CHAPTER 1
Introduction
On such a silent night a silvery jet was seen far in advance of the white bubbles at the bow. Lit up by the moon, it looked celestial; seemed some plumed and glittering god uprising from the sea. –Moby Dick
1.1 Introduction
Wildlife conservation research is currently dominated by studies into population
size and growth, genetics, seasonal movements and reproductive rates.
Hormones are key factors in reproductive rates and general health and so are
crucial to a population’s survival. Hormones control many basic functions of the
body including reproductive systems in addition to the physiological responses to
changing environmental parameters. It has been reported that measured
hormonal concentrations can offer a better understanding of factors that impair a
species’ demographic vitality or identify early warning signals for changes in
reproductive rates (Kirby and Ridgway 1984; Schoech and Lipar 1998). In order
to successfully manage any population and understand the factors that may be
influencing it, basic biological knowledge is required on internal physiology,
reproductive cycling, and effects of extraneous factors (such as increasing levels
of stress) on both individual animals and populations. The use of non-invasive
techniques to monitor hormone metabolites in wildlife is growing (Creel et al.
1991; Lasley and Kirkpatrick 1991; Wasser 1995; Robbins and Czekala 1997;
Rolland et al. 2005). These techniques can be used to determine reproductive
fitness and further our understanding of the relationship between behaviour,
physiology and the environment (Wildt and Wemmer 1999).
1 Chapter 1 – Introduction
With advances in technology, animal reproductive parameters in wildlife populations,
such as hormone concentrations and reproductive cycling, have been described for a
number of terrestrial mammal species including: bandicoots (Gemmell et al. 1985);
Asian elephants (Brown et al. 1991); black rhinoceroses (Berkeley et al. 1997);
gorillas (Czekala and Robbins 2001); feral horses (Lasley and Kirkpatrick 1991);
Siberian tigers (Seal et al. 1985); one-humped camels (Marie and Anouassi 1987);
and muriquis (Ziegler et al. 1997).
Although not as prominent as their terrestrial counterparts, there has been some
description of reproductive cycling in marine mammal species such as killer whales
(Duffield et al. 1995); polar bears (Ramsay and Stirling 1988); harbour seals
(Raeside and Ronald 1981); and captive bottlenose and common dolphins (Kirby and
Ridgway 1984). There are a number of advantages in studying terrestrial mammals
over marine mammals as they are easier to locate, can be anaesthetised, tracked,
and tagged with relative ease. In contrast, marine mammals provide researchers with
a number of complex problems when assessing their basic biology and internal
physiology. When comparing marine mammals, pinnipeds (seals and sea lions) and
polar bears and sea otters can be accessed when they come ashore to breed or rest,
but cetaceans (whales and dolphins) and sirenians (dugongs and manatees) do not
come ashore at all and novel methods must be developed.
Hormone concentrations are determined after collection of a sample through invasive
techniques such as blood (plasma or serum) sampling or through non-invasive
techniques, such as faecal or urine sampling. Obtaining samples for hormone
analysis from cetaceans can be difficult because of their aquatic locations.
Anaesthesia of cetaceans, although possible, has only been accomplished by a few
individuals (Haulena and Heath 2001) and therefore is not common practice,
2 Chapter 1 – Introduction
particularly with wild animals. Blood sampling has been achieved in captive
cetaceans (Duffield et al. 1995; Suzuki et al. 1998) and in some smaller wild
cetaceans (St. Aubin et al. 1996) but unfortunately with fatal results in great whales
(Kjeld et al. 1992; Kjeld 2001; Suzuki et al. 2001; Kjeld et al. 2004). Mansour et al.
(2002) recently developed a protocol to assess progesterone concentrations in the
blubber of minke whales using whales from the Greenland hunt. They suggest that
blubber from biopsy sampling may be another way to assess hormone
concentrations in great whales. Captive cetaceans have been trained for urine
sampling (Robeck et al. 1993; 1994; Duffield et al. 1995) although some species can
be particularly difficult to train (G. Bedford, personal communication). Faecal
sampling has been undertaken in the North Atlantic right whale (Rolland et al. 2005)
but can be difficult as great whales do not defecate at regular intervals and tend to
defecate only when in their feeding grounds. Faecal sampling has been also used to
determine stress in some seal species (Gulland et al. 1999; Hunt et al. 2004;
Mashburn and Atkinson 2004).
Most great whales migrate annually from their breeding grounds, usually in warmer
tropical waters at low latitudes, to their feeding grounds in cooler Artic or Antarctic
waters at high latitudes (Dawbin 1966). As there is little to no feeding on the breeding
grounds or along the migratory route, faecal samples can only be systematically
collected in the feeding grounds. In the Northern Hemisphere, this is in the Arctic or
Sub-Arctic areas which are readily accessible and for the coastal great whales, such
as humpback whales and right whales, close to shore. In the Southern Hemisphere,
faecal sampling is more difficult as the feeding areas are in the Southern Ocean near
Antarctica and systematic samples cannot be easily obtained.
3 Chapter 1 – Introduction
Assessment of the internal physiology (such as reproductive hormone
concentrations) of great whales and other large cetaceans is problematic without
restraint, which can endanger the animal and the lives of those that attempt it.
Currently the only reproductive hormone data of great whales comes from
commercial whaling on minke whales and fin whales (Kjeld 2001; Suzuki et al. 2001;
Mansour et al. 2002). Subsistence whaling in the Arctic on bowhead whales also
allows for further opportunity to assess hormone concentrations in some great whale
species (Reeves et al. 2001). The challenge remains to assess hormone
concentrations of large free-swimming whales without harm and to sample the
population multiple times over a given period to gain a better understanding of their
basic biology.
Reproduction and population growth are fundamental to whale conservation. In this
chapter I will present our current understanding of mammalian reproductive
hormones; review our current knowledge of cetacean reproductive hormones;
discuss the suitability of using existing non-invasive techniques to determine
reproductive hormones in cetaceans; and propose a new non-invasive method to
determine reproductive hormones in free-swimming whales.
4 Chapter 1 – Introduction
1.2. Whales and whaling
There are two suborders in the order Cetacea, the Odontoceti (toothed whales) and
the Mysticeti (baleen whales). The great whales are all those species found in the
Mystecti, including humpback, right and fin whales; and one species of Odontoceti,
sperm whales.
Great whale populations have been hunted from the time of the early Basque
whalers in the sixteenth century to the present day. Commercial whaling catch limits
were set to zero by the International Whaling Commission in 1982, coming into effect
in 1986 (IWC Convention III-11). Scientific and subsistence whaling continues today.
With the decline of whales in the Atlantic in the 1800s, many European and American
whaling ships moved to the Indian, Pacific and Southern Oceans. Until the late 1890s
whales were hunted from shore-based stations and so whalers were limited in their
catches by the number they could tow back to shore. The factory ship and the
exploding harpoon were invented in the early 1900s (Harmer 1931; Day 1992). This
meant that more whales could be caught and processed at sea, producing a sharp
decline in great whale stocks in Antarctic waters.
Humpback whales in Antarctic waters were divided into six areas designated by their
respective feeding areas (fig. 1.1). The areas were originally designated according to
whaling areas in the Antarctic and are now used to describe the different feeding and
breeding populations. Dawbin (1966) describes how those animals found in particular
feeding areas in the Southern Ocean tend to be found in specific breeding areas as
well. The whales off the west coast of Australia are known as the Area IV (70 oE-
130 oE) population and, off the east coast as the Area V (130 oE-170 oW) population
(Dawbin 1966). Area V humpback whales are thought to migrate up the east coast of
5 Chapter 1 – Introduction
Figure 1.1: World map depicting the six areas of the Antarctic as designated by the
International Whaling Commission (IWC).
6
7 Equator
Area VI Area I Area II Area III Area IV Area V
120 oW 60 oW 0o 70 oE 130 oE 170 oW
Chapter 1 – Introduction
Australia as well as New Zealand and Tonga. Prior to whaling in Australian waters it
was estimated that there were approximately 17,000 humpback whales in Area IV
(Bannister 1994) and 10,000 humpback whales off the east coast, a portion of the
Area V population (Paterson and Paterson 1984). The portion of the Area V
humpback whale stock that migrates along the east Australian coast is estimated to
have numbered 1900 ± 250 in 1992 compared with as few as 100 at the conclusion
of commercial whaling in 1962. The average annual rate of increase from 1984 to
1992 was estimated at 11.7% with a 95% confidence interval of 9.6-13.8% (Paterson
et al. 1994). Brown et al. (2003) estimated the annual rate of increase to be 8.54%
(s.e. = 0.05) from 1991 to 2000. This level of recovery is at a higher limit for a large
mammal. This suggests that there have been no detrimental environmental impacts
upon this particular stock post whaling (Paterson et al. 1994).
In 1946, the International Whaling Commission (IWC) was established to monitor
catch rates and quotas for the whaling industry. As the number of whales decreased
the IWC became responsible for the protection of the great whales. The Commission
consists of 52 member nations and focuses on the conservation of whales to allow
for an orderly development of the whaling industry. Part of the conservation program
is the comprehensive assessment of whale stocks by the Scientific Committee. In
2001, a workshop into the causes of reproductive failure in the critically endangered
North Atlantic right whale was convened. Unlike humpback and southern right whale
populations, this northern right whale population is declining and will be extinct within
200 years (Caswell et al. 1999). This decline has been attributed to a number of
different causes such as shipstrikes, entanglement and reproductive failure (IWC
2000). The workshop into reproductive failure highlighted the need to assess the
internal physiology (hormonal concentrations, disease, basal health values) of great
8 Chapter 1 – Introduction
whales. It recommended to the IWC the development of non-lethal techniques to
achieve this (Reeves et al. 2001). This study is based on these recommendations.
1.3. Hormones
Hormones are the messengers that initiate and regulate the physiological systems of
higher organisms and are the means of cell-cell communication. The study of
hormones has been limited in the past by the technology of the age. From the
beginning of the twentieth century until around the 1960s, hormones were studied
predominantly using physiological methods. These studies resulted in the discovery
of approximately 25 hormones (Norman and Litwack 1997). Improved methodology
for hormone assessment paralleled advances in biotechnology. The knowledge of
hormones in the twentieth and twenty-first centuries is due primarily to rapid
advances in technology, namely the coupling of labelled radio-isotopes with chemical
means such as chromatography, mass spectrometry and x-ray crystallography.
In mammals, hormones are responsible for specific activities, are generally
synthesised and expressed by different organs, and undergo metabolism (and
catabolism) at varying rates. Initially, hormones were divided into three classes:
polypeptide/protein, steroid and amino-acid related hormones. This review will
consider only steroid hormones. For many animal populations an important factor in
conserving a species is a greater understanding of population growth and
reproduction (Wildt and Wemmer 1999). Steroid hormones are the class of hormones
that are directly associated with mammalian reproduction.
9 Chapter 1 – Introduction
1.3.1. Steroid Hormones
In 1929 oestrone was the first steroid hormone to be isolated in the urine of pregnant
women (Butenandt and Hanisch 1935; Freeman et al. 2001). Currently there are over
225 naturally occurring steroids that have been isolated (Norman and Litwack 1997).
In mammals there are six families of steroids that can be classified on both a
structural and biological basis, they are the oestrogens (female sex steroids), the
androgens (male sex steroids), the progestins, the mineralocorticoids, the
glucocorticoids and vitamin-D (fig. 1.2). All of these steroids are biologically derived
from cholesterol. For the purposes of this review, only mammalian oestrogens,
androgens and progestins will be discussed.
Most steroid hormones have limited solubility in plasma, due to the presence of the
‘steroid’ rings which give them an intrinsically hydrophobic nature. Steroid hormones
are transported in the blood by plasma transport proteins (PTP) from their sites of
biosynthesis and secretion to the target tissues (Siiteri et al. 1982). All five of the
PTPs are synthesised in the liver (Siiteri et al. 1982). The concentration of the PTP is
subject to physiological regulation, where it can be increased or decreased. In theory,
the amount of 'free' (unbound) hormone available for biological activity is subject to
the amount of PTP present in the plasma.
1.3.1.1. Cholesterol
Cholesterol is the most commonly occurring steroid (fig. 1.2) and is found in
practically all living organisms, including blue-green algae and bacteria (Norman and
Litwack 1997). Animal products are a rich source of cholesterol, with above average
concentrations in the skin, sperm cells, and egg yolk. Virtually all cell membranes in
higher animals include cholesterol as an integral component and most organisms
also have the capacity to biosynthesise cholesterol (Popják and Cornforth 1960).
10 Chapter 1 – Introduction
Figure 1.2: Five ring cholesterol structure and the derivatives of these into the six
classes of steroids: estrogens, androgens, progestins, mineralcorticoids,
glucocorticoids and vitamin-D.
11
CH3
PROGESTINS ESTROGENS C O OH
CHOLESTEROL (C-27) Oestradiol
O HO Progesterone
VITAMIN D HO 12 ANDROGENS OH OH
CH2OH
C O O CH 2OH O HO OH Testosterone HC C O
HO
Cortisol HO OH
O
GLUCOCORTICOIDS O MINERALCORTICOIDS Aldosterone
Chapter 1 – Introduction
Since cholesterol is the precursor for the six classes of steroid hormone, it is
important to consider its biosynthetic pathway. This pathway can be divided into four
steps: 1) formation of mevalonic acid from acetate; 2) conversion of mevalonic acid
into the hydrocarbon squalene (30-carbon structure); 3) oxidation of squalene into
lanosterol; and 4) the processing of lanosterol to produce cholesterol. The early
precursors of cholesterol biosynthesis are water soluble, but after the production of
squalene the cholesterol precursors become hydrophobic. The concentration of total
body cholesterol in mammals is a dynamic interplay of biosynthetic cholesterol,
dietary cholesterol and excretion of cholesterol. It is important to note that dietary
cholesterol is not the only source of cholesterol as more than 60% of the body's
cholesterol comes from the liver and the intestines (Popják and Cornforth 1960).
1.3.1.2. Steroid synthesis
The principal tissues that synthesise the five classic steroid hormones are the ovaries
(oestrogens, progestins), testes (androgens) and the adrenal gland (glucocorticoids,
mineralcorticoids). During pregnancy, the foetal-placental unit can be a source of
oestrogen and other hormones. The production of each steroid hormone is
dependent on the stimulation of cells of origin by a specific stimulatory peptide
hormone. Luteinising hormone (LH) acts on the testes to produce androgens and on
the ovaries (corpus luteum) to produce progestins; follicle stimulating hormone (FSH)
acts on the ovarian follicles to produce oestrogens; and placental chorionic
gonadotropin (hCG) acts on pregnant female ovaries (corpus luteum) to produce
progestins (fig. 1.3).
13 Chapter 1 – Introduction
Figure 1.3: Relationship between secreted peptide hormones and the production of
steroid hormones by different endocrine glands.
14
Peptide Hormone Endocrine Gland Steroid Hormone
Luteinising male Testes Androgens Hormone (LH) (interstitial cells) (e.g. testosterone)
female
female Follicle Stimulating Ovaries Estrogens 15 Hormone (FSH) (follicles) (e.g. oestradiol)
Placental Chorionic pregnant female Ovaries Progestins Gonadotropin (hCG) (corpus luteum) (e.g. progesterone)
Adrenocorticotropin Adrenal cortex Glucocorticoids Hormone (ACTH) (zona fasciculata) (e.g. cortisol)
Chapter 1 – Introduction
1.3.1.3. Enzymes in steroid synthesis
Cytochrome P450 enzymes are involved in classical steroid hormone synthesis
(Nerbet et al. 1987; Simpson et al. 1994). They are involved in the hydroxylation
reactions that make up the metabolic pathways of steroid hormones. There are a
total of six P450 enzymes involved in steroid synthesis in the ovaries, testes and
adrenal cortex (fig. 1.4) (Norman and Litwack 1997). The first step of classical steroid
hormone synthesis is the conversion of cholesterol into pregnenolone by the
cholesterol side chain cleavage enzyme P450scc (fig. 1.4). The rate-limiting step of
adrenal and gonadal steroid hormones synthesis, however, is not this enzyme but
rather the side chain cleavage reaction. Specifically the delivery of cholesterol from
the cytoplasm across the outer mitochondrial membrane to the inner mitochondrial
membrane is actually the rate-limiting step (Black et al. 1994).
Once pregnenolone is produced it may undergo one of two conversions. It is either
converted to progesterone (fig. 1.5a) or it undergoes 17-hydroxylation, leading to
cortisol production in the adrenals. Importantly, progesterone is also an intermediate
in the synthesis of aldosterone in the adrenals. The 17-hydroxylation of pregnenolone
can also lead into the androgen/oestrogen pathways (fig. 1.5b).
