Research Article SummaryArticle’s Title Higher TSH Levels W.docx

Research Article Summary
Article’s Title: Higher TSH Levels Within the Normal Range
Are Associated With Unexplained Infertility
Studies showed that ~10%-30% of couples that have
unprotected intercourse over one year and do not succeed to
conceive have unexplained or idiopathic interfertility.
Hyperprolactinemia and thyroid dysfunction or thyrotropin are
the known causes of infertility. Both of them are associated
with irregular menstrual in women, but sometimes the normal
levels of them associated with unexplained infertility, which the
reasons are unknown. The main aim of this study is to compare
the level of prolactin and TSH in women with normal fertility
and women with unexplained infertility and with the exception
of those women who have an oligospermic male partner. The
treatment infertility is so expensive for couples.
The researchers hypothesized that unexplained infertility in
women caused by a higher level of prolactin and TSH compare
to a controlled group of women who have normal fertility, but
their partners are severely oligospermic. Understanding the
mechanisms that underlie unexplained infertility will help
couples to have less costly treatment.
The cross-sectional studies were used to obtain the data.
Researchers studied a total of 239 female patients. The female
patients were between the age of eighteen and thirty-nine that
were diagnosed with infertility and without any irregular
menstruation. They included 187 women in this study who did
not conceive over one year of unprotected intercourse
(unexplained infertility group), and 52 women that their
husband had oligospermia. They exclude women who had

hypothyroidism and hyperthyroidism.
Even though the researchers supported their findings from
previous studies, but their research was different from those
earlier studies. The other studies included various factors that
cause the level of TSH to be high or elevate and leads to
infertility. These researchers used a strict method to ensure
their findings, and the purpose of their method was to show that
even a mild difference in thyroid function can cause infertility
or unexplained infertility.
Thus, about 75% of the patients’ prolactin and TSH levels were
measured in the laboratory of Partners HealthCare, and the rest
were measured in an outside laboratory. They included only the
patients that had TSH ≤5 mIU/L. These two groups of women
were studied within the 13-year study period, and their
characteristics were compared. The unexplained infertility
group was older than the other group that their husbands had
male factor problems.
The results show that the unexplained infertility group had a
higher TSH level than the severe male factor even the
researchers excluded the UI (unexplained infertility) group that
their partners had low morphology still the results were the
same. About 27% of UI group women had ≥2.5 mIU/L TSH,
which twice the percentage of the severe male factor group
(13% mIU/L). The data showed that the prolactin level was
similar in both groups. Since the prolactin level is different
during the menstrual, the researchers performed another
analysis on only women that their prolactin level was measured
during day 3 of their menstrual, but the results were the same.
There were no significant differences between the groups.
The limitations of this study were that the researchers only
relied on the health records in a span of 13 years with the
exclusion of less severe male factors infertility. These
limitations caused to get different results; not what expected.
Thus, they were not able to measure the thyroid antibody levels
or the thyroid hormones in those two groups of women. But 19
of the 239 women were checked with the thyroid peroxidase

antibody (TPO). There were 3 elevated thyroid peroxidase
antibodies in the unexplained group and 3 in severe male factors
of those 19. When compared these two groups, the TPO median
was higher in severe male factors than the UI group, but when
the researchers removed these 6 individuals that had positive
TPO, the level of TSH got more elevated in the UI group
compare to severe male factors group. Therefore, the
researchers were not able to measure the level of thyroid
hormone on their subjects, which causes infertility in women.
The researchers were limited to the laboratory tests and health
records that were previously taken.
The interesting part of the discussion was that the previous
studies had been indicated that women who had a higher level
of prolactin and also had unknown infertility were treated with
a dopamine agonist. This treatment resulted in the conception of
16 women out of 40 with the 10 months follow-up. But it raised
the question about the treatment of thyroid hormone
replacement whether women should be treated or not, and it is
maybe a good step to treat a UI woman. Therefore, the
researchers of this article believed that further research is
needed to figure out that the treatment of a high TSH level will
decrease the time of conception in UI couples.
The researchers’ hypothesis was halfway supported by this
article and half not because the findings did not show that a
high level of prolactin can cause UI, but the article supported
that a higher level of TSH could lead to UI. The article suggests
that mild variation in thyroid function is problematic in getting
pregnant or conceiving naturally, but there are other factors too
that are associated with unexplained infertility.
Note:
UI = Unexplained Infertility
Work Cited
Tahereh Orouji Jokar, Lindsay T. Fourman, Hang Lee,

Katherine Mentzinger, Pouneh K. Fazeli
J Clin Endocrinol Metab. 2018 Feb; 103(2): 632–639. Published
online 2017 Dec 19.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800836/#s17tit
le
1. Summary
Article summary no longer than 2 1/2 pages, single-spaced.
2. Background
Sufficient background (usually 1 paragraph) to explain the
reasoning behind the research
3. Format
Summary is written in a clear and concise manner. Research
question(s) and hypotheses are stated. The methods are briefly
described including dependent variables measured and data
analysis used. Results, and their importance, were described.
Key implications of the results were explained and interpreted.
4. Competency
Summary written using complete sentences and paragraphs that
are grammatically correct. Direct quotes were avoided. No
spelling mistakes were present. All work was written in the
student’s own words.
*NOOOOOOOOOOOOOOOOO plagiarism!!!!!!!!!
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Animal Reproduction Science 124 (2011) 237–243
Contents lists available at ScienceDirect
Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
varian function in South American camelids (alpacas, llamas,
vicunas,
uanacos)�
ane Vaughan ∗
ria Genesis, PO Box 406, Ocean Grove, Victoria 3226, Australia
r t i c l e i n f o
rticle history:
vailable online 28 September 2010
a b s t r a c t
Ultrasound technology and hormone assays have provided a
better understanding of fol-
liculogenesis and ovulation in South American camelids in the
last two decades. Females
exhibit waves of ovarian follicular growth and are induced
ovulators and therefore do not
eywords:
amelid
lpaca
lama
icuna
vary

exhibit oestrous cycles in the manner of spontaneously
ovulating species such as sheep and
cattle. There is much variation in inter-wave interval among
camelid species (alpaca/llama
10–22 days, vicuna 4–11 days), within species and within
individual animals as the range of
each phase of follicular growth is wide. Ovulation occurs 24–30
h after mating and luteolysis
occurs approximately 10 days later if conception fails to occur.
© 2010 Elsevier B.V. All rights reserved.
ollicle
. Introduction
There are four South American camelids. The alpaca
Vicugna pacos) has been domesticated from the vicuna
Vicugna vicugna) and the llama (Lama glama) domesti-
ated from the guanaco (Lama guanicoe) (Kadwell et al.,
001). South American camelids resemble each other in
hape, but vary in size, fleece characteristics and geograph-
cal distribution (Novoa, 1970). Vicunas are the smallest of
he South American camelids, weighing 45–55 kg, and liv-
ng at high altitude (above 4000 m) in the central Andes.
uanacos (100–120 kg) are mainly found in the southern
ndes. Historically, alpacas (55–85 kg) were grown in Peru,
olivia, Chile and Argentina for their fleece, and llamas
130–180 kg) were used as beasts of burden and both used
or meat, hides and fuel (faeces). Alpacas and llamas are
ow found in South and North America, Europe and Aus-
ralasia and alpacas in particular, are being bred for their
oft, fine, light-weight wool that comes in a range of colours

