Ibis (2000) 142, 1-1 1

Biology of a critically endangered species, the Toki
(Japanese Crested Ibis) Nipponia nippon
JOHN C. WINGFIELD‘*, SUSUMU ISH112,MOTOSHI KIKUCHIZ, SHUlCHl WAKABAYASHI3,HIDETSUGA SAKAI3,NOBUYUKI YAMAGUCHI’, MASARU WADA4 & KOUKI CHIKATSUJF‘Department of Zoology, Box 351 800, University of Washington, Seattle, Washington 98195, USA“Department of Biology, Waseda University, Tokyo 169-50, Japan
3Department of Biology, Nihon University School of Dentistry, Surugadai, Tokyo 101, Japan
4Department of Biology, Tokyo Medical and Dental University, lchikawa-shi 272, Japan%ado Ibis Preservation Centre, Niibo-mura, Sado-gun, Niigata-ken 952-01, Japan

Populations of the Tolu, or Japanese Crested Ibis Nipponia nippon are now reduced to asingle female in Japan, and captive and free-living populations of about 80 individuals inYang Xian, Shaanxi Province and the Beijing Zoo, People’s Republic of China. Very little
has been published about the biology of this species except for Japanese journals andpopular articles. Here we outline some unique aspects of the breeding biology of thisspecies as well as introduce endocrine methods as a way of determining the source ofreproductive problems in captive breeding programmes and, tentatively, how hormone
manipulations based on these observations may help to enhance reproductive output. The
Tolu has a unique nuptial plumage that is acquired by the cosmetic application of a blacktar-like substance secreted by a well-defined patch of skin in the throat and neck region.
Apparent specialized down feathers develop in this region during a localized moult in
November. The tar-like substance is secreted in January and February and is applied to thehead, neck and upper torso by a combination of bathing and daubing behaviour.
Development of nuptial colour is thus a combination of morphological development,
secretion and a unique behaviour. This combination is found only in adults. Single plasma
samples collected from captive Tolu at known times in the breeding cycle suggest normal
hormone patterns in some individuals, and abnormal levels in those that fail to breed.
These conclusions are strengthened by endocrine investigations of a non-endangered
ibis species, Threskiornis rnefanocephalus, that reveal normal patterns of circulating
concentrations of gonadotrophins and sex steroids as expected for a seasonally breeding
bird. Measurement of faecal hormone levels may be a less invasive way of monitoring
reproductive function in critically endangered species. Recent work on Japanese Quail
Coturnix juponica shows that hormone therapy using purified avian gonadotrophins in
non-reproductive individuals results in gonadal maturation, egg-laying and production of
reproductively active offspring. Although these techniques are still in their formative
stages, results to date strongly suggest that carefully applied endocrine techniques can
indicate potential problems in captive breeding programmes and may be useful forhormone therapy in critically endangered species.

There can be few more critically endangered species
than the Toki or Japanese Crested Ibis Nipponia nippon(order Ciconiiformes, family Threskiornithidae). In the
18th century, this ibis was widely distributed through*Corresponding author.
Email: jwingfie@u.washington.edu

02000 British Ornithologists’ Union

Honshu and neighbouring small islands, part of
Hokkaido, Korea, adjacent parts of China and eastern
Siberia. In the 19th century, they may even have
expanded their range to the islands of Kyushu andShikoku, Japan (Yamashina & Nakanishi 1983). The
Toki was formerly a common sight on ponds andpaddyfields where it foraged for much of the year.

2

J.C. Wingfield et al.