1.3.1.4. Transport
The hormonally active form of steroids are free molecules, while the readily available
form of most steroids are those bound to PTP, which are transported to the target
tissues from the endocrine gland. A target tissue is defined as one that has stereo-
specific receptors permitting the accumulation of the steroid in the target tissue
against a concentration gradient. This in turn permits the generation of the
appropriate biological response in that target tissue for the specific steroid in
16 Chapter 1 – Introduction
Figure 1.4: The principal pathways of human steroid hormone synthesis, P450
enzymes which are involved in the production (italics), and tissues where the
production occurs: adrenal cortex (A), ovaries (O), placenta (P), testes (T) and skin
(S). Modified from (Norman and Litwack 1997).
17
A Cholesterol Vitamin D3
O,P,T,A Cholesterol scc
O,P,T,A A A Pregnenolone Progesterone Deoxycortisol Corticosterone C21 -hydroxylase 11 β-hydroxylase
O,P,T,A 17 α-hydroxylase/
18 C-17-C-20lyase
O,P,T,A A A 17OH-Pregnenolone 17OH-Progesterone 11 -Deoxycortisol Cortisol C21 -hydroxylase 11 β-hydroxylase
O,P,T,A 17 α-hydroxylase/ C-17-C-20lyase
O,T O,T O Dehydroepiandrosterone Androstenedione Testosterone Oe stradiol (DHEA) aromatase
Chapter 1 – Introduction
Figure 1.5: a) 21-Carbon pathway for steroid metabolism; C-21 ring is derived from
the side-chain cleavage of cholesterol. b) Androgen and oestrogen metabolism that
is a lead from the C-21 metabolic pathway; adapted from DBGET (Kanehisa 1997;
Fujibuchi et al. 1998)
19
a) 20
b) 21
Chapter 1 – Introduction
question. A key factor in determining the ability of a target tissue to bind the hormone
is the actual blood hormone concentration. The concentration of the steroid hormone
in plasma is determined by three factors: the rate at which the hormone is
biosynthesised and enters the blood; the rate at which the hormone is biologically
inactivated by catabolism or metabolism (in the case of steroid hormones) and the
‘tightness’ of the hormone binding to its PTP.
1.3.2. Androgens
Androgens (testosterone, dehydroepiandrosterone, androstenedione and 5 α-
dihydrotestosterone), commonly known as the male hormones, are the hormones
that are responsible for the development and maintenance of male characteristics
and are produced by the gonads (Norman and Litwack 1997; Squires 2003).
The two most important steroid hormones of the adult male are testosterone and 5 α-
dihydrotestosterone (DHT) (Parkinson 2001). Both are produced by the testes and
circulate in the plasma. The primary site of testosterone production is in the Leydig
cells of the testes (Braunstein 1994) and the manner in which it is produced is
analogous with other steroid hormones. These cells are under the influence of LH
which is secreted by the anterior pituitary gland (Norman and Litwack 1997). As with
other steroidogenesis, side-chain cleavage of cholesterol into pregnenolone is the
rate-limiting step in androgen production. There is significant target tissue
metabolism of testosterone into DHT.
Testosterone plays an important role in the differentiation, growth and maintenance
of the sexual reproductive tissue. In addition, testosterone, along with other
hormones, plays an important role in the maintenance of the secondary sexual
characteristics as well as skeletal and muscle growth (Parkinson 2001). The
22 Chapter 1 – Introduction
biological responses of the androgens may be divided into four categories: 1) growth-
promoting or androgenic effects on the male reproductive system; 2) development of
the male secondary sexual characteristics; 3) actions on the central nervous system
and brain; and 4) a stimulatory or anabolic effect on body weight and nitrogen
balance (Handelsman 1995; Parkinson 2001). Although it has been shown that
testosterone and DHT are the predominant androgens, it has not been determined
which is the primary initiator of the biological responses in certain tissues.
The onset of puberty is believed to occur as a result of a change in the pre-
pubescent pituitary-gonadal system. Changes in the androstenedione/testosterone
ratios with age and increased P450 enzyme activity in the testes of animals are
indicative of sexual maturity (Pineda 2003). The rate of testosterone biosynthesis is
positively correlated to the LH concentrations in the plasma (Parkinson 2001). The
secretion of LH is reduced by increasing concentrations of sex steroid in the blood.
These bind to steroid receptors in the hypothalamus and the pituitary and create a
‘suppressive negative feedback’ system (fig. 1.6).
Testosterone concentrations are highly variable among different individuals of a
species. In seasonal breeders testosterone concentrations tend to be more variable
as they vary between individuals and across seasons. Hormone concentrations are
dependent on the metabolic clearance rate, secretion and release of the hormone
and also on the circadian rhythm, season and age of the animal (Pineda 2003).
23 Chapter 1 – Introduction
Hypothalamus
Progesterone (-) GnRH
Inhibin (-) on FSH Pituitary Inhibin (-) on FSH Testosterone Oestradiol (-) FSH Oestradiol LH FSH LH (+ or -)
Testes Ovary
Seminiferous Sertoli Leydig Theca Granulosa tubules cells cells cells cells T T
Sperm
Figure 1.6: Negative feedback system of the hypothalamic-pituitary-gonadal axis in
males (testes) and females (ovaries). Dashed lines indicate negative feedback
influences and solid lines indicate positive influences.
24 Chapter 1 – Introduction
1.3.3. Progestins and oestrogens
Progestins and oestrogens are associated with foetal development, birth, growth and
female reproduction (Odell 1979). There are two distinct types of hormones involved
in the female phenotype. The first of these are the peptide gonadotropin hormones,
including LH and FSH, and both are produced by the anterior pituitary gland. The
second type is the steroid hormones which are produced by the gonads in response
to the gonadotropins. These are primarily the progestins and the oestrogens.
The ovary is the primary site for the biosynthesis of the female steroid hormones
(Norman and Litwack 1997). The two most important steroid hormones in the adult
female are progesterone and oestradiol (Squires 2003). Oestrone, oestriol and
dehydroepiandrosterone play important roles in pregnancy (Norman and Litwack
1997). Naturally occurring progesterone has a 21-carbon (21-C) ring with an oxo-
functionality on both the C-3 and C-20 (fig. 1.7a). The naturally occurring oestrogens
are 18-carbon (18-C) steroids that have an aromatic A ring with a phenolic hydroxyl
(fig. 1.7b).
The female reproductive system is governed by the secretion of FSH and LH from
the anterior pituitary gland (Hall and Crowley 1995). The secretion of both these
peptide hormones is dictated by gonadotropin-releasing hormone (GnRH). GnRH is
released in a pulsatile fashion from the hypothalamus and is a primary stimulator of
the release of FSH and LH. Once FSH and LH have been released they affect the
ovaries and the female reproductive tract. Progesterone and oestradiol function in a
negative feedback system at both the hypothalamic and the pituitary level to affect
GnRH production and release (fig. 1.6) (Hall and Crowley 1995; Squires 2003).
25 Chapter 1 – Introduction
a). CH 3 PROGESTINS C O
O Progesterone
oxo-functionality
b).
ESTROGENS O
Oestrone
HO
Aromatic A ring with
phenolic hydroxyl OH
17 β-Oestradiol
HO
Figure 1.7: a) The naturally occurring progestins have a 21-carbon ring with an oxo-
functionality on both the C-3 and C-20. b) The estrogens have a 18-carbon ring with
an aromatic ring and phenolic hydroxyl.
26 Chapter 1 – Introduction
With the onset of puberty there is an increased output of both LH and FSH (Noakes
2001). As the ovaries become more sensitive to these hormones there is a gradual
increase in the production of oestrogens and androgens. These help stimulate the
development of the vagina, uterus, external genitalia and mammary glands. The
onset of puberty is species dependent and is also influenced by external factors such
as nutrition, season and proximity of a male (Noakes 2001). In non-seasonal
polycyclic animals (such as in cows and pigs) the cyclic activity is only interrupted by
pregnancy, lactation and disease (Noakes 2001; Squires 2003). In species which are
seasonally polycyclic (such as horses, deer and dolphins) there are periods of
anoestrous (Squires 2003). The oestrous cycle and related hormonal concentrations
in animals is described in depth in Chapter 5.
1.4. Cetacean reproduction
As the analysis of hormone concentrations in cetaceans can be technically difficult
much of the current knowledge of cetacean reproductive endocrinology comes from
captive species (Sawyer-Steffan et al. 1983; Walker et al. 1988; Robeck et al. 1994;
Duffield et al. 1995; West et al. 2000).
The reproductive system works on a negative feedback system where the increase in
hormones from the gonads causes a decrease in the release of hormones from the
hypothalamus and pituitary glands (fig. 1.6). There are detailed anatomical
descriptions of both male and female reproductive anatomy in great whales
(Matthews 1937; Chittleborough 1954; b; 1955ba). However, there is little literature
on hormonal concentrations or related reproductive cycling for many great whale
species. The reproductive cycles of cetaceans were determined during the whaling
days (Chittleborough 1958) before assay techniques for the determination of
27 Chapter 1 – Introduction
hormone concentrations had been developed. Chittleborough (1958) described the
cycle by histological examination of the ovaries and the presence or absence of the
corpus albicans.
In more recent years there has been an increased interest in assisted reproductive
technologies (ART) in captive cetaceans, such as bottlenose dolphins and false killer
whales (Robeck et al. 1994; Brook 1999). Concurrent with these technological
advancements is a greater understanding of the endocrinology of cetaceans.
However, only a limited number of cetacean species (those in captivity) have been
studied and inaccuracies can occur when using one species as a model for others
(Robeck et al. 2001).
1.4.1. Females
In the dolphins (Delphinidae), females are believed to have seasonal reproduction,
yet this is from studies of a few species, the bottlenose dolphins, harbor porpoises,
killer whales and false killer whales (Kirby and Ridgway 1984; Duffield et al. 1995;
Read and Hohn 1995; Atkinson et al. 1999). Through histologic examination of the
corpus albicans, it has been shown that bottlenose dolphins reach sexual maturity
between 9.5 to 11 years of age (Sergeant et al. 1973; Perrin and Reilly 1984;
Cockcroft and Ross 1990). With the availability of newer chemical analytical
methods, baseline serum progesterone concentrations have been measured at 0.29
ng/ml for captive bottlenose dolphins during the non-cycling phase and oestrogen
concentrations at 25 pg/ml (Sawyer-Steffan et al. 1983). When serum progesterone
concentrations are higher than 3 ng/ml and then returned to less than 1 ng/ml during
one month, it is considered that the animal has ovulated but pregnancy has not
ensued (Sawyer-Steffan et al. 1983). Immature animals have progesterone
concentrations less than 1 ng/ml (Kirby 1990).
28 Chapter 1 – Introduction
In killer whales, serum progesterone concentrations range from 0.10 – 0.42 ng/ml
during non-cycling to 1 - 13.7 ng/ml during non-conceptive ovulatory cycles (Duffield
et al. 1995). During pregnancy, progesterone concentrations range from 1.5 - 68
ng/ml (Duffield et al. 1995). Oestrogen conjugate concentrations in killer whale urine
ranged from 0.5 - 55 ng/mg Creatinine (Cr) in nonpregnant animals and 0.5 - 25
ng/mg Cr in pregnant females (Walker et al. 1988). In pregnant females
pregnanediol-3α-glucuronide (PdG) range from 50 - 100 ng/mg Cr while in
nonpregnant females PdG concentrations were below the level of detectability
(Walker et al. 1988).
A small study of three captive false killer whales showed that plasma progesterone
concentrations were below 1 ng/ml in an immature female while plasma
progesterone concentrations ranged from 0.8 - 30 ng/ml in two adult females
(Atkinson et al. 1999). There were seasonal changes in plasma progesterone
concentrations. They were highest in summer and lowest in winter. Salivary
progesterone was also measured in this study. There were no seasonal trends seen
in salivary progesterone (Atkinson et al. 1999).
Using whaling carcasses, hormone concentrations have been determined for minke
whales through blubber and plasma sampling and fin whales through serum
sampling (Kjeld et al. 1992; Kjeld 2001; Suzuki et al. 2001; Mansour et al. 2002; Kjeld
et al. 2004). In minke whales a mean blubber progesterone concentration of 132.96
ng/g was an indication of pregnancy. Non-pregnant females had a blubber
progesterone concentration of 1.95 ng/g where males had a concentration of 1.86
ng/g (Mansour et al. 2002). Mean plasma progesterone concentrations were less
than 0.05 ng/ml in immature minke whales, 7.08 ng/ml in mature minke whales and
6.71 – 13.84 ng/ml in pregnant minke whales (Suzuki et al. 2001; Kjeld et al. 2004).
29 Chapter 1 – Introduction
In minke whales plasma 17 β-oestradiol concentrations were 0.63 pg/ml, 0.79 pg/ml
and 0.77 pg/ml in immature, mature and pregnant animals respectively (Suzuki et al.
2001). In fin whales plasma progesterone concentrations ranged from 0.03 to greater
than 3.14 ng/ml. Concentrations higher than 3.14 ng/ml indicated pregnancy (Kjeld et
al. 1992). Serum oestradiol concentrations varied widely in the fin whale with a mean
of 15.8 pg/ml (Kjeld et al. 1992).
1.4.2. Males
Compared to female cetaceans male hormonal concentrations have only been
described for a few species. It has been shown that male wild bottlenose dolphins
reach sexual maturity between 10 and 13 years (Sergeant et al. 1973; Perrin and
Reilly 1984; Cockcroft and Ross 1990). There is seasonal variation in testosterone
concentrations in bottlenose dolphins with concentrations being the highest during
the breeding season (Schroeder 1990a). Testosterone concentrations in plasma can
be utilised to classify dolphins as immature, pubescent or sexually mature (Kirby
1990; Schroeder 1990a). Sexually mature males are said to have testosterone
concentrations of 2 - 5 ng/ml, rising as high as 10 ng/ml in the breeding season,
whereas in pubescent animals testosterone concentrations change from 1 - 10 ng/ml.
Immature animals have plasma testosterone concentrations less than 1 ng/ml (Kirby
1990; Brook 1999). In killer whales, testosterone concentrations fluctuated
seasonally, ranging from 1.4 - 2.2 ng/ml (Robeck et al. 2001).
Testosterone concentrations have been determined for minke and fin whales through
sampling whale carcasses (Kjeld et al. 1992; Suzuki et al. 2001; Kjeld et al. 2004).
Plasma testosterone concentrations in minke whales were 0.14 - 0.81 ng/ml in
immature animals and 0.51 - 2.1 ng/ml in mature animals (Suzuki et al. 2001; Kjeld et
30 Chapter 1 – Introduction
al. 2004). In fin whales serum testosterone concentrations ranged from 0.11 - 0.5
ng/ml depending on the season when the animal was captured (Kjeld et al. 1992).
Plasma concentrations of different reproductive hormones are useful for captive
cetacean management. However plasma sampling in wild populations can be
problematic. Non-invasive methods that allow for the determination of reproductive
hormone concentrations of wild populations need to be developed initially using
captive populations to determine the likely concentration range. These validated
techniques can then be used to assess reproductive behaviour of wild cetaceans.
1.5. Current hormonal sampling techniques
The techniques that are currently used to assess reproductive hormones in
cetaceans are blood sampling (St. Aubin et al. 1996; Suzuki et al. 1998; St. Aubin
2001), urine sampling (Robeck et al. 1993; Robeck et al. 2004) and recently blubber
sampling (Mansour et al. 2002) and faecal sampling (Rolland et al. 2005). These
techniques employ either radioimmunoassay (RIA) or enzyme immunoassay (EIA) to
determine hormone concentrations. These methods are discussed in detail in
Chapter 2.
Correlations between concentrations of hormones in pooled samples (urine or
faeces) with hormone concentrations in instantaneous samples (plasma or serum)
are common place. These comparative results should be treated with caution as
there are different passage rates for hormones in urine and faeces (Whitten et al.
1998). Urinary steroids tend to be excreted within 4 to 8 hours with complete
excretion by 24 hours (Ziegler et al. 1989; Crockett et al. 1993; Wasser et al. 1994;
31 Chapter 1 – Introduction
Whitten et al. 1998) whereas faecal steroids can be excreted from 0.3 to 3 days after
they have been secreted (Ziegler et al. 1989; Heistermann et al. 1993; Wasser et al.
1994; Whitten et al. 1998). This depends on the length of the intestinal tract and the
passage rate time. Making correlations between instantaneous and pooled samples
can be further complicated by other factors, such as diet and lean body mass, which
may cause changes in the respective hormone concentrations.
Considering these variables, arguments arise as to whether correlations should be
made between plasma and other forms of sampling, or should each sampling method
describe an independent hormonal concentration range for diurnal and seasonal
variations as was done for plasma? Once the trends and variations have been
established for the new sampling methods, such as urine or faeces, the trends then
can be compared to verify if the new method is a reliable alternative and parallels the
pattern of hormone fluctuations.