� This paper is part of the special issue entitled: Reproductive
Cycles of
nimals, Guest Edited by Michael G. Diskin and Alexander
Evans.
∗ Tel.: +61 3 5254 3365; fax: +61 3 5254 3365.
E-mail address: [email protected]
378-4320/$ – see front matter © 2010 Elsevier B.V. All rights
reserved.
oi:10.1016/j.anireprosci.2010.08.031
from white, through fawn, to brown, grey and black. Phy-
logenetically, South American camelids are closely related
to dromedary (one-humped) and Bactrian (two-humped)
camels.
The reproductive physiology of camelids differs to that
of other domestic livestock. Female camelids exhibit waves
of ovarian follicular growth (Adams et al., 1990; Vaughan
et al., 2004) and are induced ovulators (San Martin et al.,
1968) and therefore do not exhibit oestrous cycles in the
manner of spontaneously ovulating species of domestic
livestock. Unmated, non-ovulatory females are sexually
receptive most of the time, regardless of the stage of ovar-
ian follicular development (Sumar, 1983). Males mate in
sternal recumbency for approximately 20 min and ejacu-
late small volumes of semen many times during this period
(Lichtenwalner et al., 1996a,b). Gestation length is approx-
imately 11.5 months and twins are rare (San Martin et al.,
1968). Generation intervals are relatively long in camelids
because males take 1–3 years to reach puberty and females
exhibit an extended gestation.
Ovarian function remains relatively poorly understood
in camelids because of lack of research funding inter-
nationally. Advances in technology have improved over

the past few decades from slaughter-house studies (San
Martin et al., 1968), laparotomy (England et al., 1971) and
dx.doi.org/10.1016/j.anireprosci.2010.08.031
http://www.sciencedirect.com/science/journal/03784320
http://www.elsevier.com/locate/anireprosci
mailto:[email protected]
dx.doi.org/10.1016/j.anireprosci.2010.08.031
uction Science 124 (2011) 237–243
Table 1
Ovarian dimensions in camelids.
Alpaca Llama Vicuna Guanaco
Right ovary
Length cm 1.6 ± 0.3 1.3–2.5 1.3 1.5
Depth cm 1.1 ± 0.2 1.4–2 0.7 n/aa
Width cm 1.1 ± 0.2 0.6–1 1.0 n/a
Left ovary
Length cm 1.6 ± 0.3 1.5–2.5 1.2 1.5
Depth cm 1.1 ± 0.2 1.5–2.5 0.7 n/a
Width cm 1.1 ± 0.2 0.5–1 1.0 n/a
238 J. Vaughan / Animal Reprod
laparoscopy (Bravo and Sumar, 1989) to the use of transrec-
tal ultrasound (Adams et al., 1989; Vaughan et al., 2004) and
the availability of hormone assays (Bravo et al., 1990a,b;
Aba and Forsberg, 1995); the latter two techniques provid-
ing a relatively non-invasive and better understanding of
folliculogenesis and ovulation.

2. Ovarian development
During embryological development, the gonads arise
from the urogenital ridges in close proximity to paired
paramesonephric (Mullerian) ducts that give rise to the
internal genitalia. The ovaries do not exhibit structural dif-
ferentiation until well after sex determination. Primordial
follicles develop some months into gestation and are seen
as oocytes surrounded by a single layer of flattened gran-
ulosa cells within a basal lamina (Parker and Schimmer,
2006).
The timing of primordial follicle development is
unknown in South American camelids but occurs at 8–12
weeks in camels (Marai et al., 1990). Oocytes are arrested
in prophase of the first meiosis and do not progress further
until shortly before ovulation in the post-pubertal camelid.
Primary follicles, characterised by an oocyte surrounded
by cuboidal granulosa cells, develop in camels from 20 to
24 weeks of gestation (Marai et al., 1990). Timing of first
appearance of primary follicles and number of primary fol-
licles present at birth is unknown in camelids. After birth,
secondary follicles develop with more than one layer of
granulosa cells and a thecal cell layer around the basement
membrane with numerous small blood vessels (Rajkovic et
al., 2006). Early folliculogenesis to the stage of pre-antral
follicles appears to be directed by signals within the ovary
and is independent of gonadotrophin stimulation. There
is also communication within the ovary amongst oocytes,
granulosa and theca cells (Parker and Schimmer, 2006).
The onset of folliculogenesis occurs immediately after the
first follicles are formed, and continues until the end of the
reproductive period (12–18 years in alpacas), even through
pregnancy and lactation (Adams et al., 1990). Follicles are
also degenerating during foetal development because a
lack of follicle stimulating hormone (FSH) does not support

further follicular development (Rajkovic et al., 2006).
Regulation of terminal follicular growth beyond the
small antral stage is a gonadotrophin-dependent pro-
cess occurring after puberty and corresponding to
initiation of follicular waves, selection of dominant
follicles and terminal maturation of pre-ovulatory folli-
cles (Monniaux et al., 1997). Gonadotrophic hormones
secreted from the pituitary gland develop a complex
feedback/feed-forward system with the ovaries, known
as the hypothalamic–pituitary–gonadal axis, allowing fol-
licles to proceed beyond the early, pre-antral stages
(Rajkovic et al., 2006). Formation of the antrum signals
transition from intra-ovarian to extra-ovarian control and
once a follicle has entered the growing pool, it is irreversibly
committed and cannot return to a quiescent state. Antral
follicles are apparent in camels at 32–36 weeks gestation
(Marai et al., 1990).
In mammalian dominant follicles, FSH stimulates gran-
ulosa cell proliferation, aromatisation of androgens to
Weight g 1–4 2.4 1.2 n/a
Adapted from Bravo (2002).
a Not available.
oestrogens, and luteinising hormone (LH) receptor expres-
sion, while LH stimulates androgen production from thecal
cells (Rajkovic et al., 2006). Inhibin, secreted by the granu-
losa cells of the dominant follicle, feeds back to the pituitary
to inhibit FSH secretion. These findings have yet to be
clearly elucidated in camelids. Granulosa cells of most
early-antral follicles undergo apoptosis and death as they
are not rescued by FSH.