Most populations underwent a short migration tobreediilg colonies in forested foothills (Yamashina &Nakanishi 1983). Probably as a result of hunting andhabitat loss, populations of the Toki began to decline a t
the end of the 19th century and by 1920 they were
thought to be extinct in Japan. Many wetland habitats
were lost and most paddyfields became surroundedby dense human habitations. It also appears that the
Tolu was rather tame and thus easily approached andcaptured (Austin 1948). As a result it was hunted andharassed to extinction over most of its range (Austin &Kuroda 1953). In 1929 and 1930, two small populations were rediscovered on the Noto Peninsula andSado Island, Niigata Prefecture. Apparently these
populations remained in forested areas and did not
migrate to open wetlands outside the breeding season.
Additionally, they nested as single pairs and not inhighly vulnerable colonies. It is assumed that for thesereasons remnant populations survived in Japan(Yamashina & Nakanishi 1983). Although the Japanese
Government gave the Toki protected status in 1934,
the Second World War prevented any conservation
measures from being applied (Anderson 1984). By
1950 there were 29, and in 1980 only five were alive(Yamashina & Nakanishi 1983, Ishii et al. 1994). In
1981 the Sad0 Japanese Ibis Preservation Centre wasestablished, and all remaining Tolu in Japan were
captured and housed here. Despite concerted efforts tobreed them in captivity, the population continued to
decline. Today, only one remains in Japan, an old
female - 'Kin' (Ishii et al. 1994).
At the time the Japanese birds were being captured
and transferred to the Preservation Centre, two nestingpairs of Tolu were unexpectedly discovered in Yang
Xian, Shaanxi, China (Ishii et al. 1994). As of 1997,
there appear to be about 44 in the wild, eight immatures and six adults in a breeding colony in Beijing Zoo,and 25 in captivity at a field station near the wild population (see Table 1). Clearly the future of the Tolu lies
with the Chinese population.
Despite the critically endangered status of the Toki,
surprisingly little has been published on this species in
the western literature. One major reason is that mostfacts about the natural history of the Toki arepublished in the Japanese literature. For example, of
639 articles cited by Yasuda (1984a, 1985) as referringto the Tok, 72 were in English, 14 in French, 12 inGerman, two in Russian, one in Spanish and one in
Latin. These comprise only 16% of the extant literature. Papers that focus entirely on the biology of theToki, and present most of what is known of thisspecies, are all in Japanese. Here we wish to review this@ 2000 British Ornithologists' Union, Ibis, 142, 1-11

information in English as a means of drawing attention
to this spectacular and biologically intriguing species.
We also review new information, including multidisciplinary approaches that combine morphology, ecology,
behaviour and endocrinology of non-threatened species,
as a way of identifying and addressing problems ofbreeding in critically endangered species. Whether theToki will survive the current population bottleneck
remains to be seen, but we hope that these data willemphasize the potential power of multidisciplinary
research in conservation biology and that this will help
other endangered species recover before their populations become so critically low.
Reproductive biology of the Toki
In historic times, the Toki was known to frequent
paddyfields (dry and wet) and ponds and swamps,
where they foraged for fish, amphibians and large
invertebrates. Nearby forested hills were preferred as
roost sites. They often formed breeding colonies,
particularly in pines Pinus spp. and chestnut trees
Castanea spp., where they built platform nests oftwigs. Many populations underwent short migrations
from low swampy areas to forested breeding grounds[Austin & Kuroda 1953, Yamashina & Nakanishi1983). Typically, four speckled eggs comprised a clutchthat was laid in April (Yamashina & Nakanishi 1983).
In the last century, it was thought that the Toki had
two colour morphs: one white and one grey. Later it
became clear that the grey morph was actually thenuptial plumage of both sexes (Yasuda 1984b). Typicalplumages of non-breeding and breeding Toki are
Table 1. Captive and wild populations of the Toki Nipponia
nippon in China in the spring of 1996.
~~

Province~~

Yang Xian

Status-~Wild
Captive

Beijing *Captive

Subtotal

Wild
Captive

Grand Total

Age~

No. of individuals--~~

AdultsYoung
Adults
Young

30
14
15
10

Adults
Young

8a
6a
44
39a

83 a

In 1997, both the Yang Xian and Beijing populations are reported to have increased.
aNot definite.

Biology of the Toki

3

Figure 1. Typical sequence of non-breeding and breeding plumages in the Toki Nipponia nippon during cosmetic acquisition of colour.
(a) Typical non-breeding plumage; (b) daubing behaviour (spreading of black tar-like substance); (c) nuptial plumage; and (d) localized
moult of contour feathers in the neck and gular region

presented in Figure 1. They are white in the nonbreeding season and grey on the head and upper bodywhen breeding. The salmon-coloured flush (Tokicolour in Japanese) to the primaries and secondaries ispresent all year, as is the red colour of the face and legs.
Development of this nuptial plumage may be a unique
phenomenon in birds. The grey colour is not acquired
through a moult, but by cosmetic application of atar-like secretion from a specialized patch of slun on
the neck and head (Uchida 1970, Yamashina &Nakanishi 1983, Yasuda 1984b). The white plumage
characteristic of the non-breeding season is acquired
through a normal postnuptial (prebasic) moult in latesummer. The sequence of plumages in the Toki isrepresented in Figure 1 (after Uchida 1970 and pers.
obs.) Note also that the plumage colour and structure,
as well as the skin colour of the legs, head (anterior to
the eyes) and eye-rings appear to be identical in males
and females. As far as we are aware, development of

nuptial colour in the Toki has not been described
previously in western journals.