1.5.1. Blood
Blood has been used in a wide range of mammalian species to ascertain hormone
concentrations: from the reproductive cycle in females to glucocorticoid production
during acute stress, to diurnal and seasonal changes in reproductive hormones
(Gemmell et al. 1985; Pietraszek and Atkinson 1994; Romero 2002; Brown et al.
2004). Although this is a practical method for assessing hormonal fluctuations in
captive management situations it is problematic when working with wild populations.
Due to its invasive nature, blood samples cannot be collected on a daily basis.
32 Chapter 1 – Introduction
1.5.2. Faecal
Faecal sampling is a non-invasive technique that has become more prominent in
recent years and particularly with wild animal studies (Wasser et al. 1988; Ziegler et
al. 1989; Berkeley et al. 1997; Goymann et al. 1999; Wasser et al. 2000; Lynch et al.
2003). Faeces are easy to collect and the steroid hormone concentrations reflect the
reproductive cycle of the animal. But as faecal hormone concentrations are pooled
over time they do not indicate instantaneous hormone concentrations. Faecal
hormones are concentrated for 0.3 to 3 days prior to defecation (Whitten et al. 1998).
This is particularly important if assessing whether glucocorticoids are affected by a
specific event. Glucocorticoids are commonly used to determine stress in animals
(Romero 2002). In order to detect if an event was “stressful” to an animal faecal
samples 1 to 3 days after the event should be collected. But would be useful in
situations of chronic stress.
It is also questionable whether faecal sampling is suitable for assessment of
hormone concentrations in seasonal feeders such as baleen whales. Since these
animals are feeding only for a portion of the year, it is not likely that faecal samples
can be collected outside of their feeding areas. These samples cannot be easily
collected during migration or on their breeding grounds. Baleen whales have a 10 -
12 month gestation (Matthews 1937; Jonsgård 1951; Chittleborough 1958; Laws
1961). Assessing hormone concentrations at the time of ovulation and fertilisation is
important. As baleen whales are migratory, and do not feed in their breeding
grounds, faecal samples may not be suitable for assessment of reproductive cycling.
1.5.3. Urine
Urine sampling has been used widely to assess hormone concentrations in
mammals. In humans the most common use for hormone assays of urine is to
33 Chapter 1 – Introduction
assess pregnancy. Urine samples have been used to determine hormone
concentrations in feral horses (Kirkpatrick et al. 1988), timber wolves (McLeod et al.
1996), gorillas (Czekala et al. 1988), and killer whales (Walker et al. 1988; Robeck et
al. 2004). Similar to faecal samples, urine is pooled in the bladder over a number of
hours before urination occurs. It is relatively easy to collect from captive animals, for
example by training in killer whales (Walker et al. 1988), or by placing trays under
cages in order to catch the voided urine, such as with primates (Ziegler et al. 1989).
In wild species, however, urine collection can be difficult. It is absorbed in soft ground
before it can be collected (Kirkpatrick et al. 1988). In species that live in snow
covered regions it is possible to obtain a urine sample from the ice or snow (McLeod
et al. 1996; Constable 2001; Parslow 2002). For wild cetaceans urine collection is not
possible.
1.5.4. Saliva
A variety of hormones and peptides have been assessed in the saliva of humans
(Riad-Fahmy et al. 1982; Vining and McGinley 1987; Lu et al. 1999; Granger et al.
1999a; Anfossi et al. 2002) and animals (Pietraszek and Atkinson 1994; Bettinger et
al. 1998; Theodorou and Atkinson 1998; Atkinson et al. 1999). Routinely RIA or EIA
is used to determine hormone concentrations but recently liquid chromatography-
mass spectrometry (LC-MS, described in Chapter 2) has been used to determine
peptide concentrations in humans (Vickers et al. 2001). Saliva is favoured over blood
sampling in many studies, particularly with children (Granger et al. 1999a). It is less
stressful and allows for daily or multiple sampling. In wildlife, saliva has been used to
determine progesterone concentrations in captive monk seals (Pietraszek and
Atkinson 1994) and Californian sea lions (Iwata et al. 2003). In addition it has been
used to determine stress in captive white-tailed deer (Millspaugh et al. 2002) and
captive gorillas (Bettinger et al. 1998).
34 Chapter 1 – Introduction
1.5.5 Other infrequently used hormone sampling methods
Milk has been used as a non-invasive alternative to plasma sampling in determining
progesterone concentrations in dairy cows (Lamming and Darwash 1998) and
captive bottlenose dolphins (West et al. 2000). This method is only possible with
female animals and needs the animal to be lactating in order to monitor changes in
the reproductive status.
Alternatively, Mansour et al. (2002) showed the feasibility of using blubber samples
from whales to determine progesterone concentrations. Although the samples in this
study were taken from dead whales it was reported that blubber samples collected
during biopsy operations could be used as a viable option to determining pregnancy
in minke whales by measuring progesterone concentrations (Mansour et al. 2002).
Although the biochemical composition of fin (Lockyer et al. 1984), sei (Lockyer et al.
1985) and minke whale (Olsen and Grahl-Nielsen 2003) blubber has been described
very little other work has been conducted on blubber. It is unknown if steroid
hormones are found uniformly throughout blubber or if there is some form of
stratification. It is difficult to use blubber biopsies as a successful measure of
hormonal concentrations until hormone distribution through the blubber is described.
That is, is the blubber biopsy an indication of the most recent hormone concentration
or is it an indication of months past?
35 Chapter 1 – Introduction
1.6. Aims of this study
Unlike terrestrial mammals, cetaceans breathe less frequently but tend to use a
greater portion of their lungs with each breath (Pabst et al. 1999). The blow
(exhalation) comes from deep inside the lungs and theoretically should carry with it
small amounts of mucus from the lungs. If sufficient mucus is collected then the
possibility to determine hormone concentrations in cetaceans is feasible. The
hypothesis of the work presented in this thesis is that blow sampling can be used to
determine reproductive hormone concentrations in cetaceans.
As no chemical assays exist for the analysis of blow samples, the primary focus of
this study was to develop validated analytical techniques to determine reproductive
hormones in blow samples. This two-part program consisted of method development
and validation; and a field program for sample collection. Due to the nature and novel
methodology employed for this study, validation and method development for
reproductive hormones in cetaceans used saliva and blow samples from captive
bottlenose dolphins.
The specific objectives of this research were to:
• develop a method for the determination of testosterone using liquid
chromatography-mass spectrometry (LC-MS) (Chapter 3);
• describe testosterone concentrations in captive male bottlenose dolphin
saliva and blow (Chapter 3);
• determine parameters of testosterone stability in saliva and blow particularly
storage conditions and enzyme inhibition (Chapter 4);
• develop a method for the determination of female reproductive hormones
(progesterone, oestradiol and oestrone) using LC-MS (Chapter 5);
36 Chapter 1 – Introduction
• describe female hormone concentrations in captive female bottlenose dolphin
saliva and blow (Chapter 5);
• determine parameters of progesterone stability in saliva and blow particularly
storage conditions and enzyme inhibition (Chapter 6);
• develop a method to collect blow samples from free-swimming great whales
(Chapter 7); and
• determine the feasibility of using blow samples to assess reproductive
hormones in humpback whales using LC-MS (Chapter 7).
Blow sampling could prove to be a powerful tool to improve our understanding of
reproductive cycling in great whales as it can be used in both the breeding and
feeding areas. This is particularly important for critically endangered species, such as
the North Atlantic right whale, as understanding reproductive cycling can lead to a
better comprehension of causes of reproductive dysfunction.
37
CHAPTER 2
General Methodology
There is no validated analytical method to determine reproductive hormone
concentrations in the blow of cetaceans. Accordingly the primary focus of this
study was to determine a method that could use the small sample volumes
that are obtained from blow sampling. This method needed to have a high
degree of selectivity and sensitivity. Immunoassays are used routinely to
assess reproductive hormone concentrations for a number of wildlife species
(Schneyer et al. 1985; Czekala et al. 1986; Schroeder and Keller 1989;
Robeck et al. 1993; Whitten et al. 1998; Hagey and Czekala 2003; Deschner
et al. 2004). Recently an alternative method, mass spectrometry, has been
used in pharmacokinetic and physiological studies (Gelpi 1995; Tiller et al.
1997; Vickers et al. 2001; Wren et al. 2003) and to assess reproductive and
adrenal hormones in human plasma (Wu et al. 2002; Nelson et al. 2004),
urine (Taylor et al. 2002; Rockwood et al. 2003) and environmental water
samples (Croley et al. 2000).
2.1. Immunoassays
The introduction of immunoassay (Ekins 1960; Yalow and Berson 1960) was
the single most important advance in biological measurement in the past 50
years (Chard 1990). Several different immunoassay methods have been
developed but radioimmunoassay (RIA) and enzyme immunoassay (EIA)
have been the most frequently used types to assay reproductive hormones.
Both are binding assays, with the quantification of the substance being
measured depending on the progressive saturation of a specific antibody by
that substance and the subsequent determination of the bound and free
38 Chapter 2 – General Methodology
phases (Chard 1990). The differentiation between the bound and free phases is
made by labelling the antibody with either a radioactive isotope (RIA) or an
enzyme (EIA). There are advantages to each method when comparing RIA with
EIA but there is very little difference between the two with respect to final
quantitative results (Chard 1990).
2.2. Mass Spectrometry
A mass spectrometer produces gas-phase ions of a molecular compound. These
ions are separated according to their mass-to-charge ratio ( m/z ) and the
proportion to which they are detected. Using mass spectrometry (MS) coupled
with gas chromatography (GC) or liquid chromatography (LC) provides a highly
selective and sensitive method of chemical analysis.
Although there is a variety of different methods that can be employed when using
a mass spectrometer, such as different ionisation techniques and the type of
mass spectrometer used, there are common fundamental analytical requirements
of the various types of MS. These requirements include: a device to introduce the
compound to be analysed; an ionising source; one or more analysers to separate
the ion; a detector to quantitate the ions and a data processing system (a
computer).
2.2.1. History
Mass spectrometry is now used routinely in many biochemical and
pharmacological laboratories. Surprisingly, the earliest recorded use of a mass
spectrometer is from 1912 with the discovery of mass spectra for oxygen (O 2),
nitrogen (N 2), carbon dioxide (CO 2), carbon monoxide (CO) and carbonyl chloride
39 Chapter 2 – General Methodology
(COCl 2) (Thomson 1913). Although there were developments in mass
spectrometers, it was not until 1958 that it was coupled with GC. In 1968, with the
beginning of the computer era, a data processing unit was added to the GC-MS
system.
The MS used for this program consisted of a quadropole analyser and an
electrospray ionisation method. The quadropole analyser was invented in 1958
but it was not until the development of the electrospray in 1988 (Yamashita and
Fenn 1984) that compounds over 20,000 Da could be analysed. Although MS
had been around for nearly a hundred years it has been only in the past fifteen
years that technological advances have allowed mass spectral analysis of large
compounds. It is anticipated that mass spectral analysis will become a more
commonly used method in the twenty-first century (De Hoffman et al. 1996).
2.2.2. Chromatography-Mass Spectrometry
There have been a number of technical issues associated with coupling MS with
LC. LC requires chemical separation compared with GC and is used with
compounds that are not volatile (i.e. not suitable for GC). In addition polar
compounds for LC do not need to be deriviatized as with GC. GC samples are
usually deriviatized to render polar substances more volatile so that they can be
eluted at reasonable temperatures without thermal decomposition or molecular
rearrangement (Scott 1998). Samples are deriviatized by either esterification or
acetylation. The problem with LC is that, unlike GC, the compounds leave the
column in a liquid state and it is more difficult to eliminate the solvent in the MS.
Different ionisation methods have been used including thermospray, ionspray or
chemical ionisation but electrospray ionisation is the preferred method for the
majority of applications (De Hoffman et al. 1996).
40 Chapter 2 – General Methodology
2.2.3. Mass spectrometric analysis
Electrospray ionisation (ESI) is produced by applying a strong electric field under
atmospheric pressure to a liquid passing through a capillary tube and is charged
by a few kilovolts. The liquid emerges as a mist of very fine charged droplets (De
Hoffman et al. 1996; Chapman 1998). A flow of drying gas (usually nitrogen to
prevent chemical interaction with the analytes) assists the solvent in evaporating,
causing the droplets to decrease in size. As the droplets get smaller they become
unstable and explode to form smaller droplets. A cascade of smaller and smaller
droplets are created until the electric field on the droplet surface is large enough
to produce the desorption of ions (De Hoffman et al. 1996).
ESI is a very mild process with almost no thermal input so there is no
fragmentation of the ions and only the molecular weight information is available
for the mass spectrum (Chapman 1998). The most important feature of ESI is
that the ions are multiple charged as MS separates ions according to their m/z .
Multiple charged ions are also advantageous as they allow for greater sensitivity
as the scanning times are reduced over the selected m/z range and allow for the
analysis of very large molecules using a low mass analyser.
There are three modes for MS: namely scan mode; selected-ion monitoring mode
(SIM); and selected-reaction monitoring mode (SRM). Scan mode measures the
complete spectra between two extreme masses, for example 50-900 Da.
Although scanning allows for detection of all m/z within the scan range, the
sensitivity for substances is decreased due to the detection of multiple ions (fig.
2.1). Using SIM increases the sensitivity by looking for pre-determined specific
masses (fig. 2.2). In SIM mode the MS acts as a sensitive and highly selective
detector that can discriminate between interference from background noise or
41 Chapter 2 – General Methodology
Int. TIC(1.00)
35.0e6
30.0e6 25.0e6 20.0e6 15.0e6 10.0e6 5.0e6
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min Time
Int. 686.35
300e3
250e3 638.60
200e3 670.00 704.70 624.95 362.45 652.70 604.00 675.55 734.15 150e3
776.60 571.60 820.10 100e3 785.30 516.25 853.40 879.50 406.40 384.30 483.00 538.25 899.00 931.25 50e3 287.40 353.10 309.15 397.40 253.00 300.25 0e3 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z Molecular weight
Figure 2.1: Chromatogram of dolphin blow in scan mode (top), the mass
spectrum (bottom) shows the different m/z s that can be found in the sample
where the line is marked.
42 Chapter 2 – General Methodology
other materials, so quantitative analysis can be achieved with a high degree of
confidence (Chapman 1998). If further sensitivity and selectivity is required then
SRM can be used with a tandem mass spectrometer (MS-MS).
The data recorder and processor show the mass spectra in an interpretive format
for analysis (fig. 2.2). The data are given as a function of time with the area under
the curve being directly proportional to the number of ions detected. A
concentration curve can be developed by analysing a number of known
concentration samples. This concentration curve shows the relationship between
the area under the mass spectral curve and the concentration of a particular
substance by using a least-squares regression analysis (Bressolle et al. 1996).
The sensitivity of the liquid chromatography – mass spectrometry (LC-MS) assay
is dependent on the choice of analyser, ion source and chromatographic
conditions (Chapman 1998). It is important to develop a replicable, simple
method for analysis, particularly if measuring multiple analytes.
2.3. Immunoassays vs. LC-MS
There are a number of advantages of LC-MS to immunoassays. Although MS
may not be as sensitive compared to some immunoassays, when coupled with
LC it provides a highly selective environment which has no cross-reactivity and
which can analyse very small sample volumes. LC-MS can analyse sample
volumes as small as 50 µl while for many immunoassays sample volumes of at
least 200 µl and sometimes higher are needed. A further advantage is that not all
of the sample is used during the LC-MS injections so it can be stored and re-used
for further analyses which is not possible with immunoassays. Collecting samples
43 Chapter 2 – General Methodology
Int. TIC(1.00) 289.20(1.00)
250e3
200e3
150e3
100e3
50e3
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 min Time
Int. 289.20 50e3
40e3 30e3
20e3
10e3
0e3 288.25 288.50 288.75 289.00 289.25 289.50 289.75 290.00 m/z Molecular weight
Figure 2.2: Chromatogram and mass spectrum of dolphin blow looking for a
specific m/z ratio of 289.20.
44 Chapter 2 – General Methodology
from wild animals is difficult and expensive so reusing samples is a significant
advantage. In addition, when using the scan mode in LC-MS, other biological
substances can be preliminarily identified which may be of interest at a later date.
Sample information is stored on CD and can be analysed or archived for future
researchers to use. The unknown m/z chromatographic peaks can be collected
after LC column elution using a splitter directing a portion of the unknown analyte
into the MS for m/z identity and purity. The other portion is collected at the same
time into a vial for amino acid sequencing providing complete information on
previously unknown analytes.
A disadvantage of LC-MS in comparison to immunoassay is the initial capital
setup cost of the equipment. Consumables are comparative in price to RIA and
EIA kits. For some analytes RIA sensitivity is higher than that achieved with LC-
MS but further work with tandem mass spectrometers may increase the sensitivity
of current LC-MS methods.