3. Adult reproductive anatomy
Camelids have a bicornuate uterus with the tips of the
horns blunt and rounded, and a single cervix, whose lumen
contains 2–3 rings/spiral folds of mucosa. The uterus is
located within the pelvic canal or at the pelvic brim in the
non-gravid state (Vaughan and Tibary, 2006). Each uterine
horn ends in a long and tortuous oviduct which joins the
uterine horn to the ovarian bursa (Sumar, 1983). There is
a prominent papilla at the uterotubal junction (Vaughan
and Tibary, 2006). The ampulla and ovarian section of the
oviduct are the most coiled parts, the isthmus less so. The
fimbria are contained within the bursa, near the ovary and
the ovarian bursa, is formed by a fold of the mesosalpinx
and completely envelops the ovary (Bravo et al., 2000).
Ovaries are round to oval and globular in shape in lla-
mas and alpacas (Sumar, 1983) and antral follicles lie over
the entire periphery of each ovary (Vaughan and Tibary,
2006). Ovarian size varies amongst the four camelid species
(Table 1) and varies within species depending on the struc-
tures present on each ovary as follicles >4 mm diameter and
corpora lutea project prominently from the surface of the
ovary (Adams et al., 1989). All growing follicles in camelids
are spherical, probably related to the prominent protru-
sion of 85% of the follicle from the surface the ovary (Del
Campo and Del Campo, 1995). Oocytes range from 172 to
200 �m in size. Immature oocytes in llamas have a distinct
and large germinal vesicle with a dark nucleolus. Mature
oocytes display a metaphase plate surrounded by a dark
area easily found at 20–40× magnification (Del Campo and
Del Campo, 1995).
4. Puberty
Time of first ovulation depends on age at first mating

as camelids are induced ovulators. Information on ovarian
follicular activity has been attained by measuring uri-
nary oestrone sulphate and indicates that follicular growth
uction Science 124 (2011) 237–243 239
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Fig. 1. Schematic representation of follicular waves in alpacas
and lla-
mas. Mating induces ovulation of the dominant follicle and
formation
of a corpus luteum. Failure to conceive leads to luteolysis of the
corpus

luteum. RF = recruited follicle, SF = selected follicle, DF =
dominant follicle,
AF = atretic follicle, O = ovulation, CL = corpus luteum.
J. Vaughan / Animal Reprod
tarts from approximately 5–6 months of age (Bravo, 1997),
ut pregnancies from 3 months of age have been recorded
Vaughan and Tibary, 2006). The age at which ovarian activ-
ty begins and conception occurs is dependent on nutrition
nd live weight. Domestic camelids are generally mated
hen they have attained two-thirds of their adult weight
Smith et al., 1994), from 12 months of age in alpacas and
8 months of age in llamas.
. Sexual behaviour
Camelids do not have regular oestrus cycles that are typ-
cal of spontaneous ovulators and therefore do not display
istinct periods of overt oestrus. Non-pregnant females
ppear receptive to males on most occasions regardless
f stage of follicular development (England et al., 1971) as
lasma progesterone levels remain low (Fernandez-Baca,
993). Time taken to adopt sternal recumbency (demon-
tration of sexual receptivity) is not a reliable indicator
f either plasma oestradiol concentration or ovarian fol-
icular diameter (Bravo et al., 1991; Vaughan et al., 2003).
either changes in the external genitalia nor vaginal cytol-
gy may not be used as an indicator of follicle size (Ferrer
t al., 1999). The sexual behaviour patterns of camelids
ay also be related to their geographic location, degree
f domestication and social structure of the herd (Novoa,
970).
In an attempt to explain continual receptivity in female

amelids, it has been proposed that the overlapping of fol-
icular waves maintains blood oestradiol concentrations at
level sufficient to maintain sexual receptivity. If asyn-
hrony occurs between successive follicle waves, oestradiol
oncentration may drop long enough for sexual receptivity
o decline (Brown, 2000) and these females appear indif-
erent to the male rather than non-receptive.
Female camelids become non-receptive in the presence
f a corpus luteum and elevated plasma progesterone. Non-
eceptive female camelids strongly reject the male when
laced in a yard together and may run away from the
ale or spit, kick and/or scream. Spitting and attempting to
scape are most indicative of reproductive status (Pollard et
l., 1994). Sexually inexperienced alpaca females are more
ikely to kick and attempt escape but less likely to spit
r threaten the male compared with experienced females
Pollard et al., 1993).
. Seasonality
Alpacas and llamas are considered non-seasonal breed-
rs as ovarian follicular activity occurs throughout the
ear and season (photoperiod, rainfall or temperature)
oes not affect the number of follicles >6 mm observed on
he ovaries (Bravo and Sumar, 1989). However, breeding
nd parturition are usually restricted by South Ameri-
an farmers to the rainy, warmer months of summer
December–April) when feed is likely to be more abundant
nd better quality (Fernandez-Baca, 1993). Vicunas breed
n the high altitude rangelands of South America in autumn
Aguero et al., 2001).

Modified from Senger (2003).
7. Folliculogenesis
Folliculogenesis, or growth and differentiation of the
oocyte and associated cells, is a highly regulated process
relying on the integration of signals from multiple organs.
Folliculogenesis is yet to be described in guanacos, how-
ever, sexually mature alpacas (Vaughan et al., 2004), llamas
(Adams et al., 1990) and vicunas (Aguero et al., 2001)
which have not been mated to or placed nearby a male
exhibit continuous renewing of terminally growing fol-
licles defined as follicular waves. The number of antral
follicles detected by ultrasonography is inversely propor-
tional to the diameter of the largest follicle (Adams et al.,
1990; Aguero et al., 2001; Vaughan et al., 2004). Other
studies have described growth of successive large anovu-
latory follicles in unmated females but did not describe a
periodic fluctuation in follicle numbers consistent with the
existence of a wave-like pattern of growth (Bravo et al.,
1990a,b; Bourke et al., 1992).
A follicle wave involves recruitment and synchronous
emergence of a cohort (8–10) of antral follicles approxi-
mately 2–3 mm diameter, followed by continued growth of
usually one (selected follicle), but sometimes two or three
follicles up to 3–5 mm diameter. The follicle destined to
become dominant continues growth, while the others in
the cohort (subordinate follicles) regress by atresia (Adams
et al., 1990; Vaughan et al., 2004) (Fig. 1).
The duration of follicular growth is unknown in
camelids but greater than that of a follicle wave observed
using ultrasonography. The first stages of follicular growth
are difficult to estimate accurately and are not consid-
ered in the estimation of the total duration of a follicular

wave. After new follicle emergence at the beginning of a
follicular wave, follicle growth may be divided into three
phases. The growth phase of the follicle in alpacas and lla-
mas takes about 5–9 days. The mature phase, when the
follicle reaches a pre-ovulatory size of 6–12 mm, is main-
tained for 2–8 days. The regression phase takes 3–8 days
(Bravo and Sumar, 1989; Adams et al., 1990; Chaves et al.,
2002; Vaughan et al., 2004). These phases are shorter in
vicunas (Aguero et al., 2001; Miragaya et al., 2004).
uction S
The interval, in days, between emergence of succes-
sive dominant follicles is known as the inter-wave interval.
There is much variation in inter-wave interval among
camelid species (alpaca/llama 10–22 days, vicuna 4–11
days), within species and within individual animals as the
range of each phase of follicular growth is wide (Adams et
al., 1990; Bravo et al., 1990a,b; Aguero et al., 2001; Vaughan
et al., 2004). Using a ‘mean inter-wave interval’ within
a particular camelid species should therefore be avoided
as it does not accurately describe what is occurring in an
individual animal nor allow prediction of the optimum
time of breeding (Vaughan et al., 2004). A longer inter-
wave interval has been associated with a larger maximum
follicle diameter in alpacas and llamas, suggesting that fol-
licles with a longer inter-wave interval remain functional
(Adams et al., 1990; Vaughan et al., 2004). There is appar-
ently no relationship between inter-wave interval and live
weight amongst alpacas (Vaughan et al., 2004).