Nuptial plumage
Development of nuptial plumage in the Tolu is bycosmetic application of colour rather than through aprenuptial (prealternate) moult. Skin from a specialized region of the head and neck (Fig. 2, after Uchida
1970, Yasuda I984b) secretes a black tar-like substanceor ‘cosmctic’ .that is then applied to feathers of thehead, neck, back and upper wing coverts resulting ina plumage that can vary from light grey to a dark ‘gunmetal’ grey. Examination of specimens (see Table 2
for a list of specimens examined for this paper) andphotographs of living Toki indicate substantial
variation in extent of the grey tint. In some individuals,
grey pigment may also extend to the primaries,
rectrices and contour feathers of the upper breast,
whereas in others it may be limited to the head, neck,@ 2000 British Ornithologists’ Union, Ibis, 142, 1-1 1

J.C. Wingfield et al

4aFigure 2. (a) Extent of the pigmented area of skin in the throat
region of the Toki Nipponia nippon. From Uchida (1970) withpermission of the author and the Yamashina Institute of
Ornithology. (b) Edge of the pigmented area of skin in the throat
region.

upper back, scapulars and some upper wing coverts(Fig. 1). Whether this variation is a function of theamount of cosmetic secreted, age and possible
experience in application, remains unknown. In the
non-breeding season, the plumage is always white.

Extent of the pigmented skin area
Measurements of specimens that are in good enoughcondition to handle indicated that the extent ofspecialized, pigmented slun (source of the blacktar-like substance) is also variable (Fig. 2a). The edge ofthe slun patch is very well defined (Fig. 2b) and its
extent can easily be measured. In one specimen, alimited throat patch is present, while another has
a small patch on the nape only. Seven other specimens
examined have large patches of black slun that extend
from the crown down to the nape but posterior to the@ 2000 British Ornithologists’ Union, Ibis, 142, 1-1 1eye, then around the sides of the neck to the throat andbelow the eye (Fig. 2a). In some individuals there may
be bilateral skm patches, but most appear to have one
patch with two connected lobes. On average, the blackskm extends along the side of the head 76 mm (rightside) and 81 mm (left side) from the base of the beak(starting from just behind the eye, range 50-100 mm
on the right side, 56 to 120 mm on the left side, coefficient of variation = 23.7% and 27.5% respectively).
With one exception, the extent of pigmented skin isgreater on the left side. On the crown, the pigmenteds h n begins a t 62 mm from the base of the culmen(range 53-67 mm, cv 9.9%). On the throat, black skinextends 47.2 mm from the base of the lower bill to its
posterior edge (range 21-96 mm, cv 58%). Others
who have examined the skin of fresh specimens (i.e.
after death and slunning) confirm the restricted andwell-defined shape of this patch (Yamashina &Nakanishi 1983, Yasuda 1984b). I t is not known if
variation in the extent and shape of the pigmenteds h n patch is related to age. One immature specimen
examined has no pigmented skin on the head and neck.
It was collected in February (Table 2), and was thus
in its first winter - a time when adults apply thecosmetic to their plumage.

Secretions of the pigmented skin area
Beginning in January, the pigmented skin patch begins
to secrete the cosmetic. Microscopic analysis revealed
that ‘chunks’ of this black substance are secreted alongfeather pores in the specialized slun region and accumulate around the rachis and on the barbules of downfeathers. Uchida (1 970) describes a black ring near theroot of the rachis. The cosmetic appears to ooze
out around the rachis and soaks into the rest of thefeather. However, the possibility that some epidermal
cells containing the black tar may slough off and burst
cannot be ruled out. Although the chemical composition of the cosmetic is unknown, it appears to consistof a black pigment (melanin?) in a non water-miscible
base (oil, wax?).
Possible rqorphological adaptation for application ofnuptial colour
Close examination of the specimens listed in Table 2
reveals a potentially specialized down feather that may
aid the application of nuptial colour. All adults withnuptial colour, or just prior to its development, havelong down feathers with short lateral barbs in theregion of pigmented skin. Down feathers from otherregions of the neck have much longer lateral barbs(Fig. 3). It is possible that these specialized down

