managers, including the Studbook keeper/SSP
Coordinator, and reproductive specialists) would keep at it until
we could find a way to be successful. That path has been long
and hard. There have been many tests, trials, and failures. Along
the way there has been little good news, but we kept learning
more about what is going wrong. That ultimately will lead to knowledge
of what we need to do to overcome the barriers to success.
Some of the things learned through failure were things like putting
semen into an immobilized female did nothing. The sperm usually
get help swimming to the egg by peristaltic movements within the
uterus. But, when the female is immobilized so is the uterus!
Knowing that, their next attempts were to place the semen at the
end of the fallopian tube. This was timed so that the semen was
deposited just before the eggs were ovulated. The sperm wouldn't
have to swim very far and the eggs would be falling down near
them very shortly. Sounds like a great plan. But we found out
later that if the females hadn't ovulated before they were immobilized,
they wouldn't because of the immobilization agents. The researchers
then decided to try IVF (in-vitro fertilization, where the sperm
is introduced to a few eggs in a petri dish. Once the sperm have
fertilized the eggs and several cell divisions have occurred,
they place the embryos back into the female). MZG along with several
other zoos participated in this effort about 6 years ago. We had
three females implanted, as well as other females around the country.
We ended up with no pregnancies! Have I mentioned that clouded
leopards are always difficult?! They discovered that even though
the females responded well to the hormonal regime to get them
to super ovulate, and then to be in the proper stage to have embryo
implantation occur, the fact that they have both types of ovulation,
(spontaneous-meaning a regular cycle, or induced- meaning when
stimulated by the presence of a male or another cat) the influence
of induced ovulation could have affected hormones necessary to
maintain a pregnancy.
After all these failures it is gratifying that anyone would be
interested in continuing to work on this project! These are an
amazing group of people. Their dedication to the species and to
the clouded leopard SSP (Species Survival Plan) and Management
Group is awesome! For the past five years the main focus for assisted
reproduction in clouded leopards was to develop a protocol to
completely shut down the female's reproductive track. The thought
being if we could quiet the system and then start it up again
with an effective dose of hormones we might eliminate the complicating
factor of the multiple ovulation systems.
Many steps were taken over that 5 yr period to identify drugs
to use, test dosages, set up trials, make sure the female's system
started up again after the test period, find out how long it took
to start up again to determine the "window" for the
final step, AI, (artificial insemination).
Every year at the Felid TAG, (Taxon Advisory Group), the researchers
would give us an update on how the trials were going. Every year
there was some bad news, and more testing to do. Two years ago,
there started to be some progress. Several drug regimes were identified
and preliminary studies looked promising. But there was still
more to do to fine tune the process before we would be ready for
the next step. Last year before the annual meeting they felt fairly
comfortable with the Lupron protocol and they were planning on
doing two cats at the National Zoo. We were all excited to hear
the news at the TAG meeting…..but again no good news. How
many times must we fail before we get a break? The researchers
went back to their studies and picked two protocols that now looked
like they had the most promise. They tested them in domestic cats
and had good results.
In August of last year MZG hosted the clouded leopard master
planning meeting. In that meeting we made recommendations for
every animal in the North American captive population. Pairs that
were together were to stay together and breeding was encouraged.
We need cubs to maintain our age pyramid as well as our overall
numbers. Moves were recommended to best meet the needs of the
individual animals and institutions. We then made recommendations
for 20 females not currently in a breeding situation, comprised
of all age levels from 18 months to 15 years, to be candidates
for an AI trial. Finally! Just getting to the point of trying
again after all these years, was a step we had all been waiting
for! We accepted two young females, (18 month old siblings) Nattie
and Nelly to come to MZG, on loan from the Central Florida Zoo.
We currently have Kuala, an 8 year old female on loan from Hexagon
Farm in California. Nellie and Kuala were on the Norplant protocol
and their AI procedures were scheduled for March 11, and 12.
The team from the National Zoo, Drs. Katey Pelican, who was in
charge of the clouded leopard portion, and JoGayle Howard, who
was working on the fishing cat project, arrived on March 10th
to spend all day setting up for the next three days of procedures.