Follicular growth rates of 0.5–0.8 mm/day (Adams et
al., 1989, 1990) and 0.9 mm/day (Chaves et al., 2002) in
llamas, 0.4 mm/day in alpacas (Vaughan et al., 2004) and
1.8 mm/day in vicunas (Aguero et al., 2001; Miragaya et
al., 2004) have been measured using ovarian ultrasonogra-
phy. In unmated alpacas, there is similar follicular growth
of the dominant follicle from Days 0 to 10 after new wave
emergence regardless of subsequent inter-wave interval
(Vaughan et al., 2004).
There is no regularly alternating pattern of dominant
follicle emergence between the left and right ovaries in lla-
mas and alpacas (San Martin et al., 1968; Fernandez-Baca et
al., 1970; Adams et al., 1990; Bourke et al., 1992; Vaughan et
al., 2004). Dominant follicles are found equally distributed
between the left and right ovary, despite the fact that 98%
of all pregnancies are located in the left uterine horn of
camelids.
7.1. Hormonal control of folliculogenesis
In mammals, gonadotrophin-releasing hormone
(GnRH) is secreted into the hypothalamo-hypophyseal
portal system in a pulsatile manner to stimulate the
episodic release of gonadotrophins into the systemic
circulation. GnRH has yet to be measured in camelids due
to the intricacies of sampling the hormone.
Further studies are required for a better understand-
ing of follicle recruitment and growth in camelids (Aba,
1995). Periodic surges in FSH and pulsatile release of LH
responsible for follicle wave emergence, follicle growth and
dominant follicle selection observed in some domestic live-
stock have yet to be identified in camelids due to poor
sensitivity of hormone assays (Aba et al., 1999). Successful

use of porcine and ovine FSH to induce follicular growth
in multiple ovulation and embryo transfer programs in
alpacas and llamas supports the hypothesis of FSH inducing
emergence of follicular waves in camelids.
Fluctuation in plasma oestradiol concentration gener-
ally reflects the follicular growth pattern in camelids, but
as mentioned earlier, has little effect on sexual behaviour.
There is a significant positive correlation between follicle
size and oestrogen concentrations in alpacas and llamas.
The emerging follicle synthesises and secretes increasing
cience 124 (2011) 237–243
levels of oestradiol during the growing phase, is maximal
just before the plateau of follicle growth is reached and
then decreases during atresia if ovulation is not induced
(Bravo et al., 1990a,b; Aba et al., 1995; Vaughan, 2001;
Chaves et al., 2002). These findings support the two-cell,
two-gonadotrophin mechanism for oestradiol biosynthe-
sis, which is based on findings from spontaneous ovulators.
The mechanism of dominant follicle selection from
among a cohort of follicles in a wave is unknown but
appears to operate systemically and is based on differen-
tial responsiveness of follicles within a wave to FSH and LH
(Adams, 1999). The ability of a developing follicle to release
high concentrations of oestrogen and inhibin, which act
locally by stimulating growth and cell differentiation of
the granulosa and by the indirect effect of feedback inhi-
bition of FSH secretion, is central to selection of a given
follicle for maturation and ovulation (Ginther, 2000). The
concentration of FSH during follicle growth decreases so
that it is inadequate for subordinate follicular growth and
delays onset of the next follicular wave, but the domi-

nant follicle still requires low concentrations of FSH for
continued growth. At a later, unknown time, the domi-
nant follicle transfers primary gonadotrophic dependence
from FSH to LH and has the ability to survive without
FSH (Ginther, 2000). Additional follicular development in
llamas is suppressed as long as the dominant follicle main-
tains its mature size (Bravo et al., 1990a,b) and presumably
its functionality as a dominant follicle. As the inhibitory
substances, such as inhibin, produced by the mature dom-
inant follicle decline prior to atresia, a new surge of FSH
occurs but the subordinate follicles from the previous wave
are unable to respond to the new stimulus.
The follicular diameter at which dominance and LH-
dependence occur in alpacas has not been reported. Bravo
et al. (1990a,b) used ultrasonography to conclude that there
was only ever one follicle with a diameter greater than
6 mm in llamas. Adams et al. (1990) found the mean max-
imum diameter of the largest subordinate follicle to be
5.3 ± 0.3 mm and observed no subordinate follicles greater
than 7 mm diameter in llamas.
8. Ovarian activity in unmated females
It is not known how long the development of primor-
dial follicle to mature oocyte takes in camelids, but could be
several months, as seen in other domestic livestock. Follic-
ular waves proceed in the absence of progesterone when
females remain unmated as camelids are induced ovula-
tors (Bravo et al., 1990a,b). Increasing plasma oestradiol
concentration during follicular growth in unmated females
does not elicit a pre-ovulatory surge of LH in camelids
(Bravo et al., 1990a,b; Vaughan, 2001).
The existing dominant follicle regresses by atresia over
a period of 3–8 days, allowing emergence of a new cohort of

follicles within 2–3 days following the first decrease in size
of the dominant follicle (Bravo et al., 1990a,b). Therefore, as
the existing dominant follicle is regressing, another follicle
destined to be the next dominant follicle has begun growth,
in such a way that the growth patterns of successive large
follicles appear to overlap when represented in pictorial
profiles. Growth and regression of successive large follicles
uction Sc
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ay overlap in camelids by 1–4 days so that as one follicle
s regressing, another is about to become dominant (Bravo
t al., 1990a,b). At any given time during non-ovulatory
ollicular waves, one would expect to find a follicle of at
east 6 or 7 mm diameter (Adams et al., 1990; Vaughan et
l., 2004).
Follicle waves continue during lactation in non-
regnant females. Lactation is associated with a smaller
aximum diameter of the dominant follicle and a shorter
nter-wave interval (Adams et al., 1990; Ratto et al., 2003).
. Ovarian activity in mated females: ovulation
The LH surge required for ovulation in camelids is stim-
lated by mating rather than by feedback of follicular
estrogen, hence the term ‘induced’ or ‘reflex’ ovulation
Fernandez-Baca et al., 1970). Males copulate in sternal
ecumbency for an average of 15–20 min (range 3–65 min)
ut there is no relationship between copulation time and
nduction of ovulation (Fernandez-Baca et al., 1970) nor
s there any difference in duration of copulation between
lpacas conceiving and those failing to conceive (Knight
t al., 1992; Vaughan et al., 2003). Males penetrate the
ervix with their penis during copulation and deposit
emen into both uterine horns during multiple ejaculations
Lichtenwalner et al., 1996a,b; Bravo, 2002). An ovulation-
nducing factor in the semen (Adams and Ratto, 2001;
anco et al., 2007) and mechanical stimulation of the cervix
y the penis during coitus are primarily responsible for