Biology of the Toki

5

Table 2. Specimens of the Toki Nipponia nippon examined: location and origins
SpecimennumberYamashina lnstitute for Ornithology
32
213
2497
2683
2684
3710
25805
25806
25808
25a09
National Museum Research InstituteNo number
1
7
12175

Location

Date

Sex and age

Korea
Korea
Korea
Korea
Korea
Korea
Echigo, Niigata, Japan
Echigo, Niigata, Japan
Kaishu, Korea
Korea

Jan 1919
12 Jan 1912
24 Mar 1929
30 Mar 1929
30 Mar 1929
4 Feb 1930
Feb 1893
Feb 1893
19 Jan 1912
Feb 4 1902

U adult
U adult
M adult
M adult
F adult
M immature
U adult
U adult
F adult
U adult

Korea/Manchuria
Sado Island, Japan
Sado Island, Japan
Korea

Pre W.W. Ila
21 Jan 1981
13 Apr 1983
Mar 1919

U adult
F adult
F adult
F immature ?

M, Male; F, female; U, unknown sex.
aSpecimenwas collected at an unknown date before the Second World War.

feathers reduce the surface area to which the cosmetic
can adhere, thus assisting application to other regionsof the plumage. The long lateral barbs of typical downfeathers may soak up most tar secreted and become
clogged with chunks thus reducing the efficiency of
application to other areas. Uchida (1970) reports thatthe cosmetic may drop off the throat and headregion onto the ground during the period of heaviest
secretion. This is consistent with the presence ofspecialized down feathers with short barbs thatprovide much less surface area to soak up thecosmetic secretion allowing more copious applicationelsewhere. By analogy, given equal volumes of paint on
a brush, the one with short bristles drips more easily
than one with long bristles.
Museum specimens examined in post-breedingplumage have normal down feathers with long barbs inthe pigmented (but not actively secreting) slun area
(Fig. 3). The one immature bird examined also does
not have the specialized down feathers on the throat
and head regions where pigmented, secretory skinwould eventually have developed. These observations
support the possibility that specialized down feathers
occur in the pigmented skin area, but also suggest thatthey are not always present, and must develop prior tothe cosmetic application of pigment. Recently, it has
been observed that adult Tolu undergo a highly
restricted moult of feathers in the throat and headregions in November (Fig. 1d). We suggest that it is a t
this time that many normal down feathers are shed,
and the specialized ‘short-barbed’ down grows - but
only in the pigmented slun area. Thus it appears that
there is a rhorphological change associated with acquisition of nuptial plumage in the Toki, which involves
development of a ‘tool’ for cosmetic application.

Application of nuptial pigment
In addition to a morphological change related to develFigure 3. Specialized down feathers in the throat region of theToki Nipponia nippon. Right, normal down feather; top left, downfeather from the throat of an immature; bottom left,
pigmented down feather from an adult with nuptial colour.

opment of nuptial colour, and physiological changes
associated with secretion of the cosmetic, there are also
distinct behaviour patterns (‘daubing’) associated withcosmetic application. These behaviours usually occur

02000 British Ornithologists’ Union, Ibis, 142, 1-1 1

6

J. C. Wingfield et al.

in association with bathing, and the grey tint becomesdarker with each application. Five or six bathingdaubing sessions are usually needed to complete development of nuptial colour (Uchida 1970). Observationsof Toki during daubing sessions reveal two major
components to the behaviour patterns (Fig. 1). Firstthe throat and sides of head are rubbed on the upperback and wings thus depositing cosmetic on the outerfeathers (Fig. 1). Secondly, the head is turned so thatthe crown and back of the head (as well as the beak)are rubbed over the back and wings to spread th,epigment (Fig. 1). The neck also becomes coated withpigment picked up from the back and wings. The
daubing behaviour clearly serves to apply pigment tocontour feathers and then to spread it uniformly.