Recent hormone concentration studies have shown that there are considerable
variations between immunoassay results from different laboratories but there is
sufficient reproducibility within a laboratory to be able to compare hormone
concentrations of individuals (Gail et al. 1996; McShane et al. 1996).
Comparative studies have been conducted to highlight the differences between
immunoassays and mass spectrometry. Further to this Dorgan et al. (2002)
showed that although the absolute concentrations between immunoassay and
mass spectrometry may differ for some hormones, the two methods showed
similar estimates of between-subject differences in serum concentrations of most
sex steroid hormones. In summary there is a range of established hormone
analysis techniques to choose from as an analytical method.
45 Chapter 2 – General Methodology
2.4. General Method Development
Due to the small sample volumes collected in this study immunoassay was not a
viable technique. The preferred method for this study was high performance liquid
chromatography (HPLC) coupled with a mass spectrometer due to sample
volume constraints. As blow samples yielded volumes between 50 and 100 µl LC-
MS would allow for a higher degree of accuracy and specificity to determine
reproductive hormone concentrations.
The LC-MS was located at the research laboratories of the Department of
Anaesthesia and Pain Management, University of Sydney at Royal North Shore
Hospital, Sydney. The system compromised of a Shimadzu LC-10AD liquid
chromatograph (Shimadzu, Japan) and Shimadzu SIL-10Axl autoinjector,
attached to a Shimadzu LCMS-2010. The system was controlled by Shimadzu
LC-MS solution 1.0 software. An Alltech Macrosphere 300Å, C8, 5µm, 150 mm x
2.1 mm HPLC column (Alltech Associates Ltd., Sydney, Australia) was used. The
larger pore columns were found to be more economical than the traditional
columns (100Å) as they tended to use less solvent, 0.2 ml/min (300Å) as
opposed to 1 ml/min (100Å). Different LC mobile phases were used for
testosterone and the female hormones due to conflicting m/z s.
LC-MS analysis was performed on a Shimadzu LC directly connected to a
quadrapole Shimadzu LCMS-2010 equipped with an electrospray interface. The
solvent delivery system consisted of LC10AD dual pumps. The system was
conducted in positive SIM mode using the m/z determined for each of the
following hormones: testosterone, progesterone, oestrone and oestradiol and in
negative SIM mode for the internal standard (Fmoc-L-Gln(Trt)-OH).
46 Chapter 2 – General Methodology
The MS instrumental parameters for all hormones were: detector gain, 2kV;
capillary voltage, 4.5kV; drying gas was nitrogen at 4.5 L/min; drying gas
temperature, 250 oC; nebulizer pressure, 6.89 MPa; ionization source at 200 oC.
At the commencement of this study it was anticipated that a single LC-MS
method could be developed to determine reproductive hormones in cetaceans.
However, during the preliminary developmental stage, it was found that
testosterone had the same m/z as formic acid (fig. 2.3) which was being used in
the separation of hormones through liquid chromatography. Formic acid is added
to the mobile phases to allow for the protonation of the hormones in the mass
spectrometer. Acetic acid was used as an alternative to formic acid to overcome
the difficulty posed by common m/z values of formic acid and testosterone but
this caused analytical problems with oestradiol (fig. 2.4). Hence, separate
methods were developed using acetic acid in the mobile phase for testosterone
(Chapter 3) and formic acid for the remaining female reproductive hormones
(Chapter 4).
In order to validate the liquid chromatographic method the following criteria were
met (Bressolle et al. 1996):
i. Long-term and short-term stability of the compound in the biological matrix
(Stability);
ii. Identification and inclusion of a suitable internal standard (Internal Standard);
iii. Limit of detection of the compound;
iv. Limit of quantification of the compound;
v. Concentration curve;
vi. Ruggedness of the method; and
vii. Intra- and inter-assay variability (Accuracy and Precision).
47 Chapter 2 – General Methodology
a)
Int. 289
10.0e6 Testoster 7500e3 357 5000e3
330 105 237 130 377 2500e3 196 213 243 146 256 345 125 178 307 317 391 0e3 165 210 271 399 100 125 150 175 200 225 250 275 300 325 350 375 m/z
b)
Int. 217 300e3 250e3 Formic 200e3 330 150e3 100e3 289 200 50e3 235 264 279 255 305 325 353 367 381 395 0e3 399 200 225 250 275 300 325 350 375 m/z
Figure 2.3: Formic acid has the same m/z as testosterone.
48 Chapter 2 – General Methodology
a) Int. 255 1750e3
1500e3
279 1250e3 151 Oestradiol 190 1000e3
750e3 296 500e3 217 197 305 223 250e3 173 321 349 245 271 332 373 379 0e3 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 m/z
Int. b) 255
150000 Acetic acid 125000
100000 271 297 237 75000 213 279 171 234 50000 195 153 179 25000 339 315 353 369 387 409 429 435 455 462 476 488 0 150 175 200 225 250 275 300 325 350 375 400 425 450 475 m/z
Figure 2.4: Acetic acid has the same m/z as oestradiol.
49 Chapter 2 – General Methodology
2.4.1 Stability
Early experimental data of testosterone analysis showed the stability of
testosterone in saliva and blow was markedly variable. Recovery of testosterone
in saliva during the extraction process was 203 ± 55%, and in blow was 582 ±
66% (n = 10). During the extraction process samples were at room temperature
(21 oC) for approximately eight hours. Due to the dramatic increase in
testosterone concentration, a comprehensive study on the stability of the
hormones at 21 oC, -20 oC and -80 oC was conducted. The majority of the literature
which discusses stability of reproductive hormones has been conducted using
human matrices of plasma and serum. To determine wildlife reproductive
hormone concentrations a number of biological matrices are used, such as
plasma, faeces and urine. However there is a paucity of information concerning
reproductive hormone stability over both short-term and long-term storage. As
stability of reproductive hormones in cetacean samples has not been validated a
stability program was investigated (Chapter 4 & 6).
2.4.2. Internal Standard
GC-MS or LC-MS requires inclusion of an internal standard to control for
variability in the extraction process, during chromatographic injection and from
ionisation of the compound. Ideally the internal standard should have similar
physical chemical properties to the substance under investigation, good
resolution, and be close to the elution time of the substance under investigation
(Kawakami and Montone 2002). In steroid hormone analysis internal standards
are chosen because they are substances that are stable isotopes or close
derivatives of the substance under investigation, such as trideuterated
testosterone for testosterone (Wang et al ., 2004) or 5 α-cholestane for
testosterone (Kawakami and Montone 2002).
50 Chapter 2 – General Methodology
However as the exact metabolic pathways for steroid hormones in dolphin saliva
and dolphin and whale blow were unknown, it was inappropriate to choose an
internal standard that was similar to these substances. For example, an internal
standard that was labelled testosterone or a testosterone analogue would likely
alter the testosterone concentration (or other hormone concentrations) in the
highly complex androgen metabolic pathway (fig. 1.5). An internal standard was
needed which would not interfere in any part of this pathway. As the research aim
was to yield the maximum information possible from free-swimming whale blow
samples a distinct neutral internal standard was investigated that would not alter
the steroid hormones of interest.
A number of different compounds that were eluted in a similar manner to
reproductive hormones were considered as internal standards (table 2.1). The
retention times given in table 2.1 are for a 55% mobile phase B isocratic gradient
(mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90%
acetonitrile). Initially a number of local anaesthetics and anti-depressants were
tested. As synthetic compounds they were unlikely to occur in dolphin saliva and
blow. The local anaesthetic, lidocaine, eluted off the liquid chromatography
column close to the steroid hormones but the peak shape grossly changed when
added to dolphin saliva (fig. 2.5).
A number of anti-depressants, such as trimipramine and carbamazepine, were
tested but were found to bind to proteins in the saliva (Ulrich and Martens 1997)
and were eluted off during solid phase extraction, e.g. trimipramine recovery was
3.5 % (n = 5).
51 Chapter 2 – General Methodology
Table 2.1: Substances tested as internal standards, molecular weight (mw),
mass-to-charge ratio ( m/z ) and retention time on the column (RT) when using a
55% isocratic mobile phase B method (mobile phase A = 0.5% acetic acid, mobile
phase B = 0.5% acetic acid, 90% acetonitrile).
Substance mw m/z RT
Fentanyl 336.46 337.25 8.8 min
Lidocaine 234.30 235.15 6.5 min
Caffeine 194.20 195.05 3.0 min
Theophylline 180.20 181.25 2.4 min
Carbamazepine 236.26 237.00 2.8 min
Trimipramine 294.42 295.10 14.0 min
Sufentanyl 386.5 387.2 11.2 min
Cortisone 360.4 302.85 2.8 min
Penta-Substance P 631.0 613.25 3.4 min
635.15
Fmoc-L-Gln(Trt)-OH 610.7 609.15* 9.2 min
* negative ion SIM
52 Chapter 2 – General Methodology
a) Int. 3:235.15(1.00)
1750e3 Lidocaine 1500e3
1250e3
1000e3
750e3
500e3
250e3
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Int. 1:289.20(1.00) b) 2:235.15(1.00) 35000 Lidocaine
30000
25000 Testosterone 20000
15000
10000
5000
0 1 2 3 4 5 6 7 8 9 min
Figure 2.5: a) Chromatogram of lidocaine standard, using 55% mobile phase B
isocratic method (mobile phase A = 0.5% acetic acid, mobile phase B = 0.5%
acetic acid, 90% acetonitrile), retention time = 6.5 min, m/z = 235.15. b)
Chromatogram of lidocaine, testosterone standard and mixed dolphin saliva,
using 55% mobile phase B.
53 Chapter 2 – General Methodology
Fmoc-Glutamine (Fmoc-L-Gln(Trt)-OH, Auspep, Melbourne, Australia) is a
synthetically produced substance that is used in the synthesis of peptides. It was
found to be the most appropriate internal standard. It fulfilled all the requirements
of an internal standard for this program, as it was not endogenous in dolphin
saliva or blow, it did not bind to proteins and had approximately 80% recovery in
both saliva and blow (n = 20). In addition it demonstrated ideal retention times of
9.2 minutes using a 55% mobile phase B isocratic method and 11.5 minutes
using a 50% mobile phase B isocratic method.
2.4.3. Other validation criteria
The remaining validation criteria: limit of detection of the compound (LOD); limit of
quantification of the compound (LOQ); concentration curve; ruggedness of the
method; and intra- and inter-assay variability (accuracy and precision) (Bressolle
et al. 1996) are addressed in Chapter 3 (testosterone) and Chapter 5 (female
reproductive hormones).
54
CHAPTER 3
Testosterone
3.1. Introduction
Testosterone is an androgenic steroid hormone produced by the gonads (fig.
1.1). It plays a key role in the growth, differentiation and maintenance of sexual
tissues, and the development of secondary sexual characteristics (Norman and
Litwack 1997; Squires 2003). In mammalian males, testosterone is one of the
principal androgens and is secreted by the testes. In females, testosterone is
produced in limited quantities by the ovarian follicles (Norman and Litwack 1997).
Testosterone is required for the production of sperm in the testes and for sperm
maturation in the epididymis (Parkinson 2001). In addition, an animal’s libido,
which is responsible for mating and aggressive behaviour, is primarily dependent
on the androgenic hormones such as testosterone and 5 α-dihydrotestosterone
(Parkinson 2001). Although the correlation between testosterone concentration
and libido has been debated, a positive correlation has been shown in some
domestic species (Boyd and Corah 1988; Chenoweth 1997).
Human testosterone research has been well documented but there is less known
about testosterone concentrations in wildlife. With the present focus on captive
breeding programs there is a priority to understand wildlife reproductive hormonal
cycles to improve management programs of potentially threatened species.
55 Chapter 3 – Testosterone
Historically, problems of declining populations or those which may be exhibiting
reproductive dysfunction were assessed by studying female reproductive cycles.
Little attention has been paid to male infertility as it was assumed that permanently
sterile males would leave no offspring, and so would be a generational and
evolutionary redundant component of the species survival (Malo et al. 2005).
However, males exhibit varying degrees of fertility as a result of their genetic
makeup, or may be temporarily infertile due to stress, poor nutrition or disease. In
many mammalian species, male fertility is determined by semen traits or sperm
motility (Malo et al. 2005). Yet in those species where semen samples cannot be
collected easily, such as cetaceans, alternatives are needed to measure male fertility.
Determining testosterone concentrations in relation to semen traits in captive
cetaceans would allow for a clearer understanding of male fertility in wild populations.
The extensive and enduring presence of endocrine disrupting chemicals (EDCs) in
the environment has been of concern for the last decade (Colburn et al. 1993). These
chemicals are known to affect the endocrine system and so reproductive capacity is
at risk (Baillie et al. 2003). Although studies into humans and laboratory rodents show
that EDCs have an effect on male fertility, there is little knowledge of the effects on
wild mammals. Testosterone concentrations are rarely reported in reproductive
biology. However, concerns about the decline in male mammalian fertility highlight
the need to determine baseline fluctuations in testosterone concentrations in different
species (Baillie et al. 2003). Novel non-invasive methods are required to describe
baseline testosterone concentrations in wild mammals. Without this background
knowledge it will be difficult to determine if reproductive dysfunction in a population is
due to female and/or male infertility.
56 Chapter 3 – Testosterone
This chapter describes a novel non-invasive sampling technique to determine
testosterone concentrations in captive bottlenose dolphin saliva and blow samples.
The aim of this chapter was to:
• develop a non-invasive method to determine testosterone concentrations in
saliva and blow samples using LC-MS; and to
• determine a preliminary biological range of salivary and blow testosterone in
captive bottlenose dolphins of different ages.
This chapter has been published in the Journal of Chromatography B - Analytical
Technologies in the Biomedical and Life Sciences in January 2005 (Appendix 1).
3.2. Materials and Methods
3.2.1. Chemicals and solutions
All chemical reagents were of HPLC grade: acetonitrile (BioLab Scientific, Melbourne,
VIC, Australia), acetic acid 17.5M (BDH Laboratory Supplies, Poole, England), water
was purified by a Milli-Q system (Millipore®, Sydney, NSW, Australia) and nitrogen
gas (5.0, BOC, Sydney, NSW, Australia) was of ultra-high purity.
Crystalline testosterone (Sigma-Aldrich, Sydney, NSW, Australia) was >99% purity
as tested by thin layer chromatography by the manufacturer. Stock solutions were
made fortnightly by dissolving testosterone in 60% acetonitrile (1 mg/ml) and stored
at 4 oC away from light. From the stock solution, working concentrations of
testosterone at 100, 10, 1 µg/ml and 100 ng/ml, were prepared volumetrically by
serial dilution with 60% acetonitrile. Stock solutions of the internal standard (Fmoc-L-
Gln(Trt)-OH) were prepared in 60% acetonitrile at a concentration of 10 µg/ml. To
maintain consistency with testosterone analysis in samples, 100 mM manganese
57 Chapter 3 – Testosterone
chloride (MnCl 2; Clyde Industries, NSW, Australia), stabilising agent for testosterone,
was also added to each working concentration (Chapter 4). Thus, each working
concentration was 60% acetonitrile in 100mM MnCl 2.
3.2.2. LC analysis
The LC elution conditions for testosterone analysis were as follows: mobile phase A,
0.5% acetic acid and mobile phase B, 90% acetonitrile, 0.5% acetic acid, at isocratic
conditions of 55% B and a flow rate of 0.2 ml/min. The HPLC column was
equilibrated for 30 mins prior to a series of runs and maintained its performance for
over 500 injections.
3.2.3. LC-MS methods
The LC-MS and settings used for the analysis are described in Chapter 2. Data
acquisition was set for 11 mins using a m/z 289.20 [M + H] + (fig. 3.1a) for
testosterone with zero variability of detector range. A second m/z 330.25 [M + H +
+ CH 2CN] was the acetylated adduct of testosterone and was used to further confirm
the retention time RT (Bressolle et al. 1996). The acetylated adduct of 330.25 was
present in both biological samples and testosterone standard with a ratio of
approximately 30% (fig. 3.1a) compared to the m/z 289.2 peak. The Fmoc-Glutamine
m/z was 609.15 [M – H] +.
The data acquisition time was lengthened to 27 mins when analysing biological
samples due to an interfering peak at 16 - 24 mins RT (fig. 3.2). Isocratic conditions
were used as gradient conditions and equilibration of the column were found to have
a longer run time.
58 Chapter 3 – Testosterone
a) Int. 289 10.0e6
7500e3 [M + [M+H+CH2CN]+ 357 5000e3
330 105 237 130 377 2500e3 196 213 243 146 256 345 125 178 307 317 391 0e3 165 210 271 399 100 125 150 175 200 225 250 275 300 325 350 375 m/z
Figure 3.1: Determination of testosterone m/z by direct infusion into the MS (5 µl
injection of 100 µg/ml in 60% acetonitrile), testosterone ( m/ z 289.20, 100% relative
intensity), acetylated adduct ( m/z 330.25, approximately 30% relative intensity).