he neuro-endocrine reflex of ovulation, presumably begin-
ing with a sudden and large release of GnRH (Kauffman
nd Rissman, 2006). Visual, auditory, olfactory, physical
nd pheromonal cues, including vocalisation by the male
known as ‘orgling’) also contribute to transmission of neu-
al signals to the brain of the female, as some unmated
emales in the presence of a mating pair can ovulate with-
ut coitus (Fernandez-Baca et al., 1970).
The first significant rise in plasma LH in alpacas and lla-
as occurs 15–40 min after the initiation of mating (Bravo
t al., 1991). Peak LH occurs 2–3 h after mating, and is basal
y 4–7 h up to 12 h after joining (Bravo, 1990; Bravo et
l., 1991; Aba and Forsberg, 1995; Aba, 1998). LH concen-
rations do not differ in amplitude or duration between
emales that conceive and those that fail to conceive (Aba,
998).
The LH surge triggers resumption of meiosis in the
ocyte, disruption of cumulus cell cohesiveness, rupture
f the follicular wall to release the cumulus-oocyte com-
lex and a decline in plasma oestradiol levels over a period
f approximately 24 h (Bravo et al., 1990a,b; Vaughan,
001). Granulosa cells remaining in the post-ovulatory fol-
icle luteinise and form a corpus luteum, which produces
rogesterone necessary for uterine preparation and main-
enance of pregnancy (Rajkovic et al., 2006).
The ability to ovulate in response to mating depends
artly on the diameter and developmental status of the
ominant follicle at the time of mating: follicles <6 mm
iameter follicles in alpacas, llamas and vicunas fail to ovu-

ate; dominant follicles 6–15 mm diameter are capable of
vulation (Adams et al., 1989, 1990; Bravo et al., 1991;
guero et al., 2001; Chaves et al., 2002; Ratto et al., 2003;
ience 124 (2011) 237–243 241
Vaughan et al., 2003). Ovulatory capability is not necessar-
ily related to the fertility of the oocyte contained within the
ovulating follicle. It is likely that growing and early static-
phase follicles contain oocytes more likely to be fertilised
successfully (Ratto et al., 2003; Vaughan et al., 2003).
The LH surge in females in response to copulation may
be dependent on follicle size in alpacas and llamas. Females
with follicles 4–5 mm diameter released less LH over a 6-h
post-mating period and ovulation failed to occur compared
with females with follicles >5 mm diameter in one study
(Bravo et al., 1991). However, another study did not show
any correlation between plasma oestradiol and the amount
of LH released after GnRH stimulation in alpacas and llamas
(Aba and Forsberg, 1995). Repeated copulatory periods at 6
or 24 h after the initial event do not apparently increase LH
significantly, suggesting that the hypothalamus or pituitary
gland may undergo a period of refractoriness, possibly due
to depletion of pituitary LH or down-regulation of GnRH
receptors in the pituitary gland (Bravo et al., 1992).
The ovulation-inducing factor found in the seminal
plasma of male alpacas and llamas also plays a role in induc-
ing ovulation but effects on post-coital LH secretion in the
female are yet to be studied. The ovulation-inducing factor
has a dose-dependent effect on ovulation rate and corpus
luteum form and function in llamas (Tanco et al., 2007).
The interval between mating and ovulation is approx-
imately 30 h (range 24–36 h) in the alpaca and llama (San

Martin et al., 1968; Bourke et al., 1995; Adams and Ratto,
2001; Ratto et al., 2006) and is not affected by follicle diam-
eter at the time of mating (Adams et al., 1990). There is
no effect of lactational status or ability to conceive on the
interval from mating to ovulation (Adams et al., 1990).
Ovulation occurs from the surface of the ovary at any
point apart from the hilus, with equal frequency from the
left and right ovaries even though most pregnancies are
located in the left uterine horn (Fernandez-Baca et al., 1970;
Adams et al., 1989; Vaughan and Tibary, 2006). The origin
of the oocyte from the left or right ovary has no effect on the
likelihood of pregnancy (Vaughan et al., 2003). Generally,
there is only one dominant follicle but occasionally there
are two (5–15%), or very rarely three, dominant follicles
(Fernandez-Baca et al., 1970; Bravo et al., 1993).
Two to 5 days post-coitus, a corpus luteum develops at
the site of ovulation on the ovary and is associated with ris-
ing plasma progesterone concentrations from 4 to 6 days
after mating (Aba et al., 1995; Ratto et al., 2006). There
is a close temporal relationship between corpus luteum
diameter, measured by ultrasonography or rectal palpa-
tion, and plasma progesterone while the corpus luteum is
growing. The corpus luteum reaches a maximum diame-
ter of 8–15 mm with maximum progesterone output 7–9
days after mating in alpacas and llamas (Aba et al., 2000).
There is a decrease in plasma progesterone 1–3 days before
the morphological decrease in corpus luteum diameter
(Adams et al., 1991). The progesterone output of the cor-
pus luteum decreases from 9 to 11 days after mating and
corpus luteum diameter is halved by 12 days after mat-
ing (Adams et al., 1990; Ratto et al., 2006). The presence
of a corpus luteum in llamas and alpacas is usually asso-
ciated with a circulating progesterone level greater than

1–2 ng/mL (3.2–6.4 nmol/L) (Sumar et al., 1988; Aba et al.,
uction S
1995). Females that fail to conceive become sexually recep-
tive approximately 12–14 days after mating as plasma
progesterone levels decline below 6 nmol/L (2 ng/mL).
Recruitment of follicles and a new follicular wave starts
soon after ovulation (Adams et al., 1990). The dominant fol-
licle in camelids in the first wave after mating is detected
via ultrasound approximately 2 days after ovulation (Ratto
et al., 2003). The presence of a corpus luteum, and there-
fore elevated plasma progesterone, alters follicular wave
dynamics in llamas, alpacas and vicunas by shortening
the inter-wave interval and reducing maximum follicu-
lar diameter attained during each follicular wave (Adams
et al., 1990; Vaughan, 2001; Chaves et al., 2002; Aba et
al., 2005). Peak values of plasma oestradiol in alpacas and
llamas can be up to three times higher during follicular
growth in the absence of a corpus luteum in non-pregnant
females compared with peak plasma oestradiol concentra-
tions measured in pregnant females. These results suggest
that progesterone from the corpus luteum exerts a negative
influence on follicle activity in animals that have ovulated
(Aba et al., 2000) possibly by effects on LH. The effect
that progesterone exerts on follicular growth described in
camelids is most likely mediated via negative feedback of
progesterone on the hypothalamic–pituitary axis. Suppres-
sion of hypothalamic GnRH pulses reduces pituitary LH
secretion which reduces follicular diameter and inter-wave

interval.
9.1. Luteolysis
Regression of the corpus luteum in camelids is under
the influence of pulsatile secretion of prostaglandin F2�
(PGF2�) secretion from the uterine horns (Aba et al., 2000;
Vaughan and Tibary, 2006) with repeated pulses of great-
est concentration being secreted from 8 to 9 days after
mating to 12 days after mating (Aba et al., 1995, 2000).
The role of oxytocin in luteolysis remains unknown. It has
been postulated that PGF2� secretion from the left uter-
ine horn may induce luteolysis of a corpus luteum in the
right ovary via a local veno-arterial pathway (Fernandez-
Baca et al., 1979; Del Campo et al., 1996) and may explain
why embryos derived from right-sided ovulations migrate
to the left uterine horn for successful gestation.
10. Ovarian activity in pregnant females
The corpus luteum is the major source of progesterone
throughout pregnancy and its presence is required to main-
tain pregnancy (Sumar, 1988). The embryonic signal for
maternal recognition of pregnancy remains unknown, but
must be transmitted as early as 8–10 days after mating in
order to rescue the corpus luteum of pregnancy (Aba et al.,
1997). There is a temporary decline in plasma progesterone
8–12 days after mating during the period of maternal
recognition of pregnancy, levels reach a peak approxi-
mately 20 days after mating, then concentrations vary
throughout gestation but remain greater than 6 nmol/L
(2 ng/mL) (Adams et al., 1991; Aba et al., 1995). Proges-
terone is higher in pregnant than non-pregnant females 8
days after mating but is not known whether this occurs
because there is an embryo present in pregnant females