Why is daubing behaviour associated with bathing?
Because of its composition, the cosmetic cannot be
diluted by water. Uchida (1970) showed that the tar
becomes lodged as small fragments on the proximal
barbules of the feathers (i.e. those projecting backfrom the lateral barbs) rather than the distal barbules(i.e. those projecting forward from the lateral barbs
and interlocking with the proximal barbules). I t ispossible that bathing in water may help emulsify thecosmetic as it is daubed onto wet feathers. Very small
fragments of pigment may result, that would thenadhere to the surface of barbules to give a more
uniform finish to the colour. This hypothesis could betested by applying cosmetic to dry versus wet feathers.
Note, that the feathers are not ‘stained’ in the sensethat the keratin structures become pigmented themselves, but small fragments of pigment adhere to theirsurface.
After breeding, the T o h undergoes a complete postnuptial (prebasic) moult. At this time, the pigmented
skin area on the head and throat is not actively secreting pigment, and birds show no daubing behaviour.
Thus all contour feathers develop as pure white orsalmon-coloured structures (Uchida 1970). Bathing
continues to occur all year, but secretion of thecosmetic, and daubing behaviour following bathing,
are restricted to a discrete period in January andFebruary just prior to the first courtship behaviours
and onset of breeding.
Development of nuptial colour in the Tolu appears
to be unique among birds. Cosmetic application ofcolour may occur in a few other species (see van Tyne
& Berger 1959), but the combination of a morphological change (restricted autumn moult and developmentof a specialized down feather), physiological change(secretion of the cosmetic) and behavioural changes

02000 British Ornithologists’ Union, Ibis, 142, 1-1 1(the bathing-daubing patterns) is truly unique.
Given the unusual breeding biology of this criticallyendangered species, we consider below the possible
factors leading to the failure of the current captive
breeding programme to re-establish the species inJapan. Specifically we address how endocrine investigations of non-threatened species might be used as atool to facilitate breeding programmes of endangered
species by identifying problems, as well as by increasing the likelihood of successful reproduction.

Reproductive endocrinology of an endangered
species
Following capture in 1981 of all six remaining Toh inJapan, two females died within a year. Another female
was crippled with arthritis and eventually died in 1986
without breeding. A remaining female paired with amale displayed breeding behaviour and copulation but
suddenly died and was found to be egg-bound (i.e. an
egg had lodged in the oviduct, eventually causing
death). After 1983, only the male and an old female
remained, and although the female remained sexuallyactive (as judged by behaviour and development of
nuptial colour) no breeding occurred (Ishii et al.
1994). Even the introduction of a male from the
population in China failed to result in breeding. Now
the old female is the only Toki left in Japan andcurrently she fails to develop nuptial colour, suggestingthat she is now too old to breed.
Given the critically low numbers of Toh it is clearly
important to determine as accurately as possible thereasons for reproductive failure. Anderson (1984)
reports that the old female and the only remaining
male were not compatible and they fought. However,
it is difficult to determine exactly what such observations mean. Were these birds reproductively mature(i.e. were the testes and ovaries developed to a state
where breeding could commence) or had one individual failed to mature and thus failed to respond tocourtship from the other, resulting in conflict?
Assessment of circulating reproductive hormone levels
would be one way to gain further critical information
on reprodictive status. Individuals that apparently
have failed to mature and show deficits in endocrine
secretions or patterns might then be brought into
breeding condition by hormone treatment. To do thisrequires considerable background information onchanges in plasma levels of appropriate reproductive
hormones during gonadal development, onset of laying
and termination of breeding. Obviously, this is notpractical for critically endangered species where only

Biology of the Tokia handful of individuals remain. An alternative is tocollect samples from a closely related, but not endangered, species as a model. Then single spot samples
from the endangered species can be compared to thetemplate developed for the related species to identify
possible deficits of endocrine function that couldin turn point to revisions of the captive breeding
programme, or even suggest therapy with reproductive
hormones.