59 Chapter 3 – Testosterone
Int. TIC(1.00) 289.20(1.00)
250e3
200e3
150e3
100e3
50e3
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 min
Figure 3.2 : MS chromatogram ( m/z 289.20) of saliva sample from an adult male
dolphin (Sirius) showing the native testosterone peak at 3.6 mins and peak at 16 - 24
mins requiring duration of isocratic conditions to have a 27 mins assay time.
60 Chapter 3 – Testosterone
3.2.4. Sample collection and preparation
Saliva and blow samples were collected just prior to the breeding season from four
male captive bottlenose dolphins, two adults: Sirius (23 years) and Delbert (36
years); and two sub-adults: Zac, (10 years) and Buck (4.5 years). The dolphins were
trained to open their mouths on cue and the saliva was collected from the oral cavity
by wiping fresh cotton gauze (Smith & Nephew, Sydney, Australia; 7.5 x 7.5 cm x 4
ply) along the roof of the mouth. Saliva was then expressed from the gauze by
placing it inside a 6 ml syringe and applying pressure to the plunger. Blow samples
were collected using 50 ml polypropylene tubes (Sigma-Aldrich, Sydney, Australia).
The tube was held inverted over the animal’s blowhole at a distance of 2 - 3 cm and
the dolphins were trained to exhale on cue. Two exhalations were made per tube.
There was no apparent stress to the dolphins observed by the dolphin trainers from
either collection method.
To prevent degradation of testosterone in the saliva samples, 200 µl of 100mM MnCl 2
was added to each sample. For blow samples 500 µl of MnCl 2 was added and the
tube was shaken vigorously to ensure that all blow mucus was collected in the
solution.
Saliva samples were stored in 1.8 ml round bottom cryo-storage tubes (Sigma-
Aldrich, Sydney, NSW, Australia) and blow samples were stored in 50 ml
polypropylene tubes. Samples were stored at -20 oC for two days before transporting
on dry ice and further storage for one week at -80 oC.
Testosterone was extracted from unspiked dolphin saliva and blow using a ‘Envi-
Chrom P’ solid phase extraction (SPE) cartridge (Sigma-Aldrich, Sydney, NSW,
61 Chapter 3 – Testosterone
Australia) and was adapted from the method described by Vickers et al . (2001) for
SPE of human saliva. The SPE procedure was as follows: (1) 5 ml Milli-Q water
added to 200 µl of sample (saliva or blow) in a 10 ml polypropylene centrifuge tubes
(Crown Scientific, Sydney, NSW, Australia), vortexed for 30 secs and then
centrifuged for 10 mins at 3000 rpm to separate particulate matter before being
loaded onto the SPE cartridge; (2) SPE cartridges were preconditioned using 20 ml
acetonitrile and then 5 ml Milli-Q water (higher than the manufacturer’s
recommendations, to completely remove interfering extraneous material) (Vickers et
al. 2001); (3) samples were loaded onto the cartridges at 5 ml/min; (4) cartridges
were then washed with 7.5 ml of Milli-Q water to remove salts and the eluant
discarded; (5) elution of testosterone was carried out with 5 ml acetonitrile and eluant
dried under nitrogen; (6) samples were reconstituted in 50 µl 60% acetonitrile (to
prevent testosterone precipitation) for LC-MS analysis.
3.2.5. Validation
The method was validated for specificity, linearity, accuracy, precision, limits of
quantification and detection, and stability. A calibration curve was calculated for
testosterone in 60% acetonitrile for: 50, 20, 10, 5, 1 and 0.5 ng/ml, four curves were
run on four separate non-consecutive days. The calibration curves were calculated
with unweighted least-squares regression method (Bressolle et al. 1996). Three
different runs of six repeats on separate non-consecutive days were performed to
evaluate intra- and inter-batch accuracy and precision respectively using 1, 20 and
50 ng/ml spiked saliva and blow. Precision was determined by calculating the relative
standard deviation (RSD, %) and accuracy by calculating the difference between the
nominal and spiked values for the spiked saliva and blow samples (RE, %) (Starcevic
et al. 2003). Ruggedness was ascertained by assaying stock solutions at two
different concentrations: 50 ng/ml and 5 ng/ml, using the same method but with two
62 Chapter 3 – Testosterone
different 300Å C8 columns (Lot #: 02110886-1 and 02110885-1). Recovery of the
SPE extraction was determined by spiking samples with high (50 ng/ml) and low (1
ng/ml) concentrations of testosterone. Samples were separated into 2 x 200 µl
aliquots, one 200 µl aliquot was used for the pre-extraction assay (n = 6). The other
was extracted and reconstituted in 200 µl 60% acetonitrile, and used for the post-
extraction assay (n = 6).
Cotton-based sampling materials have been known to affect immunoassay results for
some salivary steroid hormones (Dabbs 1991; Shirtcliff et al. 2001). To observe if this
was also the case with LC-MS, a series of direct infusions with the gauze were
conducted to identify possible interfering m/z values. Direct infusions were conducted
in scanning mode, scan range 100 – 500 m/z .
Biological samples of saliva and blow were spiked with 50 ng/ml and 5 ng/ml
testosterone standard and stored at 21 oC, -20 oC and -80 oC. Stability experiments
and results are presented in Chapter 5.
3.2.6. Statistical Analysis
Pearsons r correlation and residual analysis were used to assess linearity of the
calibration curves.
63 Chapter 3 – Testosterone
3.3. Results
3.3.1 Validation
Retention time (RT) for testosterone was 3.6 min and the internal standard (IS) was
9.2 min (fig. 3.3). RTs for column A (Lot # 02110886-1) and B (Lot # 02110885-1)
were 3.62 min and 3.71 min, respectively. The RSD between the results of the two
columns was 8.4% (n = 12) at 5 ng/ml and 10.1% (n = 12) at 50 ng/ml. The equation
for the calibration curve for testosterone (0.5 - 50 ng/ml) was y = 0.01x + 0.0045 and
was linear (r 2 = 0.959, r = 0.979, p < 0.001). The residuals were normally distributed
and centred around zero. The LOD (S/N 3:1, ± 5% RT) was 0.2 ng/ml, and the LOQ
(S/N 5:1, ± 5% RT) was 0.5 ng/ml. Table 3.1 shows intra-batch and inter-batch
precision and accuracy data.
Recovery of the SPE for saliva at 50 ng/ml testosterone was 93 ± 7.9% and at 1
ng/ml was 91.5 ± 3.72%. Recovery of 50 ng/ml of testosterone in blow was 83.3 ±
6.8% and for 1 ng /ml was 85.8 ± 4.6%. Recovery of the internal standard in saliva
was 73.0 ± 14.2% and in blow was 78.63 ± 4.29%.
There were no m/z ratios in the cotton gauze that interfered with testosterone
identification (fig 3.4).
64 Chapter 3 – Testosterone
Int. 1:289.20(1.00) 4:609.15(1.00)
25000
20000 Testo
15000
10000
5000
0 1 2 3 4 5 6 7 8 9 10 min
Figure 3.3 : Retention times of testosterone 3.59 mins (20 ng) and the IS Fmoc-Gln
9.22 mins (20 ng). HPLC conditions: 55% isocratic B, mobile phase A 0.5% acetic
acid, mobile phase B 0.5% acetic acid 90% acetonitrile, 0.2 ml/min.
65 Chapter 3 – Testosterone
Int. 143
250e3
83 200e3
150e3 100 100e3 208
192 114 50e3 59 123 229 157 171 180 281 42 235 267 289 325 349 392 77 246 305 359 379 403 425 433 448 456 469 489 498 0e3 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 m/z
Figure 3.4: Direct infusion of cotton gauze into the MS. No m/z coincides with
testosterone ( m/z = 289.20).
66 Chapter 3 – Testosterone
Table 3.1 : Intra-batch and inter-batch precision and accuracy for testosterone in
dolphin saliva and blow.
Intra-batch Sample Concentration Mean Precision Accuracy n type (ng/ml) (ng/ml) (RSD, %) (RE, %) Saliva 1 6 0.9 1.6% 4.4% 20 6 20.0 5.3% 0.1% 50 6 48.7 14.1% 2.7% Blow 1 6 1.0 2.5% 2.6% 20 6 20.0 5.3% -0.1% 50 6 49.4 10.9% -1.2%
Inter-batch Sample Concentration Mean Precision Accuracy type (ng/ml) n (ng/ml) (RSD, %) (RE, %) Saliva 1 18 1.0 4.5% 0.2% 20 18 20.2 5.5% 1.2% 50 18 49.5 12.0% 1.0% Blow 1 18 1.0 4.9% 0.8% 20 18 20.2 7.3% 1.1% 50 18 49.7 9.8% 0.7%
67 Chapter 3 – Testosterone
3.3.2. Dolphin samples (endogenous testosterone concentrations)
Testosterone could be measured by LC-MS in all male saliva samples (fig. 3.5a)
ranging from 9.7 - 23 ng/ml (n = 10) (table 3.2). The two adult dolphins had a mean
testosterone concentration of 15.2 ± 5.7 ng/ml (n = 5) and the two sub-adult dolphins
had a mean testosterone concentration of 13.5 ± 3.5 ng/ml (n = 5). Testosterone in
blow samples (fig. 3.5b) ranged from 14.7 - 86.2 ng/ml (n = 11) (table 3.2). The two
adult dolphins had a mean testosterone concentration of 42.5 ± 21.8 ng/ml (n = 6)
and the two sub-adult dolphins had a mean testosterone concentration of 59.9 ± 23.9
ng/ml (n = 5) in the blow.
68 Chapter 3 – Testosterone
(a) Saliva
Int. TIC(1.00)
40000 Testosterone
35000
30000
25000
20000
15000
10000
0 1 2 3 4 5 6 7 8 9 min
(b) Blow
Int. TIC(1.00)
35000 Testosterone 30000
25000
20000
15000
10000
1 2 3 4 5 6 7 8 9 min
Figure 3.5: a) MS chromatogram of endogenous testosterone (8.9 ng/ml) in an adult
male dolphin (Sirius) saliva, (m/z 289.20, RT = 3.6 mins). (b) MS chromatogram of
endogenous testosterone (4.9 ng/ml) in an adult male dolphin (Sirius) blow.
69 Chapter 3 – Testosterone
Table 3.2: Endogenous testosterone concentrations (ng/ml) in saliva and blow of
male adult and sub-adult bottlenose dolphins. Three different samples were collected
over three consecutive days.
Saliva testosterone Blow testosterone (ng/ml) (ng/ml) Adults Sirius 19.0 48.2 23.0 59.9 Not obtained 30.5 Delbert 9.9 14.7 14.4 73.1 9.9 28.6 Mean ± SD 15.2 ± 5.7 42.5 ± 21.8
Sub-adults Buck 17.2 86.2 16.2 Not obtained 14.4 84.7 Zac 9.9 48.1 Not obtained 34.1 9.7 46.7 Mean ± SD 13.5 ± 3.5 59.9 ± 23.9
70 Chapter 3 – Testosterone
3.4. Discussion
3.4.1 Validation
A reproducible, accurate LC-MS method has been validated for testosterone in saliva
and blow in bottlenose dolphins. The intra- and inter-batch precision results were less
than 15% which was acceptable for chromatographic analyses (Dadgar et al. 1995).
High recoveries for both 50 ng/ml and 1 ng/ml spiked testosterone in both saliva and
blow samples indicated that the SPE is suitable for both matrices. SPE was chosen
over liquid-liquid extraction as the global objective of this program was to develop a
universal method that could be used to determine reproductive hormones (male and
female) in great whale blow. The primary reason that SPE was chosen over the
liquid-liquid extraction is that SPE allows retention and elution of virtually all analytes
in the saliva and blow matrices. This method optimises the data available from these
matrices particularly concerning detection of steroid hormones and in the future
protein hormones (such as LH and FSH). Although liquid-liquid extraction is more
rapid than SPE, it only allows retention and elution of the steroid hormones in the
non-polar liquid (e.g. ether). As the composition of blow is unknown, it was
considered that liquid-liquid extraction would result in loss of valuable compounds.
Biological samples collected with cotton gauze or cotton salivettes have artificially
elevated concentrations of reproductive hormones when using immunoassays
(Dabbs 1991; Shirtcliff et al. 2001). 'Cotton-based' sampling materials, such as
gauze, can alter the results of hormonal RIAs (Dabbs 1991; Shirtcliff et al. 2001), but
do not appear to affect ‘male’ hormonal results with LC-MS as demonstrated in this
study.
71 Chapter 3 – Testosterone
3.4.2. Dolphin samples (endogenous testosterone concentrations)
It is possible to determine endogenous testosterone concentrations in bottlenose
dolphin saliva and blow. Although sample sizes for this study were small, expected
trends were observed in saliva where the two adult male dolphins had higher
concentrations of testosterone than the two sub-adult males. The variability of both
adult and sub-adult samples may be explained due to age. One of the adult males
(Delbert) had lower concentrations of testosterone in both saliva and blow samples.
Delbert at 36 years old may have fluctuating testosterone concentrations, as male
and female bottlenose dolphins are known to have slowing reproductive rates from
the mid-thirties onwards (Duffield et al. 1999). One of the sub-adult males (Zac) had
lower testosterone concentrations due to the presence of a contraceptive implant.
Concentrations of testosterone in blow samples were higher than those in saliva. As
blow sampling has not been trialled previously a comprehensive and longitudinal
study of this species using the method described will provide a greater understanding
of the fluctuations in testosterone concentrations in bottlenose dolphins. The
measurement of hormone concentrations in captive dolphins using a non-invasive
and easily trainable technique provides an outstanding opportunity to further
understand the physiology of wild marine mammals. Saliva and blow samples allow
for multiple daily sampling, and demonstrate no observable stress to the animals,
allowing for diurnal and seasonal data measurements.
72 Chapter 3 – Testosterone
3.5. Conclusion
An accurate, reproducible and rapid LC-MS method has been developed and
validated to determine testosterone concentrations in biological matrices (saliva and
blow) obtained from bottlenose dolphins. Future longitudinal studies will determine
diurnal and seasonal fluctuations in testosterone concentrations in saliva and blow.
Used in conjunction with semen sampling, a greater understanding of the relationship
between testosterone concentration and male fertility in captive dolphins could be
established. This may provide guidelines to understanding male fertility in great
whales.
73
CHAPTER 4
Testosterone Stability
4.1. Introduction
Saliva has been used for a number of years to assess steroid hormone
concentrations in humans (Riad-Fahmy et al. 1982; Luisi and Franchi 1984;
Vining and McGinley 1987; Dabbs 1991; Lu et al. 1999; Granger et al. 1999a)
but it is only more recently salivary hormones have been measured in animals
such as the monk seal (Pietraszek and Atkinson 1994; Theodorou and
Atkinson 1998); western lowland gorilla (Grassi 2003); false killer whale
(Atkinson et al. 1999) and bottlenose dolphin (Hogg et al. 2005).
As early as the 1980s, assessing the stability of steroid hormones in saliva
became paramount (Luisi and Franchi 1984; Banerjee et al. 1985; Vining and
McGinley 1987; Lipson and Ellison 1989). These studies showed that leaving
saliva samples at room temperature for a number of days did not seem to
have any adverse effects on the stability of different reproductive hormones
(Luisi and Franchi 1984; Dabbs 1991). There are, however, differing results in
reference to the stability of testosterone in saliva. Vining and McGinley (1987)
found that testosterone in male salivary samples did not change over time but
that testosterone in female salivary samples increased over time for no
apparent reason. Other studies (Luisi and Franchi 1984; Banerjee et al. 1985;
Granger et al. 1999b) have assessed the stability of testosterone in saliva at
lower temperatures (–20 oC and –80 oC) and found that samples could be
stored for six to twelve months at these temperatures with no adverse change
in hormone concentrations. Studies have tended not to include independent
hormone stability experiments. Granger et al. (2004), however, have recently
74 Chapter 4 – Testosterone Stability
shown a 330.8% increase in testosterone concentrations in human saliva after
four weeks storage at 4 oC but found very little change in testosterone
concentration at -20 oC or -80 oC. It is becoming a requirement for researchers to
assess steroid hormone stability under different storage conditions in order to
optimise sampling regimes (Khan et al. 2002; Washburn and Millspaugh 2002;
Lynch et al. 2003; Granger et al. 2004).
Analytical chemists determine the stability of compounds differently to biologists
and a number of articles have been published that discuss how to assess stability
of compounds in biological matrices (Dadgar et al. 1995; Bressolle et al. 1996).