cience 124 (2011) 237–243
or there is a reduced ability to secrete progesterone in
females that fail to conceive (Sumar, 1999). Lactation does
not appear to affect progesterone levels during gestation
(Adams et al., 1990).
Ovarian follicular activity continues in a wave-like fash-
ion during pregnancy in camelids. Progesterone appears
to exerts a negative effect on folliculogenesis throughout
gestation as there is a decrease in the number of follicles
detected, smaller maximum diameter of the dominant fol-
licle, reduced inter-wave interval and less prominent day
to day growth and regression profiles of dominant follicles
up until 6 months of gestation (Adams et al., 1990; Aba,
1995). From 7 months gestation, only 3–4 mm diameter fol-
licles are present on the ovaries of alpacas indicating waves
become less prominent towards the end of gestation (Bravo
and Varela, 1993). The first follicular wave post-partum is
usually observed within a week of parturition (Aba et al.,
1998).
Conflict of interest statement
The author, Jane Vaughan, does not have a financial
or personal relationship with other people or organisa-
tions that could inappropriately influence or bias the paper
entitled “Ovarian function in South American camelids
(alpacas, llamas, vicunas, guanacos)”.
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Ovarian function in South American camelids (alpacas, llamas,
vicunas, guanacos)IntroductionOvarian developmentAdult
reproductive anatomyPubertySexual
behaviourSeasonalityFolliculogenesisHormonal control of
folliculogenesisOvarian activity in unmated femalesOvarian
activity in mated females: ovulationLuteolysisOvarian activity
in pregnant femalesConflict of interest statementReferences
Research communication — Navorsingsberig
The measurement of glucocorticoid concentrations in the serum
and faeces
of captive African elephants (Loxodonta africana) after ACTH
stimulation
S K Stead
a
, D G A Meltzer
a
and R Palme
b
INTRODUCTION
In 1998, 30 juvenile elephants were
captured in Botswana and transported to
t r a i n i n g f a c i l i t i e s a t Af r i c a n G a m e
Services (AGS) ( 25°47’ S, 27°46’E) in South
Africa. The move ignited a heated debate

between animal rights and conservation
organisations concerning the welfare of
the so -called ‘Tuli Elephants’. On a
broader scale, the psychological well-
being of elephants maintained in zoos
and circuses was highlighted, and the
need to optimise husbandry conditions
for behaviour, health and well-being was
reiterated. As Garaï7 noted, it is reason-
able to assume that juvenile elephants
separated from their families, captured
and translocated have experienced a
certain degree of stress. Stress is a subjec-
tive experience, and thus the extent to
which individuals are ‘stressed’ is difficult
to quantify5.
Although there is no universal scientific
agreement on the definition of stress,
stress responses cause an increase in
glucocorticoids, primarily cortisol and
corticosterone, in the blood9. Conven-
tionally, the assessment of adrenal re-
sponses to stress relies on collection of
blood samples and measurement of
corticosteroids10. However, the process of
blood collection is impossible without the
use of capture drugs when studying
free-ranging wild animals and will, in
itself, elicit elevated cortisol levels16.
Metabolism of glucocorticoids occurs
primarily in the liver3. There are large
inter-species differences with respect to
the metabolites formed and their route of

excretion1,11–13,18, therefore the efficacy of
measuring faecal cortisol metabolites
should be evaluated for each species.
Palme and Möstl13 have established an
11- oxoaetiocholanolone enzyme im-
m u n o a s s a y ( E I A ) t h a t m e a s u r e s
11,17-dioxoandrostanes (11,17-DOA), a
group of faecal cortisol metabolites. The
biological relevance of this method has
been proven in ruminants following
ACTH stimulation of cortisol release by
the adrenal cortex15 and used to monitor
transport stress in cattle14. This non-
invasive technique has been applied to a
number of domestic, zoo and wildlife
species1,11,18,19.
In elephants, cortisol has been mea-
sured in the saliva from 2 Asian elephants4
and in urine from 1 African and 1 Asian
elephant2 as a means of assessing adreno-
cortical activity in a non-invasive manner.
However, the collection of faecal samples
is more practical, especially when dealing
with free-ranging animals, and provides
measurements that are independent of
short-term fluctuations2,15. The aim of our
study was to validate a method for
measuring glucocorticoid metabolites in
elephant faeces, and to conduct a prelimi-
nary investigation into the method’s
biological relevance.
MATERIALS AND METHODS

Animals
Twenty elephants at AGS made up a
large proportion of the animals studied.
Of these, 14 animals, referred to as Group
1, were being trained by Indonesian
mahouts and were kept in a relatively
large enclosure of approximately 2500 m2.
The remaining 6 animals, Group 2, were
kept some distance away and out of sight
and sound from the main hub of activity
centred around the training process. Two
of these animals were in an enclosure of
150 m2 and 4 in an enclosure 350 m2 in
extent. Both groups had been in their
respective enclosures for 12–14 months
when the study was undertaken. The
elephants were of a similar age, estimated
between 5 and 7 years.
Three further elephants, Group 3,
which were kept on the farm of the Glen
Afric Lodge, Broederstroom (25°49’S,
27°51E), approximately 10 km distant
from the AGS premises, were included in
192 0038-2809 Tydskr.S.Afr.vet.Ver. (2000) 71(3): 192–196
aWildlife Unit, Faculty of Veterinary Science, University of
Pretoria, Private Bag X04, Onderstepoort, 0110 South
Africa.
bInstitute of Biochemistry and Ludwig Boltzmann Institute
of Veterinary Endocrinology, University of Veterinary
Medicine, Veterinärplatz 1, A-1210 Vienna, Austria.

Received: February 2000. Accepted: August 2000.
ABSTRACT
Conventionally, the assessment of adrenal responses to stress
relies on blood sample collec-
tion. However, blood collection from animals is impossible
without restraint or immobilisa-
tion that influences results. This study was undertaken to
validate recently established
enzyme immunoassays that measure faecal glucocorticoid
metabolites in elephants, and to
perform a preliminary investigation into the biological
relevance of this non-invasive
method for use in assessing the degree of stress in this species.
Four juvenile African
elephants were injected i.m. with 2.15 mg synthetic
adrenocorticotrophic hormone
(Synacthén, Novartis, Switzerland). Blood and faecal samples
were collected over 4 h and
7 d respectively. Concentrations of serum cortisol and faecal
cortisol metabolites were
determined using immunoassay. Variability of basal and peak
values in blood and faeces
was observed among the elephants. After ACTH injection,
serum cortisol concentrations
increased by 400–700 %. An 11-oxoaetiocholanolone enzyme
immunoassay (EIA) proved
best suited to measure cortisol metabolites (11,17-
dioxoandrostanes) when compared to a
cortisol and corticosterone EIA in faecal samples.
Concentrations of faecal
11,17-dioxoandrostanes increased by 570–1070 %, reaching
peak levels after 20.0–25.5 h.
Greater levels of glucocorticoid metabolites were measured in
faecal samples from

elephants kept in small enclosures compared to levels in the
faeces of animals ranging over
a larger area. The results of this preliminary study suggest that
non-invasive faecal monitor-
ing of glucocorticoid metabolites is useful in investigating
adrenal activity in African
elephants.
Key words: ACTH, animal welfare, cortisol, EIA, elephant,
faeces, glucocorticoids,
non-invasive.
Stead S K, Meltzer D G A, Palme R The measurement of
glucocorticoid concentrations in
the serum and faeces of captive African elephants (Loxodonta
africana) after ACTH stimu-
lation. Journal of the South African Veterinary Association
(2000) 71(3): 192–196 (En.). Wildlife
Unit, Faculty of Veterinary Science, University of Pretoria,
Private Bag X04, Onderstepoort,
0110 South Africa.
the study. These animals had been reared
from an early age on the farm, were habit-
uated to humans and roamed about in a
750 ha enclosure during the day accom-
panied by a game guard. One animal, a
male, was approximately 10 years old and
the 2 females were 4 and 6 years of age.
None of the elephants studied were
pregnant.
Management of the elephants