Comparisons of circulating hormone levels.
As a template for the reproductive cycle of the Toki,
Ishii (1983, 1984) and Ishii et al. (1994) collected aseries of blood samples from captive populations ofWhite Ibises Tlzreskiornis melanocephalus breeding in
the Tama Zoo, Tokyo. They then measured plasma
levels of gonadotrophic hormones as an indicator of
normal reproductive development in ibises.
Gonadotrophins are glycoprotein hormones secretedby the anterior pituitary gland and control gonadal
function. These are: (a) luteinizing hormone (LH)which acts primarily on the endocrine gonad to regulate synthesis and release of sex steroid hormones andalso induces ovulation; and (b) follicle-stimulating
hormone (FSH) which acts primarily on the gametogenic gonad but can influence sex steroid hormone
release as well. It was found that both male and female
White Ibises show a dramatic rise in plasma levelsof LH and FSH during late winter and early springcoincident with gonadal development and eventual
onset of nesting (Fig. 4a, 4b). Levels of these hormones
are minimal in late summer and early autumn when
the breeding season is over (Ishii 1983, 1984, Ishii
et al. 1994). Furthermore, changes in sex steroid(androgens) levels parallel the patterns of LH (Fig. 4c).

7

These patterns are typical of many species of birds thathave been studied both in the field and in captive
populations (see Wingfield & Farner 1993 for review).
Furthermore, comparisons of LH, FSH and sex steroid
patterns in individuals that are breeding normally,
versus those that are not, show marked differences(Wingfield & Farner, 1993), suggesting that monitoring
hormone levels can provide information on hormone
deficits in individuals that fail to breed. Adjustments
can then be made in the breeding programme tocorrect the deficit, or hormone treatment may be
applied to induce breeding.
Changes in plasma levels of LH and FSH in the Toki
(Table 3, Fig. 5) are consistent with the data obtainedfor White Ibises. The individual code-named Blue (Ao)showed higher levels of FSH and LH between
December and March - the period of presumed
gonadal development prior to nesting in April.
However, this bird failed to breed and this is reflectedin the generally low and erratic levels of the sex steroid
hormone oestradiol-17P (Table 3) compared withmany other avian species breeding naturally (Wingfield& Farner 1993). Ao was the Toki crippled witharthritis. She never developed full nuptial plumage andnever laid eggs, although some gonadal development
probably occurred. Three other birds (Table 3) had
variable levels of reproductive hormones in October.
The bird code-named Gold (Kin) is the old female that
did not breed and she had lower levels of hormones,
particularly LH (Table 3, Fig. 5). White (Shiro) had
the highest levels of LH and also elevated FSH. This
bird initiated egg-laying but died because she became
egg-bound.
These data, although sparse because of the very few
data points available, are nonetheless encouraging andTable 3. Plasma gonadotrophin and sex steroid concentrations in the plasma of Toki Nipponia nippon.

Hormones (ng/ml plasma)
Individual
identification
Blue (Ao)White (Shiro)Gold (Kin)
Green (Midori)

~

Date

FSH

LH

E2

2 Feb 1982
Oct 1982
21 Dec 1982
25 Mar 1983
19 Oct 1983
29 Feb 1984
22 May 1984
Oct 1982
Oct 1982
Oct 1982

1.99
3.86
3.34
0.39
2.90
2.31
1.51
1.22
1.94

0.67
0.61
1.89
2.34
0.99
2.02
1.31
2.52
0.06
1.45

0.23
0.15
0.1 1
0.12
0.08
0.02
0.20
0.12

T

-

-

FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, oestradiol-17p; T, testosterone. All concentrations of hormones are
expressed as ng/ml of plasma. From lshii eta/. (1994) and unpubl. data.

@ 2000 British Ornithologists' Union, Ibis, 142, 1-1 1

8

J.C. Wingfieid et at.

3.5 -

a

3 h

E

\

2.5 -

0,
L

v

a

2

E
- 1.5
c
._

3

1

0.5

0
M

J

J

A

S

O

N

D

J

F

M

M

J

J

A

S

O

N

D

J

F

M

A

A

S

O

N

D

J

F

M

A

A

M

h

.-c
LL

0.5
0

C

rl

M

J

J

M

Month

Figure 4. Hormone levels in two individual White Ibis Threskiornis melanocephalus males (0 and M). (a) Luteinizing hormone (LH); (b)follicle-stimulating hormone (FSH); (c) androgen levels. From lshii el a/. (1983, 1984).

suggest that hormone levels in single spot samples canhelp to indicate reproductive status when compared@ 2000 British Ornithologists’ Union, Ibis, 142, 1-1 1with a template derived from multiple samples ofclosely related, non-threatened species.