It was going to be a very busy several days. On Monday March 11,
tension was high as we coordinated the immobilizations of both
the male 5661, for fresh semen donation, and female, Nellie for
her laparoscopic exam. Katey would perform this exam to determine
if the ovaries had responded correctly to the protocol (this was
the first clouded leopard to be done on this protocol). Sadly,
she had not ovulated. Instead her ovaries appeared to have been
hyper stimulated and were full of many immature follicles. The
disappointment in the room was palpable. Here we had a young,
prime candidate, our best hope to date, and once again failure!
There was no point in inseminating her. Measurements and descriptions
of the uterus and ovaries, along with photographs and videotape
to document what Katey was seeing, was recorded. This information
is vital when the team tries to figure out what to change for
next time.
All of us involved with this project know the probability of
failure is high. We try to always keep that in mind as we approach
these procedures, but it is in our nature to hope that this time
might be the time when every thing will go right. So despite our
disappointment, we put on our professional faces and looked at
the rest of the procedure as a learning experience. Tomorrow is
another day.
Tuesday, March 12th, we had Kuala scheduled for her laparoscopic
exam and at the same time would have male 6376 on the table with
a second team collecting his very valuable semen. He is at the
top of the genetic wish list. He has never reproduced. He never
bred with his first mate of seven years, and killed his second
mate during introductions. He is twelve years old and has some
medical issues. He does have good quality semen but it starts
to die as soon as it leaves his body. The researchers made a few
changes to his collection protocol that morning and the improvement
was substantial. His sperm count was good and it stayed at the
70% motile level throughout the prep time. Kuala had been on the
same Norplant protocol as Nellie had been, so we were anticipating
similar results from her laparoscopic exam. The room fell silent
with guarded anticipation as Katey looked through the scope. Finally,
we heard those wonderful words, "we have ovulation"!
There was literally dancing and hugging going on in that surgical
suite. Clouded leopard history was being made in that room that
day. We actually had our best shot in ten years at a successful
artificial insemination procedure! I have to say it was one of
the highlights of my professional career.
Does that mean that we are expecting clouded leopard cubs this
summer? Well, we can hope so, but these are clouded leopards after
all! Lots of things can go wrong. The sperm still needs to find
the ovulated eggs and fertilize them. Semen was placed in both
uterine horns where fresh eggs were waiting. If the eggs were
fertilized the embryos would still have to successfully implant
into the uterine lining to be nourished throughout the gestation
period of 90 days. We do not plan on testing for pregnancy since
that would require an immobilization. We will manage Kuala as
if she is indeed pregnant. Probably the first sign of pregnancy
would be a day or two before delivery when females often den up
and refuse food. Even if Kuala would give birth, neonatal mortality
is high in first time mothers. We will continue to monitor her
and provide every possible management skill we know to provide
the best possible opportunity for success.
I do want to emphasize that even if we do not end up with cubs,
this was a huge accomplishment for clouded leopard assisted reproduction.
I would be thrilled to have cubs at MZG again, it has been 12
years since our last litter, but I am very gratified to have had
the opportunity to have been a part of putting good quality semen
from a very valuable, un-represented male, into a female in an
excellent reproductive state for our first real shot at a successful
artificial insemination.
We have Nattie on an oral Regu-mate protocol. She is scheduled
for her AI procedure in mid May.
We were also able to attempt the Norplant protocol on our two
female fishing cats. Both are getting up in years and have had
no opportunity for breeding. These were the first fishing cats
the protocol was performed on. Excellent quality semen was brought
from males housed at the National Zoo. Both these females had
laparoscopic procedures done, one on March 12, the other on March
13. Both females responded well to the regime. Both ovulated and
were inseminated. What a difference from clouded leopards, success
on the first two attempts! Again insemination does not guarantee
cubs, but the National Zoo team feels we have a pretty good shot
at it with them. I am excited about the prospect. Our exhibit
female 8330 is genetically the second most important female in
the captive population. To get surviving cubs from her would do
the population a lot of good as well as being a wonderful addition
to the Tropics Trail.
Submitted by Beth Jo Schoeberl, Tropics Zoologist
Institutional representative to the Felid TAG
Clouded Leopard SSP Management Team Member (recently elected to
my third three year term).
Fishing cat SSP Management Team Member
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