Husbandry of all the AGS elephants,
Groups 1 and 2, was the responsibility of
AGS under the supervision of the National
Council of the Society for the Prevention
of Cruelty to Animals. The Group 1 ele-
phants were tethered by a front and a
back leg in a barn overnight (between
17:00 and 10:00) and provided with fresh
fruit, vegetables, Eragrostis curvula, tef or
oat hay, lucerne and bedding. During
the day they were released into their
enclosure and had free access to a similar
variety of feed.
The elephants in Group 2 remained free
in their respective enclosures. They had
little contact with humans, who only
attended to them when providing feed
and when the enclosures were cleaned.
Group 3 elephants spent the day wan-
dering about in the 750 ha enclosure feed-
ing as they pleased and were tethered by
1 leg in a barn overnight between 16:00
and 07:00. They were given 2–3 kg horse
cubes, lucerne and bedding during the
evening.
ACTH administration and sample
collection
Only 4 elephants were available for the
purposes of this experiment. As a result,
an experimental design in which control
animals would have been given an injec-

tion of saline solution instead of ACTH
could not be used.
Each elephant was injected intramuscu-
larly with 300 mg azaperone. After 15
min, an 18-gauge catheter was inserted
into an ear vein and a blood sample col-
lected using a 10 m syringe. Two further
blood samples were collected at 15 min
intervals before the intramuscular admin-
istration of 2.15 mg ACTH (Synacthén,
Novartis, Switzerland). Thereafter, a
venous blood sample was collected every
30 min for 4 h.
Faecal samples from almost all defaeca-
tions were collected for 3 days before and
4 days after the ACTH injection.
Blood samples
Five-millilitre samples of whole blood
were placed in plain vacutainer tubes
(Becton Dickenson, USA). Blood was
allowed to clot for 1 h and centrifuged at
1700 × g. Serum was placed in cryotubes
(Amersham, Johannesburg) and stored at
–20 °C until analysis.
Measurement of serum cortisol
Serum concentrations of cortisol were
determined using a Clinical Assays™
G a m m a C o a t ™ C o r t i s o l 1 2 5 I R a d i o-
immunoassay kit (DiaSorin; SA Scientific,

Johannesburg).
Faecal samples
Samples were collected within 30 min of
defaecation. A single faecal bolus was
mixed by hand and then a handful of
faeces was placed into a plastic freezer
bag and stored at –20 °C until the prepara-
tion for extraction and EIA analysis.
Faecal samples were collected from
each group of elephants.
Analysis of faecal cortisol
Frozen faecal samples were oven-dried
at 100 °C. Each sample was powdered and
mixed thoroughly. A 0.5 g subsample was
mixed with 10 m 80 % ethanol, shaken
for 30 min and centrifuged at 1700 × g for
15 min. One millilitre of the supernatant
was drawn off and stored at –20 °C until
EIA analysis. Aliquots of the extract were
analysed with 3 EIA systems (cortisol,
corticosterone and 11-oxoaetiocholano-
lone) as described by Palme and Möstl13.
High-performance liquid
chromatography (HPLC)
HPLC of the faecal metabolites was per-
formed at the Institute of Biochemistry,
Vienna, as described by Teskey-Gerstl
et al.18. Faecal extracts containing peak

11,17-DOA concentrations were sub-
jected to a clean-up procedure (Sep-Pak
C18). Separation was performed on a
reverse-phase Nova-Pak C18 column
(3.9 × 150 mm, Millipore Corporation,
Milford, Massachusetts, USA) using a
linear gradient starting at 50 % methanol.
Three fractions per minute were collected,
dried under a stream of nitrogen, and
reconstituted in assay buffer. Immuno-
reactive glucocorticoid metabolites were
quantified with the cortisol, corticos-
terone and 11-oxoaetiocholanolone EIAs
as previously described13.
RESULTS
HPLC analysis
HPLC separations revealed a number of
immunoreactive substances present in
elephant faeces. They showed a chro-
matographic mobility between cortisol
a n d 1 7 , 2 0 - d i h y d r o x y p r o g e s t e r o n e
(Fig. 1). The main metabolite determined
with the 11- oxoaetiocholanolone-EIA
eluted around cortisol. Lower amounts of
i m m u n o r e a c t i v e s u b s t a n c e s w e r e
detected by testing the HPLC fractions
with the corticosterone-EIA and negligi-
ble amounts with the cortisol-EIA (detec-
tion limit = 2 nmol/kg faeces).
ACTH challenge
Injection of ACTH resulted in an in-

crease of serum cortisol (Fig. 2) and faecal
cortisol metabolite concentrations (Fig. 3).
Serum cortisol levels began to rise after
0038-2809 Jl S.Afr.vet.Ass. (2000) 71(3): 192–196 193
Fig 1: HPLC separation of immunoreactive glucocorticoid
metabolites in 1 faecal sample
from an elephant as tested in a cortisol-, corticosterone- and 11-
oxoaetiocholanolone-EIA.
Fractions marked with represent the approximate elution time of
respective standards
(17a,20aP = 17a,20a-dihydroxy-progesterone).
injection with azaperone and insertion of
catheters. Following ACTH administration
serum cortisol increased between 4- and
7-fold, reaching highest recorded values
(526–652 nmol/ ) after 2 h. No distinct
peaks were observed.
Individual differences in basal and peak
values of faecal cortisol metabolites were
observed. Basal values of faecal 11,17-
DOA and corticosterone equivalents
ranged from 21 to 168 nmol/kg (median:
48 nmol/kg) and 33 to 133 nmol/kg
(median: 50 nmol/kg) respectively.
ACTH-induced peaks were between
572–1104 % (11,17-DOA) and 160–353 %
(corticosterone) higher than basal values.
These peak concentrations occurred
20–25.5 h after the injection. Additional