Biology of the Toki

Midori (male)

Shiro (female)

Ao (female)Kin (female)

Figure 5. Plasma levels of luteinizing hormone (m) and folliclestimulating hormone (0) in blood of individual captive Toki,
Nipponia nippon. From lshii eta/. (1994).

9

These data suggest that changes in faecal levels ofsteroid hormones can be used as indicators of reproductive development. These studies may then be usedas templates to compare changes in faecal levels ofsteroids in endangered species such as the Toki (Fig. 6).
The male code-named Green (Midori) had elevated
LH and FSH levels (Table 3 , Fig. 5) and showed anormal pattern of faecal androgen levels (Fig. 6)
compared with the pattern of circulating androgens inWhite Ibises (Fig. 4c). Faecal testosterone levels
increase in December and January coincident with
beginning gonadal maturation and when nuptial colour
is developed. Midori, the only male in captivity, developed full nuptial colour. We do not know whether thesecretion of the cosmetic and its application are testosterone-dependent, but in other species in which males
160

Measurements of faecal steroids as indicators of
reproductive status
Frequent blood sampling of critically endangered
species is clearly out of the question, particularly ifthese animals are in the field. Recent work has shownthat changes in faecal (i.e. excreted) steroid hormone
levels may be an accurate measure of gross changes incirculating levels. Obviously, faecal samples can becollected without handling the individual, and they
represent a non-invasive way of obtaining criticalendocrine information. It may also be possible tomeasure gonadotrophins in faecal samples, althoughmethods have yet to be validated.
Measurement of steroid levels in faeces is not a newtechnique, but particular attention has recently been
paid to validating the methods by comparing changes
in plasma levels of steroid hormones with faecal levels
collected from the same animal. This has proved
useful for determining if and when female mammals
ovulate or become pregnant (Wasser 1995). It is
important to validate the faecal steroid procedure inthis way so that precise conclusions can be drawn fromthe data. In female Japanese Quail Coturnix japonica,
faecal levels of oestradiol parallel ovarian development
and plasma oestradiol levels. Similarly in male Japanese
Quail, plasma and faecal levels of testosterone increasein parallel during reproductive development and are
extremely low in castrated individuals (Ishii 1985, Ishii
et al. 1994). Further studies show that if female
Japanese Quail are given subcutaneous implants ofoestradiol or progesterone in capsules that release thehormones at constant rates, then both plasma andfaecal levels of oestradiol and progesterone increase inparallel (Kubodera et al. 1988, Ishii et al. 1994).

140

120

h

$ 100
a,

m
m
m

c

--.
80
c
m
a,

2

U

2

60

40

20

0

Jul

Sep

Aug

Dec-JanMonthFigure 6. Faecal levels of androgen in 'Midori', a male Toki

Nipponia nippon. Each point represents the mean level fromfaeces collected on four consecutive days in each month.
Vertical bars are the standard errors. From Kikuchi and lshii(1997).

@ 2000 British Ornithologists' Union, Ibis, 142, 1-1 1

10

J. C. Wingfield et al

and females develop a nuptial plumage, testosterone
regulates the process in both sexes (e.g. many speciesof the genus Lams, Witschii 1961, Terkel et al. 1976,
Groothuis & Meeuwissen 1992). We shall not be able
to determine by experiment whether development of
nuptial plumage in the Toki is testosterone-dependent,
but deductions made from other species as well asfrom changes in faecal steroid levels allow us to makepredictions. For example, if individual Toki fail todevelop nuptial colour, or do so only partially, thentreatment with testosterone may help to complete theprocess and enhance the possibility of successful
breeding. This could easily be tested by feeding low
doses of testosterone in food (steroids can be absorbed
from the gut).
Faecal steroid analysis may have further uses.
Yamaguchi and Ishii (1991) have shown that faecal
levels of progesterone can be used to determine
whether female Japanese Quail have ovulated. Females
that laid eggs showed a large surge of progesterone infaeces during the late evening. In non-laying females,
no such surge in faecal progesterone levels is observed.
Further analyses of this type will undoubtedly shedlight on reproductive function that can be used tomonitor breeding status in endangered species.