The fundamental difference is that chemically-based studies spike biological
samples with a known concentration of the compound being measured (Dadgar
and Burnett 1995; Bressolle et al. 1996). This is done to ensure that observed
changes in hormone concentration are at the true rate of change. Measurement
of endogenous hormone concentrations, without comparable spiked standards, in
the matrix to be investigated is inadvisable as the state of degradation is
unknown.
In order to determine the stability of a compound in a biological matrix the
following should be assessed: long-term stability of the compound in the
biological matrix, stability of the reference standard, short-term stability of the
biological matrix stored at different temperatures, in-process stability during the
extraction process, stability of the processed sample over the maximum time from
collection to analysis and the freeze-thaw stability of compounds (Dadgar and
Burnett 1995). If these factors are not determined then changes that occur due to
storage can mask the biological significance of a study’s results (Lynch et al.
2003).
75 Chapter 4 – Testosterone Stability
The structures of steroid hormones are generally consistent across mammals but
there are other influencing factors in each biological matrix, whether it is plasma,
saliva, urine or faeces. These affect hormones in different species in various
ways. Each of these matrices contains different enzymes, bacteria and proteins
which may have diverse effects on hormones. So the stability of steroid
hormones in each biological matrix of a species should be assessed prior to that
matrix being used to measure different life history parameters or behavioural
patterns. The aim of this chapter was:
• to determine the short-term stability of testosterone in saliva and
blow; and
• to determine long-term stability of testosterone in saliva and blow of
captive bottlenose dolphins.
4.2. Materials and Methods
4.2.1. Sample preparation
Saliva and blow collection from four male bottlenose dolphins was described in
Chapter 3. Samples were stored at –20 oC for two days; shipped on dry ice; and
stored at –80 oC for two months prior to analysis. As this was not a kinetic study,
the saliva samples from different individuals were pooled. The same was done for
blow. A series of experiments was set up to determine stability of testosterone in
both saliva and blow.
4.2.2. LC-MS assay
LC-MS was used to determine testosterone concentrations as described in
Chapter 3.
76 Chapter 4 – Testosterone Stability
4.2.3. Inhibitors
Preliminary analysis of spiked saliva and blow at room temperature (21 oC)
showed changes in testosterone concentrations. At the commencement of this
program a cocktail of inhibitors consisting of leupeptin, pepstatin A (Auspep,
Melbourne, Australia) and a mini-tab (Roche Diagnostics, Sydney, Australia) was
used to prevent the degradation of the hormones from oral bacteria (Dadgar and
Burnett 1995; Vickers et al. 2001). However, during the pilot study it was noted
that when spiking samples for stability analysis at 21 oC, hormone concentrations
were still changing even with the cocktail mix. Two alternative inhibitors were
trialled to combat any degradation that may be occurring, manganese chloride
(MnCl 2, Clyde Industries, NSW, Australia) and amoxycillin/potassium clavulanate
(Augmentin ®, SmithKline Beecham, Sydney, Australia).
In order to determine the most suitable concentration of MnCl 2 to be used, a
series of test experiments were run using 400 mM, 100 mM and 1 mM MnCl 2 with
a 5 ng/ml spike of testosterone in saliva. Testosterone concentration was
measured at time 0,1,2,3 and 6 hours.
4.2.4. Freeze-thaw experiments
Pooled saliva and pooled blow were spiked with either 50 ng/ml, 20 ng/ml or 1
ng/ml of testosterone (n = 5). Due to the difficulties associated with weighing
small sample volumes of crystalline testosterone the following method was used:
stock standard solutions (1 mg/ml) were made in 60% acetonitrile. Serial dilution
was used to obtain the desired concentrations of hormone required for spiking
and the acetonitrile was dried off under air. Dry standards were then reconstituted
in the equivalent volume of pooled saliva or blow. Samples were spiked and
stored at -80 oC between sample runs. Each spiked sample was measured after
77 Chapter 4 – Testosterone Stability
initial spiking and then after each thaw cycle, three freeze-thaw cycles were
measured in total. Samples were not extracted but were injected directly onto the
column.
4.2.5. Short-term storage
Samples were spiked with 50 ng/ml or 5 ng/ml of testosterone standard in order
® to determine which inhibitor, MnCl 2 or Augmentin , would be more suitable to
prevent degradation of testosterone in saliva and blow. Measurements at 21 oC
were made at time 0, 6, 9, 12, 15, and 18 hours after initial spiking. In addition to
testosterone and the internal standard, either 100 mM MnCl 2 or 100 µg/ml
Augmentin ® was added to each sample.
4.2.6. Long-term storage
From this room stability experiment, MnCl 2 was deemed to be the superior
inhibitor for testosterone and was used as the inhibitor for the -20 oC and -80 oC
experiments. After initial spiking, samples were stored at -20 oC and analysed at
weeks 1, 2, 3 and 4; and at weeks 4, 6, 8, 10 and 12 when stored at -80 oC.
All spiked samples were analysed according to the time schedules given until
they exhibited a significant change to the initial spiking concentration.
4.2.7 Statistical analysis
Analysis of variance (ANOVA) was used to determine any significant differences
in the freeze-thaw and stability studies. If the data did not meet the assumptions
of ANOVA, Mann-Whitney U or Kruskal-Wallis tests were used where
appropriate.
78 Chapter 4 – Testosterone Stability
4.3. Results
4.3.1. Inhibitors
During the initial validation of the methods for this study, saliva and blow spiked
with 50 ng/ml or 5 ng/ml of testosterone showed a marked increase in
testosterone concentration over an eight hour period. The cocktail inhibitor mix,
consisting of leupeptin, pepstatin A and a mini-tab, did not prevent the changes in
testosterone concentration at both high and low spiking levels (fig. 4.1).
Testosterone concentrations increased in saliva by 203 ± 55% (n = 5), and in
blow by 582 ± 66% (n = 5) after eight hours.
The concentration of 100 mM MnCl 2 was used for all stability experiments as it
had the best stabilising effect on testosterone concentrations (fig. 4.2).
o Comparative analysis of testosterone stability at 21 C using 100 mM MnCl 2 or
® 100 µg/ml Augmentin as inhibitors showed MnCl 2 to be the superior inhibitor for
testosterone in both saliva and blow (fig. 4.3).
79 Chapter 4 – Testosterone Stability
Figure 4.1 : a) Changes in 50 ng/ml spiked testosterone in bottlenose dolphin
saliva and blow after eight hours at 21 oC with first cocktail inhibitor used
(leupeptin, pepstatin A and mini-tab). b) Changes in 5 ng/ml spiked testosterone
in bottlenose dolphin saliva and blow after eight hours at 21 oC with first cocktail
inhibitor used (leupeptin, pepstatin A and mini-tab). Mean ± SE.
80 Chapter 4 – Testosterone Stability
a) 400
350
300
250
200
150
Testosteroneconcentration (ng/ml) 100
50
0 Saliva 0 1 2 3 4 5 6 7 8 Blow Hours
b) 40
35
30
25
20
15
10 Testosteroneconcentration (ng/ml)
5
0 Saliva 0 1 2 3 4 5 6 7 8 Blow Hours
81 Chapter 4 – Testosterone Stability
10
9
8
7
6
5
4
Testosterone concentration (ng/ml) 3
2
100 mM MnCl 2 1 400 mM MnCl 2 0 1 2 3 6 1 mM MnCl 2 Time (hours)
Figure 4.2: Differences in testosterone concentration in saliva over six hours,
following a 5 ng/ml spike of testosterone using different MnCl 2 concentrations
(400 mM, 100 mM and 1 mM). Mean ± SE. (Data points are slightly offset in
graph due to the software used).
82 Chapter 4 – Testosterone Stability
Figure 4.3: (a) Stability of saliva spiked with testosterone (5 ng/ml) at 21 oC with
® amoxycillin/potassium clavulanate (Augmentin ) and MnCl 2 over 18 hours. Mean
± one SE. (b) Stability of blow spiked with testosterone (50 ng/ml) at 21 oC with
® amoxycillin/potassium clavulanate (Augmentin ) and MnCl 2 over 18 hours. Mean
± SE. Data are not continuous; lines linking points are shown to allow for easy
interpretation of the observed trends. (Data points are slightly offset in graph due
to the software used).
83 Chapter 4 – Testosterone Stability
(a) 20
18
16
14
12
10
8 Testosterone concentration (ng/ml) 6
4
MnCl 2 2 ® Augmentin 0 2 4 6 8 10 12 14 18 Hours
(b)
140
120
100
80
60
40 Testosteroneconcentration (ng/ml)
20
0 MnCl 2 0 2 4 6 8 10 12 14 16 18 ® Augmentin Hours
84 Chapter 4 – Testosterone Stability
4.3.2. Freeze-thaw experiments
When saliva was spiked with 50 ng/ml testosterone there was no significant
change in testosterone concentration over the three freeze-thaw cycles (fig 4.4a).
Yet when blow was spiked with 50 ng/ml testosterone, concentrations increased
significantly (ANOVA: F = 221.6, df = 3, p <0.001) after each cycle of freeze-
thawing (fig. 4.4a). When saliva was spiked with 5 ng/ml testosterone,
concentrations decreased significantly (ANOVA: F = 13.4, df = 3, p < 0.001) with
each freeze-thaw cycle (fig. 4.4b). Yet when blow was spiked with 5 ng/ml
testosterone, concentrations increased significantly after the first thawing and
then decreased after subsequent thawing (ANOVA: F = 7.56, df = 3, p = 0.002;
fig 4.4b).
4.3.3. Short-term storage
4.3.3.1. Saliva at 21 oC
With no inhibitors added, testosterone concentrations in saliva samples at 21 oC
changed significantly within one hour if concentrations were low and within three
hours if concentrations were high (table 4.1). The presence of MnCl 2 or
® Augmentin slowed the rate of change (fig. 4.3). MnCl 2 proved to be the superior
inhibitor as significant changes did not occur until 15 hours for 5 ng/ml spiked
testosterone and at 18 hours for 50 ng/ml spiked testosterone (table 4.1).
Although Augmentin ® improved the stability of testosterone at 21 oC, significant
changes were observed at 18 hours for 50 ng/ml spiked testosterone and at 12
hours for 5 ng/ml spike testosterone (table 4.1, fig. 4.3).
85 Chapter 4 – Testosterone Stability
a) 300
280
260
240
220
200
180
160
140
120 Testosterone concentration (ng/ml) 100
80
60 Blow 40 Saliva Fresh Thaw 1 Thaw 2 Thaw 3
b) 18
16
14
12
10
8
Testosteroneconcentration (ng/ml) 6
4
Blow 2 Saliva Fresh Thaw 1 Thaw 2 Thaw 3
Figure 4.4 : a) Three cycles of freezing and thawing of saliva and blow with 50
ng/ml testosterone. b) Three cycles of freezing and thawing of saliva and blow
with 5 ng/ml testosterone. Mean ± SE.
86 Chapter 4 – Testosterone Stability
Table 4.1: Changes in spiked testosterone concentration in bottlenose dolphin
o ® saliva and blow at 21 C with no inhibitors or inhibitors (MnCl 2 or Augmentin )
added. Storage time is the time when changes were observed.
SALIVA Hormone Storage concentration Inhibitor Statistical test, statistic df p time (ng/ml) 50 No inhibitor 2 hours ANOVA F = 2.7 2 NS 50 No inhibitor 3 hours ANOVA F = 16.6 3 <0.001 5 No inhibitor 1 hour Mann-Whitney U: Z = 2.3 0.021
50 MnCl 2 15 hours ANOVA F = 2.2 4 NS 50 MnCl 2 18 hours ANOVA F = 7.8 5 <0.001 5 MnCl 2 15 hours K-W: chi-square = 2.6 4 NS ® 50 Augmentin 15 hours ANOVA F = 1.2 4 NS ® 50 Augmentin 18 hours ANOVA F = 6.3 5 0.005 ® 5 Augmentin 6 hours ANOVA F = 0.2 3 NS ® 5 Augmentin 12 hours ANOVA F = 7.6 3 0.004
BLOW Hormone Storage concentration Inhibitor Statistical test, statistic df p time (ng/ml) 50 No inhibitor 1 hour Mann-Whitney U: Z = -2.6 0.009 5 No inhibitor 3 hours ANOVA F = 0.9 3 NS 5 No inhibitor 4 hours ANOVA F = 5.5 4 0.003 50 MnCl 2 15 hours ANOVA F = 2.2 4 NS 50 MnCl 2 18 hours ANOVA F = 4.2 5 0.007 5 MnCl 2 9 hours ANOVA F = 2.7 3 0.08 5 MnCl 2 13 hours ANOVA F = 13.5 4 <0.001 ® 50 Augmentin 15 hours ANOVA F = 1.2 4 NS ® 50 Augmentin 18 hours ANOVA F = 6.3 5 <0.001 ® 5 Augmentin 9 hours K-W: chi-square = 4.0 3 NS ® 5 Augmentin 13 hours ANOVA F = 86.8 4 <0.001
87 Chapter 4 – Testosterone Stability
4.3.3.2. Blow at 21 oC
Blow spiked with testosterone without inhibitors exhibited significant changes in
testosterone concentrations at 21 oC. Changes occurred within one hour for 50
ng/ml testosterone and within 4 hours for 5 ng/ml testosterone (table 4.1). There
® was no difference between MnCl 2 and Augmentin in blow samples. Both
inhibitors stabilised spiked testosterone concentrations for 18 hours at 50 ng/ml
and for 13 hours at 5 ng/ml (table 4.1). However, the absolute increase in
testosterone concentration was higher for samples with Augmentin ® than those
with MnCl 2:
Augmentin ®
50ng/ml: t (0) hrs = 51.51 ng/ml 5ng/ml: t (0) hrs = 5.07 ng/ml
t (18) hrs = 100.55 ng/ml t (13) hrs = 11.09 ng/ml
MnCl 2
50ng/ml: t (0) hrs = 51.51 ng/ml 5 ng/ml: t (0) hrs = 5.10 ng/ml
t (18) hrs = 91.10 ng/ml t (13) hrs = 8.00 ng/ml)
Interestingly, at 21 oC testosterone concentrations in both saliva and blow
increased over a period of 12 to 18 hours before they began to decline. This was
observed with both 50 and 5 ng/ml testosterone concentrations.
4.3.4. Long-term storage
4.3.4.1. Saliva at -20 oC
Saliva samples spiked with 50 ng/ml testosterone showed a significant increase
(ANOVA: F = 34.7, df = 4, p < 0.001) after four weeks at -20 oC. Those samples
spiked with 5 ng/ml testosterone showed a highly significant decrease in
concentration after four weeks at -20 oC (ANOVA: F = 36.0, df = 4, p < 0.001).
88 Chapter 4 – Testosterone Stability
4.3.4.2. Blow at -20 oC
Blow samples spiked with 50 ng/ml or 5 ng/ml of testosterone showed no
significant changes in concentration after four weeks at -20 oC.
4.3.4.3. Saliva at -80 oC
When spiked with 50 ng/ml testosterone and stored at -80 oC saliva samples
showed a significant increase in testosterone concentration after ten weeks
(ANOVA: F = 3.8, df = 4, p = 0.02). Saliva samples spiked with 5 ng/ml
testosterone showed no significant change in testosterone concentration after
twelve weeks at -80 oC.
4.3.4.4. Blow at -80 oC
Blow samples stored at -80 oC showed no significant change in testosterone
concentration when spiked with 50 ng/ml for 12 weeks. Blow samples spiked with
5 ng/ml testosterone significantly decreased after six weeks at -80 oC storage
(ANOVA: F = 5.5, df = 3, p = 0.007). Testosterone concentrations in blow spiked
with 5 ng/ml were as follows: week 0 = 5.13 ± 1.29ng/ml, week 4 = 4.78 ± 0.78
ng/ml; week 6 = 3.07 ± 0.87 ng/ml; and week 8 = 4.07 ± 0.53 ng/ml (n = 5).
89 Chapter 4 – Testosterone Stability
4.4. Discussion
Testosterone was not stable in bottlenose dolphin saliva and blow samples.
Changes were observed in testosterone concentrations at a number of different
stages of sample storage. Average concentrations for both saliva and blow over
an eight hour period at room temperature (21 oC) were two to five-fold greater
than the original concentration. Although testosterone is thought to be stable at
room temperature (Chattoraj and Watts 1987; Vining and McGinley 1987; Lipson
and Ellison 1989), Granger et al . (2004) showed a 330.8% increase in
testosterone concentrations in human saliva over 4 weeks at 4 oC. This current
study showed similar results for testosterone in dolphin saliva at 21 oC (room
temperature). For blow samples kept at 21 oC with no inhibitors added,
testosterone concentrations increased over time regardless of the initial spiking
concentration.