Fig. 2: Time course of serum cortisol concentrations (nmol/ ) in
4 elephants before and after intramuscular injection of 2.15 mg
ACTH at
time zero. Individual elephants were identified as SW, BB, TZ
and SH.
Fig. 3: Time course of concentrations of faecal 11,17-
dioxoandrostanes and corticosterone equivalents (nmol/kg) in 4
elephants before and
after intramuscular injection of 2.15 mg ACTH at time zero.
Individual elephants were identified as SW, BB, TZ and SH.
peaks of varied height were observed for
both groups of metabolites before and
after the ACTH-induced peaks.
Faecal glucocorticoids in elephant
groups
The results of faecal glucocorticoid analy-
ses are summarised in Table 1.
DISCUSSION
There have been few studies to investi-
gate the possibility of using non-invasive
methods to assess adrenocortical activity
in elephants2,4. Methods to identify and
measure faecal glucocorticoid metabo-
lites have been successfully used in a
number of domestic livestock11,13,15 and

some wild species1,7,18,19. The aim of this
study was to assess whether the measure-
ment of faecal cortisol metabolites is more
suitable than using blood cortisol values
to monitor adrenocortical activity in
elephants.
Serum cortisol levels began to rise be-
fore ACTH was injected, suggesting that
handling or the injection of azaperone
affected levels of serum cortisol within
30 min. These findings are in accordance
with Sire et al.1 7 and Fulkerson and
Jamison,6 who reported that physical
restraint and blood-sampling can pro-
duce an increase of blood cortisol levels
within 15 min of handling. The lowest
values recorded before ACTH injection
(73–131 nmol/ ) were similar to those
found by Hattingh et al.8 in plasma from 5
undisturbed adult female elephants that
had been shot (mean = 111 nmol/ ). How-
ever, it must be noted that their results are
associated with a high standard deviation
(24.8). The results are not directly compa-
rable to those in this study owing to the
elephants’ age, unknown reproductive
status and undefined environmental
stressors at the time of sample collection.
Morton et al.10 collected blood samples
from 27 elephants immobilised with etor-
phine/xylazine. A mean plasma cortisol
concentration of 347± 0.95 nmol/ was es-
timated. It is likely that these values were
a result of capture procedures and are not
representative of baseline cortisol levels.

After ACTH injection, serum cortisol
levels continued to rise, and in 3 of 4 cases
reached a plateau. No further blood sam-
ples were taken, as it was regarded as un-
ethical to collect blood for longer than 4
h o u r s . T h e t r a n q u i l l i s i n g e f f e c t o f
azaperone began to wear off and it
became increasingly difficult to prevent
the elephants from pulling the indwelling
catheters out of the ear veins. Sampling
from other sites, such as the tail, had been
attempted previously and proved unsuc-
cessful.
The highest recorded cortisol concen-
trations were within the range of those
found in 5 elephants that had been
herded for 6–20 minutes and darted with
succinyldicholine before sample collec-
tion (mean: 688 nmol/ , SD: 269.5)8. This
gives an indication of the type of stressor
that may produce such elevated levels.
A number of glucocorticoid metabolites
were detected in elephant faeces using
HPLC analysis. Although the exact iden-
tity of the metabolites was not deter-
mined due to the cross-reactions of the
EIA, a group of them may be collectively
described as 11,17-dioxoandrostanes
(11,17-DOA)13,15. As reported in domestic
livestock 1 1 , 1 3 , negligible amounts of
cortisol and low amounts of cortico-
sterone were found in elephant faeces.

These findings support the suggestion
that the recently-developed 11- oxo-
aetiocholanolone EIA, which measures
11,17-DOA, is the most suitable EIA to use
for the non-invasive monitoring of
adrenocortical activity in elephants.
As observed in other species, the time
course of faecal cortisol metabolite con-
centrations reflected the ACTH-induced
stimulation of glucocorticoid produc-
tion11,15. Concentrations peaked 20–25.5 h
after the ACTH injection. Similar times
were found by Möstl et al.11 in ponies. It
has been suggested that the delay in faecal
glucocorticoid excretion is correlated
with the transit time of digesta from the
duodenum to the rectum12. Our findings
fit well with the total passage time from
mouth to rectum of 33 h reported for
Indian elephants20. Differences in diet
and individual adaptations in hepatic or
gastrointestinal function may explain
differences in excretion rates21.
T h e l a r g e i n c r e a s e o f 1 1 , 1 7 D OA
(572–1104 % above basal levels) after
ACTH injection was higher than that
observed in ponies (200–660 %) by Möstl
et al.11, but within the range of reported
increases in cattle during transport
(400–1100 %)14. Lower percentage in-
creases in corticosterone were measured,
supporting the earlier suggestion that
11,17-DOA is a more suitable group of me-

tabolites to measure.
Additional peaks after the ACTH-
induced peak could be due to an entero-
hepatic circulation of the metabolites12.
Alternatively, additional peaks may have
been caused by stressful events approxi-
mately 24 h before they were recorded.
More prolonged periods of behavioural
observations conducted before, during
and after the trial may have made it possi-
ble to identify events that had caused
these responses.
As reported in other species11,13,18, indi-
vidual variation in basal and peak values
was observed. This may be due to differ-
ences in previous experiences, body mass,
metabolism, age, diet or sex. Further
investigations with greater numbers of
animals are necessary to identify the
influence of these confounding factors.
Preliminar y investigations into the
application of the technique to assess
w e l f a r e s h o w e d g o o d c o r r e l a t i o n
between behavioural observations, envi-
ronmental stressors and faecal gluco-
corticoid metabolite concentrations.
Concentrations of 11,17-DOA from
elephants allowed to range in 750 ha fell
within the lower end of the range of basal
values measured in Group 1. These Glen
Afric elephants were exposed to fewer
stressors than those housed at AGS
and had more opportunity to perform
species-specific behavioural activities

such as foraging in a large area. The
elephants kept in the small enclosures at
AGS, Group 2 animals, had 11,17-DOA
concentrations 450 % higher than the
elephants at Glen Afric.
The primary advantages of faecal sample
collection are that the collector does not
require special skills and there is no need
to handle the animals. Secondly, concen-
trations of faecal glucocorticoid metabo-
lites probably more closely reflect the
amounts of cortisol produced and ex-
creted than cortisol measured in blood,
which only reflects a point in time during
a dynamic process of absorption, metabo-
lism and excretion15. We conclude that
measuring faecal 11,17-DOA is a valuable
0038-2809 Jl S.Afr.vet.Ass. (2000) 71(3): 192–196 195
Table 1: The effect of enclosure size on faecal 11,17-
dioxoandrostane concentration.
Group Number of elephants Enclosure size Range of 11,17-DOA
Median Number of samples
(m2) (nmol/kg) analysed
1 14 2 500 21–168 48 42
2 6 350 62–1000 176 6
3 3 7 500 000 15–47 39 6

tool for non-invasive monitoring of
adrenocortical activity in African ele-
phants. This could help to optimise the
capture, transport and husbandry of
Afrian elephants and be useful in investi-
gating stress in free-ranging situations.
ACKNOWLEDGEMENTS
We thank Mrs A Kuchar-Schulz, Mrs M
Stark, Mrs A Human and Mrs M Mulders
for excellent assistance in the laboratory
and Dr C Speedy for her assistance in the
field. The financial support of World
Wildlife Fund and the Taeuber Manage-
ment Trust Pty (Ltd) is gratefully ac-
knowledged. The study was conducted
with the permission of Mr R Giazza (Afri-
can Game Services), Mr J Brooker (Glen
Afric Country Lodge) and the National
Council of the Society for the Prevention
of Cruelty to Animals.
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