Using reproductive hormones to enhance
reproductive output
Plasma and faecal levels of steroids coupled withbehaviour and morphological observations can be used
to pin-point deficits in reproductive function. The firststep is to adjust the captive breeding programme tocorrect those deficits without invasive treatment, sothat the birds breed normally. However, if this fails
then the next step is to provide hormone therapy
as a means of enhancing reproductive output. As
mentioned above, brief hormone treatment may besuccessful in completing development of nuptial
colour or triggering courtship behaviour. More extensive treatment may be necessary if individuals still do
not breed.
Hormone therapy of this type is fraught with difficulties because endocrine manipulations may affectother processes as well. For example, hormone treatment may disrupt parental behaviour so that youngdie, or may influence the phenotype, even sex, thatdevelops. It is possible that many offspring generated
following hormone therapy of adults may not be viable
for reintroduction into the wild but they may,
nonetheless, be useful as captive breeding stock to
induce natural breeding without further hormone
treatment. In cases where populations are reduced to02000 British Ornithologists' Union, Ibis, 142, 1-1 1

critical levels there may be few options other than totry hormone therapy.
It has been more than 20 years since avian
gonadotrophins (from chickens) were first isolated
and purified but, even now, preparations of these
hormones are not generally available and certainly notin sufficient amounts to support a major hormone
therapy programme. Molecular techniques will
provide the answer as genes for these hormones arecloned, sequenced and recombinant techniques used
to generate large quantities of gonadotrophins fortherapeutic purposes. Recent research suggests thattreatment with avian gonadotrophins can indeed
induce reproductive maturation and even ovulation.
Much work remains to be done, but results areencouraging.
Multiple injections of hormones over a period ofmany days or even weeks are likely to be counterproductive because of the stress of handling. However,Wakabayashi et al. (1987, 1989, 1992) used ALZET
osmotic pumps filled with a mixture of avian LH andFSH to treat female quail kept on short, winter-like,days that normally do not support reproductive
development. These pumps can be implanted
intraperitoneally through minor surgery and deliver
hormone preparations at a constant rate for up to two
weeks. Measurement of plasma levels of LH and FSH
in these females revealed that the pumps successfully
elevated circulating gonadotrophin levels overcontrol females that received pumps filled with saline
only. Then some of the treated females were given
additional injections of avian gonadotrophins and threeout of seven laid a total of eight eggs. These females
were housed with males and, when the eggs wereincubated, two out of eight hatched. Both chicks weremales, grew normally and at least one proved to be
fertile (Wakabayashi et al. 1987). The yield may not be
impressive, but for an initial experiment the results
are encouraging. Since some of the female Toki incaptivity failed to lay at all, the possibility certainly
exists that in the future it may be possible to enhance
reproductive output with gonadotrophin treatment
and produce viable offspring.
Wakabayashi et al. (1987) also applied the osmotic
pump techniques to administer chicken gonadotrophins to a passeriform, the Chinese Grosbeak
Eophona migmtoria. Treatment resulted in marked
development of the ovary and oviduct suggestingthat chicken gonadotrophins may be used even in
unrelated avian species. Ishii et al. (1994) obtained
preliminary evidence that chicken gonadotrophin
preparations may also stimulate ovarian development

Biology of the Tokiin the Sacred Ibis and Scarlet Ibis Eudocimus ruber.
However, we should be cautious because over longer
periods (weeks to months) it is likely that the recipientof chicken gonadotrophins may eventually form
antibodies (as a response to alien chicken protein) that
could neutralize biological effects.
The Tolu is a critically endangered species with afascinating and novel breeding biology that enriches
our knowledge of reproductive biology in general. I t isa typical example of how biodiversity includes not only
a web of many species intertwined in a habitat, butalso diversity of morphology, physiology and behaviour. All of these aspects are important to consider
when attempting to bring back such criticallyendangered species from the brink of extinction. It is
possible that, by incorporating endocrine techniques,
we may be able to identify reproductive malfunction
and then use hormone therapy in a limited way toenhance reproductive output. Studies of closely
related species to provide a template and investigationsof hormone therapy procedures in other species may
be new ways in which to pursue conservationendocrine techniques.
Preparation of this manuscript was aided by a US/Japan
Cooperative Research grant (INT-9 1 19072), and the Russell
F. Stark University Professorship to J.C.W.

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Received 24 February 1998; revision accrpted 8 March 1999

@ 2000 British Ornithologists’ Union, Ibis, 142, 1-1 1