All steroid hormones are derived from cholesterol (Norman and Litwack 1997)
and are involved in a complicated metabolic pathway (fig. 1.5). The exact
metabolic pathway of steroid hormones in bottlenose dolphins is unknown. Yet as
certain steroid hormones metabolise, their structure is subsequently modified to
other related hormones before further metabolism or catabolism occurs. The
increases in hormone concentration seen at room temperature in the present
study can be attributed to this complex system. It is believed that compounds
higher up in the metabolic pathway are degrading through testosterone and so
initially showing increases in concentration before decreasing. If there is no
biosynthesis of hormones in saliva or blow, once compounds higher up in the
pathway have degraded, no further increases in concentration should occur.
90 Chapter 4 – Testosterone Stability
When inhibitors were added the rate of change slowed but did not cease. Some
wildlife studies that use faecal samples have tested ethanol (Wasser et al. 1988;
Khan et al. 2002) and sodium chloride (Wasser et al. 1988) as inhibitors. Sodium
chloride was noted to alter extraction recovery (Wasser et al. 1988). Glacial
acetic acid and boric acid have been recommended as inhibitors for urine,
although no stability studies have been reported for them (Young and Bermes
1987). Sodium azide has been shown to stabilise testosterone in human saliva
(Vining and McGinley 1987; Lipson and Ellison 1989) but was not tested as an
inhibitor for this program due to its toxicity, particularly when it comes into contact
with water. This study investigated marine animals and samples were transported
by commercial airline so sodium azide was deemed unsuitable.
Manganese chloride was examined as an inhibitor for testosterone in this study
as it is known to affect testosterone steroidogenesis in rat Leydig cells (Cheng et
al. 2003). Cheng et al. (2003) showed that different concentrations of MnCl 2 had
differing affects on testosterone steroidogenesis. The present study showed that
a 100 mM concentration was required to stabilise testosterone in dolphin saliva
and blow for a suitable length of time (more than six hours). The long-term focus
of this program was to determine the feasibility of assaying reproductive hormone
concentrations in whale blow. A suitable time for testosterone stability was
deemed to be longer than ten hours. This takes into account samples being
collected in the morning in the field, and not being put into -20 oC until the end of
the research day. Augmentin ® was also tested as an inhibitor as it was thought
that bacteria in the mouth and lungs may have an influence on testosterone
concentrations. Bacterial and enzyme activity in urine and faeces are known to
have an influence on steroid hormones (Chattoraj and Watts 1987; Wasser et al.
1988; Millspaugh et al. 2002). In order to maximise the stabilising power of
91 Chapter 4 – Testosterone Stability
Augmentin® 100 µg/ml concentrations were used as an inhibitor. It was felt that
anything less than this would prove ineffective.
Saliva and blow samples spiked with 50 ng/ml testosterone and blow samples
spiked with 5 ng/ml testosterone showed no differences between the stabilising
® affects of MnCl 2 compared with Augmentin . MnCl 2 was the superior inhibitor
when saliva samples were spiked with 5 ng/ml testosterone. In addition, changes
in the absolute testosterone concentrations in blow samples were higher with
® Augmentin than with MnCl 2. Therefore, MnCl 2 was deemed the superior inhibitor
and used for all subsequent long-term storage experiments.
Interestingly, at 21 oC testosterone concentrations in both saliva and blow
increased over a period of 12 to 18 hours before they began to decline. Yet at -
20 oC, the concentration of testosterone in saliva increased over time after spiking
with 50 ng/ml testosterone whilst it decreased after spiking with 5 ng/ml
testosterone. After six weeks at -80 oC the blow testosterone concentration was
3.07 ± 0.87 ng/ml (a 40.16% decrease from the original concentration). Yet the
concentration of the same sample after eight weeks at -80 oC was 4.07 ± 0.53
ng/ml. The causes of this decrease and then increase of concentration are
unknown. Further analysis of testosterone and its precursors and metabolites is
required. This will determine if the increases and decreases in concentration are
due to some form of metabolism.
Other studies have found no change in testosterone concentrations when
samples are stored at -20 oC and -80 oC but those studies did not spike the
samples with a known testosterone concentration, so it is unknown if there was
hormone degradation when the initial sample was assayed (Luisi and Franchi
92 Chapter 4 – Testosterone Stability
1984; Banerjee et al. 1985; Granger et al. 1999b; Granger et al. 2004). In
addition, those studies assayed samples stored at lower temperatures for six to
twelve months. As seen at the lower temperature analyses here, testosterone
concentrations fluctuate over very short periods of time. Hormone concentrations
decreased and then increased within the initial six month period. We assayed the
samples every week at -20 oC and initially at four weeks and every two weeks
thereafter when stored at -80 oC.
Further, small fluctuations in hormone concentration when samples are spiked
with high concentrations of testosterone give results which may be explained by
simple assay variation. The same fluctuations in hormone concentrations in
samples that were spiked with low concentrations of testosterone showed
significant changes. Vining and McGinley (1987) found that salivary testosterone
concentrations in men reflected free hormone concentrations in plasma. However
in women salivary testosterone concentrations varied widely and were often
considerably higher than free plasma concentrations. Vining and McGinley (1987)
did not report whether the fluctuations seen in male salivary testosterone and
female salivary testosterone were similar. Small changes in concentration in low
concentration samples can affect the apparent stability of a sample. This may
explain why saliva and blow samples with low testosterone concentrations appear
to be less stable than those samples with high concentrations.
The freeze-thaw experiments showed changes in hormone concentrations with
each freeze-thawing cycle. Repetitive thawing is known to cause error with
steroid hormones (Chattoraj and Watts 1987). For steroid hormones that are
bound to proteins in a matrix, thawing may break these bonds causing an
increase in “free” hormone concentrations. If the assay detects “free” hormone
concentrations there will be an increase with each freeze-thaw cycle.
93 Chapter 4 – Testosterone Stability
Alternatively, if the assay is designed to measure bound hormones, then breaking
the hormone-protein bond would be reflected in a decrease in concentration with
each freeze-thaw cycle. Caution should be taken to ensure that freeze-thawing is
kept to a minimum with non-invasively and invasively collected samples prior to
thawing for extraction and assay.
This study not only assessed short-term testosterone stability but long-term
stability at -20 oC and -80 oC. This is because many wildlife studies that use non-
invasive samples (Kirkpatrick et al. 1988; Wasser et al. 1988; Schoech and Lipar
1998; Goymann et al. 1999; Millspaugh et al. 2002; Terio et al. 2002; Monfort
2003) store samples at -20 oC or -80 oC prior to assay. Unfortunately the length of
storage is not commonly mentioned in the literature. In addition, there is very little
comment on the use of inhibitors to slow or cease changes in testosterone
concentrations.
94 Chapter 4 – Testosterone Stability
4.5. Conclusion
Few studies (Khan et al. 2002; Lynch et al. 2003; Granger et al. 2004) have
looked at the changes of steroid hormone concentrations at varying temperatures
for varying lengths of time, regardless of the biological matrix. It is now apparent
that each hormone in every matrix from different species has the potential to
change. Comprehensive stability studies, which include spiking the biological
matrix with the hormone to be analysed, should be conducted at the different
storage temperatures and lengths of storage. The complexity of the metabolic
pathway of steroid hormones can complicate the manner in which hormone
concentrations change over time. Concentrations can increase before they
decrease and have the potential to increase again. Simply measuring the
endogenous hormone concentrations for stability studies may not be indicative of
hormone stability as it is unknown what stage of degradation is being measured.
As non-invasive hormone analysis is becoming more widely used in wildlife
studies (Monfort 2003) it is imperative that any changes in the hormone
concentrations (due to: collection substrate, storage time and temperature,
freezing and thawing, and extraction) are understood for the species studied and
the matrix used. Unless comprehensive stability studies have been conducted
caution should be used when formulating biological conclusions from a program
that is based on non-invasive sampling.
95
CHAPTER 5
Female Reproductive Hormones
5.1. Introduction
Progesterone and oestradiol are the two most important female reproductive
hormones. Progesterone is produced by the ovary (corpus luteum) and the
placenta and function to prepare and maintain the body for pregnancy (Norman
and Litwack 1997; Squires 2003). The major oestrogens are oestradiol, oestrone
and oestriol. Oestrogens are produced by the female’s ovaries, the placenta and
the foetus during pregnancy (Norman and Litwack 1997; Squires 2003). Under
some circumstances the testes of males can produce significant amounts of
oestradiol. For purposes of this study, progesterone, oestradiol and oestrone are
discussed due to their major involvement in the oestrous cycle and pregnancy.
5.1.1. Oestrous cycle
The oestrous cycle can be divided into a number of phases: follicular, luteal and
anoestrous (fig. 5.1) (Noakes 2001). The primary events of the follicular phase
are the growth and development of the follicle and the maturation of the uterine
endometrium. The primary events of the luteal phase are the growth and
development of the corpus luteum (CL), and in the absence of pregnancy,
regression of the CL (Squires 2003). Anoestrus is the prolonged period of sexual
rest where there is minimal follicular growth and the corpora lutea, although
identifiable is present, are non-functional. Anoestrus is generally confined to
seasonal breeders (Squires 2003), although it also occurs post-partum and
during lactation in many species.
96 Chapter 5 – Female Reproductive Hormones
During the follicular phase the dominant follicle(s) produce(s) oestradiol. This acts
by positive feedback on the pituitary to increase luteinising hormone (LH)
production (fig. 1.6). The ovarian follicle consists of a large round oocyte that is
surrounded by follicular cells and when mature, follicular fluid that increases in
volume prior to ovulation. The surge of LH causes the mature follicle to rupture
releasing the oocyte (ovulation) (Norman and Litwack 1997; Squires 2003). After
ovulation the granulosa cells of the follicle give rise to lutein cells which are
responsible for the formation of the CL (Noakes 2001). The CL remains during
most of the cycle. The lutein cells produce progesterone which inhibits
gonadotropin releasing hormone (GnRH) by the hypothalamus which in turn
decreases the release of LH by the pituitary (fig. 1.6) (Squires 2003).
During the luteal phase of the cycle, the CL grows quickly and produces
progesterone, which increases rapidly at the beginning of the cycle (Norman and
Litwack 1997). Progesterone concentrations remain high until luteolysis, which
occurs 10 - 20 days after ovulation depending on the species and in the absence
of pregnancy or pseudopregnancy. If fertilisation does occur then the embryo
implants and generally produces proteins which prevent the regression of the CL
(Squires 2003). Progesterone is required for the maintenance of pregnancy and
is produced by the ovary, the placenta and the adrenal gland (Norman and
Litwack 1997). The source of progesterone and the length of gestation is species-
dependent.
As the CL regresses it produces a small white ovarian scar known as the corpus
albicans. If the released ovum is fertilised the corpus luteum continues to grow for
the first three months of pregnancy (Norman and Litwack 1997). At three months,
in humans, the corpus luteum regresses and leaves a corpus albicans.
97 Chapter 5 – Female Reproductive Hormones
Pregnancy if fertilisation occurs
Corpus luteum regression Luteal phase
Follicular
phase
Anoestrous phase if
Oestrous seasonal breeder
Ovulation
Figure 5.1: Outline of the oestrous cycle, this cycle is interrupted by periods of
pregnancy and lactation and anoestrus in the case of seasonal breeders
(modified from Squires 2003).
98 Chapter 5 – Female Reproductive Hormones
In other species, such as goats and horses, the CL is present throughout
pregnancy (Squires 2003). So it is difficult to determine if the scars on an ovary
are due to an ovulation resulting in pregnancy or an ovulation with no fertilisation.
This is particularly important if histological examination is being used to determine
the number of ovulations per cycle for a species.
In many species the onset of oestrus is determined by the acceptance of a male.
As the onset and end of oestrus tend to be the only measurable points of the
cycle it is used for determining cycle length (Noakes 2001). Cycle length and
frequency vary among species, in domestic animals the cycle lasts for 21 days in
cattle, pigs and goats (Squires 2003); 19 to 25 days in horses (Squires 2003) and
16 to 17 days in sheep (Squires 2003). In wildlife the cycle length is not as
intensively studied but tends to be longer than domestic animals. Cycle length in
chimpanzees is 25 to 84 days (Tutin and McGinnis 1981); in river buffalo 21 days
(Ahmad 2001); in Asian elephants 52 to 64 days (Brown et al. 1991); in Nile
hippopotami 29 to 40 days (Graham et al. 2002); and in killer whales 41 days
(Robeck et al. 1993).
Seasonal differences in the anoestrus of many species are dependent on
environmental conditions (Squires 2003). During the anoestrous period, low
concentrations of oestradiol inhibit the secretion of LH by the pituitary. In some
species follicular development continues during anoestrus but LH concentrations
are insufficient for maturation of the follicle. In other species follicular
development ceases entirely during anoestrus (Squires 2003).
Female steroid hormone production is more complex than male testosterone
production. Female hormones are released to optimise reproductive capacity and
99 Chapter 5 – Female Reproductive Hormones
a variety of hormones, including progestins and oestrogens, are involved.
Changes in progesterone concentration during human reproductive cycles have
been well documented and concentrations have been determined using a
number of biological matrices, such as plasma (Meulenberg and Hofman 1989;
Brown et al. 1991; Wren et al. 2003), urine (Czekala et al. 1988; Li et al. 2001;
O'Connor et al. 2003), and saliva (Pietraszek and Atkinson 1994; Lu et al. 1997;
Laine and Ojanotko 1999; Lu et al. 1999; Groschl et al. 2001; Graham et al.
2002; Ishikawa et al. 2002) .
More recently, the oestrous cycle in a number of wildlife species has been
described. Changes in progesterone concentrations have been determined by
RIA in plasma (Kjeld et al. 1992; Pietraszek and Atkinson 1994), urine (Czekala
et al. 1988; Walker et al. 1988; Lasley and Kirkpatrick 1991), faeces (Kirkpatrick
et al. 1988; Graham et al. 2002) and saliva (Pietraszek and Atkinson 1994;
Atkinson et al. 1999). As progesterone plays a vital role during ovulation and
throughout pregnancy it is used by conservation management agencies and
captive institutions to monitor pregnancy and oestrous cycles. Knowledge of
baseline progesterone concentrations and the naturally occurring changes that
make up the oestrous cycle are important as unusual events or fluctuations can
lead to a better understanding of reproductive dysfunction. This is particularly
important in captive animal and endangered species management.
The reproductive cycle of a bottlenose dolphin can be described as polyoestrous
to seasonally polyoestrous (Kirby and Ridgway 1984; Schroeder 1990a). Periods
of anoestrus have been documented in this species (Brook 1997). Most cycling,
however, appears to occur from spring until autumn, although calves are born all
year round (Robeck et al. 2001). Detailed analysis of the bottlenose dolphin’s
100 Chapter 5 – Female Reproductive Hormones
cycle through blood collection has not been achieved due to ethical restrictions
associated with multiple daily blood sampling (Robeck et al. 1994). Weekly
samples collected over a 3-year period showed that bottlenose dolphins are
spontaneous ovulators and have a 21 to 42 day oestrous cycle (Kirby and
Ridgway 1984; Schroeder 1990a). Pregnancy is indicated by oestrogen
concentrations remaining elevated and progesterone concentrations higher than
3 ng/ml for a period of longer than six weeks (Schroeder 1990a).
The aims of this chapter were to:
• determine progesterone, oestradiol and oestrone concentrations in
female bottlenose dolphin saliva and blow samples using LC-MS; and
• describe differences in hormone concentrations for females at different
stages of the oestrous cycle.
5.2. Materials and Methods
5.2.1. Chemicals and solutions
All chemical reagents were of HPLC grade: acetonitrile (BioLab Scientific,
Melbourne, VIC, Australia), formic acid, 90%, 23.0M (Ajax Chemicals, Clyde
Industries, Australia), water was purified by a Milli-Q system (Millipore ®, Sydney,
NSW, Australia) and nitrogen gas (5.0, BOC, Sydney, NSW, Australia) was of
ultra-high purity.
Crystalline progesterone (Fluka, Switzerland) was >97% purity as tested by
HPLC by the manufacturer. Crystalline oestrone (Sigma-Aldrich, Sydney,
Australia) was of ultra high purity (99%), tested by HPLC by the manufacturer.
Crystalline β-oestradiol (Sigma-Aldrich, Sydney, Australia) was of ultra high purity
101 Chapter 5 – Female Reproductive Hormones
(98%), tested by the manufacturer. Stock solutions were made fortnightly by
dissolving crystalline compounds in 60% acetonitrile (1 mg/ml) and stored at 4 oC
away from light. From the stock solutions, working concentrations of
progesterone at 10 and 1 µg/ml and 100 ng/ml, oestradiol at 100 and 10 µg/ml
and oestrone at 100, 10 and 1 µg/ml, were prepared volumetrically by serial
dilution with 60% acetonitrile. Stock solutions of the internal standard (Fmoc-L-
Gln(Trt)-OH) were prepared in 60% acetonitrile at a concentration of 10 µg/ml.
5.2.2. LC Analysis
The LC elution conditions for the female hormone analysis were as follows:
mobile phase A, 0.5% formic acid and mobile phase B, 90% acetonitrile, 0.5%
formic acid, at isocratic conditions of 50% B and a flow rate of 0.2 ml/min. The
HPLC column was equilibrated for 30 mins prior to a series of runs and
maintained its performance for over 500 injections.
5.2.3 LC-MS methods
LC-MS analysis and instrumental parameters used for MS analysis have been
described in Chapter 2. Direct infusions of 5 µl of 100 µg/ml of standard were
used to determine the m/z of progesterone, oestradiol and oestrone. Calibration
curves were determined using serial dilutions of stock standard solutions. Data
acquisition for all hormones was 12 mins.
5.2.4 Sample collection and preparation
Saliva and blow samples were collected from eight female bottlenose dolphins,
during the peak of the austral breeding season (February in Australia). There
were seven adults and one sub-adult (Nila). Of the seven adults, two were
lactating (Moki and Stormy); one was pregnant (Ava); one was possibly
102 Chapter 5 – Female Reproductive Hormones
reproductively senescent at 36 years old (Splash) and the remaining three were
believed to be cycling (Gemma, Scooter and Squeak).
Saliva and blow collection for the female dolphins was the same as for the males.
Nylon stocking was used in place of cotton gauze as it was more inert (Chapter
7). The stocking was pre-cleaned by sonicating in 100% acetonitrile for 15 mins
and then sonicating in Milli-Q water for 15 mins, changing the water at 5 min
intervals.
200 µl of inhibitor mix (100 mM MnCl 2 + 100 µg/ml amoxicillin/potassium
clavulanate) was added to each sample to prevent degradation of progesterone
in saliva samples (Chapter 6). For blow samples 500 µl of inhibitor mix was
added and the tube shaken vigorously to ensure that all blow mucus was
collected in the solution. Saliva samples were stored in cryo-storage tubes and
blow samples in 50 ml polypropylene tubes. Samples collected at the dolphin’s
location were stored at -20 oC overnight, transported on dry ice and stored for two
weeks at -80 oC prior to analysis.
Samples were extracted using the method previously developed to determine
testosterone concentrations in male dolphin saliva and blow (Chapter 3).
5.2.5 Validation
The method was validated for specificity, linearity, accuracy, precision, limits of
quantification and detection, and stability. Calibration curves were calculated for:
1. progesterone in 60% acetonitrile at 50, 20, 10, 5, 1, 0.5 and 0.2 ng/ml;
2. oestradiol in 60% acetonitrile at 200, 100, 50 and 20 ng/ml; and
3. oestrone in 60% acetonitrile at 100, 50, 20, 10 and 5 ng/ml.
103 Chapter 5 – Female Reproductive Hormones
Four curves were run on four separate non-consecutive days. The calibration
curves were calculated using an unweighted least-squares regression method
(Nelson et al. 2004).
For progesterone, three different runs of six repeats on separate non-consecutive
days were performed to evaluate intra- and inter-batch accuracy and precision,
using 0.5, 5 and 50 ng/ml spiked saliva and blow. Ruggedness of the method
was ascertained by assaying stock solutions at two different concentrations (50
ng/ml and 0.5 ng/ml) using the same method but with two different 300Å C8
columns (Lot #: 02110886-1 and 02110885-1). Recovery of the SPE was
determined by spiking samples with high (50 ng/ml) and low (0.5 ng/ml)
concentrations of progesterone. Samples were separated into 2 x 100 µl aliquots.
A 100 µl aliquot was used for the pre-extraction assay (n = 6) and the other was
extracted and reconstituted in 100 µl 60% acetonitrile, and used for the post-
extraction assay (n = 6).
Saliva and blow samples were spiked with 50ng/ml or 0.5 ng/ml progesterone
and assessed for stability at 21 oC (room temperature), -20 oC and -80 oC. Stability
experiments and results are presented in Chapter 6.
5.2.6 Statistical Analysis
Pearsons r correlation was used to assess linearity of the calibration curves.
104 Chapter 5 – Female Reproductive Hormones
5.3. Results
5.3.1 Progesterone validation
The m/z for progesterone was 315.20 [M + H] + (fig. 5.2) and a second m/z 356.25
+ [M + H + CH 2CN] was the acetylated adduct of progesterone and was used to
further confirm the retention time (RT) (Bressolle et al. 1996). The acetylated
adduct of 356.25 was present in both biological samples and progesterone
standard with a ratio of approximately 70 % (fig. 5.2) compared to the m/z 315.20
peak. The Fmoc-Glutamine m/z was 609.15 [M – H] +.
The calibration curve equation for progesterone (0.2 - 50 ng/ml) was y = 0.076x –
0.0123 and was linear (r 2 = 0.995; r = 0.997; p < 0.001). The residuals were
normally distributed and centred around zero. The LOD (S/N 3:1, ± 5% RT) was
0.1 ng/ml, and the LOQ (S/N 5:1, ± 5% RT) was 0.2 ng/ml. RT for progesterone
was 5.0 mins and the internal standard was 8.3 mins (fig. 5.3). RTs for column A
(Lot # 02110886-1) and B (Lot # 02110885-1) were 4.9 mins and 5.0 mins,
respectively. The RSD between the results of the two columns was 13.2% (n =
12) at 0.5 ng/ml and 8.3% (n = 12) at 50 ng/ml. Intra and inter-batch accuracy
and precision of the method are given in Table 5.1.
SPE recovery of 0.5 ng/ml progesterone in saliva was 96.9 ± 0.1% and for 50
ng/ml was 89.2 ± 3.8%. Recovery of 0.5 ng/ml progesterone in blow was 98.7 ±
0.2 % and for 50 ng /ml, 87.7 ± 5.2%. Recovery of the internal standard in saliva
was 74.0 ± 3.2% and in blow was 76.3 ± 3.4%.
105 Chapter 5 – Female Reproductive Hormones
Int. 315.20
[M + H] + 500e3 356.25 + [M + H + CH 2CN]
250e3
224.05 151.10 279.55 208.05 333.20 171.20 239.05259.10 297.25 330.25 365.05395.80 408.70 427.15 0e3 273.10 378.45 449.40 463.10 481.05 497.15 150 175 200 225 250 275 300 325 350 375 400 425 450 475 m/z
Figure 5.2: Determination of progesterone m/z by direct infusion into the mass
spectrometer (5 µl injection of 100 µg/ml in 60% acetonitrile), progesterone ( m/ z
315.20, 100% relative intensity), acetylated adduct ( m/z 356.25, approximately
70% relative intensity).
106 Chapter 5 – Female Reproductive Hormones
Int. 1:315.30(1.00) 2:356.25(1.00) 3:609.15(1.00) IS 60e3
50e3 Progesterone
40e3
30e3
20e3
10e3
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 min
Figure 5.3: RT of progesterone 5.0 mins (5 ng) and the internal standard (IS)
Fmoc-Gln 8.3 mins (5 ng). HPLC conditions: 50% isocratic B, mobile phase A
0.5% formic acid, mobile phase B 0.5% formic acid 90% acetonitrile, 0.2 ml/min.
107 Chapter 5 – Female Reproductive Hormones
Table 5.1: Intra- and inter-batch precision and accuracy for progesterone in
dolphin saliva and blow.
Intra-batch Sample Concentration Mean Precision Accuracy n type (ng/ml) (ng/ml) (RSD, %) (RE, %) 0.5 6 0.5 3.8% 10.4% Saliva 5 6 4.9 11.4% -0.1% 50 6 49.3 1.4% -1.5% 0.5 6 0.5 7.0% -6.4% Blow 5 6 5.8 0.1% 0.2% 50 6 53.3 1.2% 6.6%
Inter-batch Sample Concentration Mean Precision Accuracy type (ng/ml) n (ng/ml) (RSD, %) (RE, %) 0.5 18 0.5 10.8% 1.3% Saliva 5 18 4.8 9.5% -4.4% 50 18 50.7 5.7% 1.4% 0.5 18 0.5 9.1% -3.4% Blow 5 18 4.9 0.2% -0.1% 50 18 53.1 3.9% 6.2%
108 Chapter 5 – Female Reproductive Hormones
5.3.2. Oestradiol validation
The m/z for oestradiol was 255.20 [M - OH] +, MW = 272.39 (fig. 5.4a). The
calibration curve for oestradiol (20 – 200 ng/ml) was y = 3524x – 15688 and was
2 linear (r = 0.997; r = 0.998; p < 0.001). The residuals were normally distributed
and centred around zero. For oestradiol the LOD was (S/N 3:1, ± 5% RT) was 10
ng/ml, and the LOQ (S/N 5:1, ± 5% RT) was 20 ng/ml. RT for oestradiol was 3.60
mins (fig. 5.5a).
5.3.3. Oestrone validation
The m/z for oestrone was 271.25 [M + H] +, MW = 270.37, (fig. 5.4b). The
calibration curve equation for oestrone (5 - 100 ng/ml) was y = 8157x – 20616
and was linear (r 2 = 0.996; r = 0.998; p < 0.001). The residuals were centred
around zero and normally distributed. The LOD for oestrone (S/N 3:1, ± 5% RT)
was 2 ng/ml, and the LOQ (S/N 5:1, ± 5% RT) was 5 ng/ml. The RT for oestrone
was 3.95 mins (fig. 5.5b).
109 Chapter 5 – Female Reproductive Hormones
a) Int. 255 1750e3
1500e3 + [M - OH] 279 1250e3 151 190 1000e3
750e3 296 500e3 217 197 305 223 250e3 173 321 349 245 271 332 373 379 0e3 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 m/z
b) Int. 151
2500e3 271 2000e3 [M + H] + 1500e3 305 223 279 190 1000e3 330 213 500e3 237 261 195 297 332 349 165 370 376 0e3 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 m/z
Figure 5.4: a) Determination of oestradiol m/z by direct infusion into the MS (5 µl
injection of 100 µg/ml in 60% acetonitrile), oestradiol (m/z 255.20, 100% relative
intensity). b) Determination of oestrone m/z by direct infusion into the MS (5 µl
injection of 100 µg/ml in 60% acetonitrile), oestrone ( m/z 271.25, 100% relative
intensity).
110 Chapter 5 – Female Reproductive Hormones
a) Int. 255.20(1.00) 273.20(4.89) 700e3
600e3 Oestradiol
500e3
400e3
300e3
200e3
100e3
0 1 2 3 4 5 6 7 8 9 10 11 12 min
b) Int. 271.20(1.00)
1000e3 Oestrone 750e3
500e3
250e3
0 1 2 3 4 5 6 7 8 9 10 11 12 min
Figure 5.5: RTs of a) oestradiol (3.60 mins) and b) oestrone (3.95mins), using a
50% isocratic B, mobile phase A 0.5% formic acid, mobile phase B 0.5% formic
acid 90% acetonitrile, 0.2 ml/min.
111 Chapter 5 – Female Reproductive Hormones
5.3.4 Dolphin samples (endogenous progesterone concentrations)
Progesterone was quantified in the pregnant female dolphin (Ava) in both saliva
and blow samples (fig. 5.6). Salivary progesterone concentration was 14.9 ± 0.7
ng/ml (n = 3) and in blow was 30.9 ± 16.2 ng/ml (n = 3).
Progesterone peaks were qualified in five of the eight remaining dolphins. Four of
these animals were adults and one was the sub-adult (Nila). The progesterone
peak height in the four adults was greater than the sub-adult female, but none
were quantifiable (i.e. S/N ratio < 5:1, fig 5.7). Three of the adult animals were the
females that were cycling, Gemma, Scooter and Squeak. The last adult was a
lactating female (Stormy). No progesterone peaks were observed in the oldest
adult female, Splash, or in the other lactating female, Moki.
112 Chapter 5 – Female Reproductive Hormones
a).
Int. 1:315.30(1.00) 14000 Progesterone 13000
12000
11000
10000
9000
8000
7000
6000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
b). Int. TIC(1.00) 315.30(1.00) 15000
12500 Progesterone 10000
7500
5000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.6: a) MS chromatogram of endogenous progesterone (15.4 ng/ml) in
pregnant dolphin (Ava) saliva (arrowed), ( m/z 315.30, RT = 5.0 mins). b) MS
chromatogram of endogenous progesterone (19.5 ng/ml) in pregnant dolphin
(Ava) blow (arrowed).
113 Chapter 5 – Female Reproductive Hormones
a)
Int. TIC(1.00) 315.30(1.00) 7500
7000 Progesterone 6500
6000
5500
5000
4500
4000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
b) Int. TIC(1.00) 315.30(1.00)
11000
10000
9000 Progesterone
8000
7000 6000
5000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.7: a) MS chromatogram of endogenous progesterone in a sub-adult
female dolphin (Nila) saliva (arrowed), ( m/z 315.30, RT = 5.0 mins), peak too
small to quantify. b) MS chromatogram of endogenous progesterone in a sub-
adult female dolphin (Nila) blow (arrowed) peak too small to quantify.
114 Chapter 5 – Female Reproductive Hormones
5.4. Discussion
The LOD for oestradiol and oestrone with this LC-MS method was 10 ng/ml and 2
ng/ml, respectively. As the LOD was relatively high it was felt that the sensitivity
would not be low enough to determine these hormones in dolphin saliva and
blow. This is because plasma oestrogen concentrations in non-cycling bottlenose
dolphins are 25 pg/ml (Sawyer-Steffan et al. 1983). The LC-MS method was only
fully validated for determining progesterone concentrations in saliva and blow.
The progesterone method developed here can be used to determine pregnancy
in bottlenose dolphins using saliva or blow samples. Currently pregnancy is
determined in captive dolphins when plasma progesterone concentrations are
greater than 3 ng/ml (Schroeder 1990a) or through ultrasonography (Williamson
et al. 1990). In most wild studies it is impossible to determine pregnancy and so
reproductive rates are determined by the presence of a calf (Whitehead and
Mann 2000). As seen with testosterone in male dolphins (Chapter 3), the
concentration of progesterone in the blow sample was greater than in saliva. The
reasons for this are currently unknown and further analysis of the composition of
blow is warranted.
Unfortunately, only the presence or absence of progesterone can be determined
in samples from cycling females as the developed method did not achieve the
required sensitivity for determining precise concentrations. The presence of
progesterone in saliva and blow samples shows promise for this method. Further
analysis using tandem mass spectrometry should lower the limit of detection for
progesterone improving the sensitivity of the method.
115 Chapter 5 – Female Reproductive Hormones
The progesterone peak in adult cycling females was higher than that observed in
the sub-adult. The sub-adult female (Nila) was eight years old and had only
recently reached puberty. Bottlenose dolphins are sexually mature at five to
seven years of age (Schroeder 1990b) and reach reproductive maturity at 9.5 to
11 years (Cockcroft and Ross 1990). It is important to distinguish between sexual
maturity (puberty) and reproductive maturity. Reproductive maturity is when a
female is cycling regularly and has reached full adult size (Schroeder 1990b).
Further, it has been theorised that bottlenose dolphins may exhibit adolescent
sterility (Whitehead and Mann 2000). That is, females will begin cycling without
ovulating or ovulate without conceiving. Evidence of female adolescent sterility
has been observed in other dolphin species, such as killer whales and common
dolphins. In many mammals the first oestrous cycle tends to be short and ‘silent’
i.e. the animal does not exhibit open signs of oestrous, but it does have
increasing concentrations of progesterone (Noakes 2001). Lower detection limits
of the method and longitudinal sample collection will indicate whether Nila is
cycling regularly.
The complete lack of progesterone in the old female (Splash) was to be expected
as she was over 36 years old. Duffield et al. (1999) showed that female
bottlenose dolphins have slowing reproductive rates from mid-thirties onwards.
Bottlenose dolphins are believed to live as long as 45 years (Whitehead and
Mann 2000). Studies on short-finned pilot whales (Marsh and Kasuya 1984) and
killer whales (Olesiuk et al. 1990) show clear evidence of menopause but data of
reproductive senescence in bottlenose dolphins are inconclusive (Marsh and
Kasuya 1984).
116 Chapter 5 – Female Reproductive Hormones
Interestingly, one of the lactating females (Stormy) showed the presence of
progesterone and the other female (Moki) did not. Suckling stimuli generally
suppresses the secretion of LH in lactating animals, which prevents normal
follicular growth and ovulation (McNeilly 1988). However this is not the case with
bottlenose dolphins. Calves are generally weaned during the first ten months of
their mother’s next pregnancy (Mann et al. 2000) indicating that females begin
cycling while lactating. As the calving interval for females is between three and
six years (Connor et al. 1996) the difference between the two lactating females in
the present study was likely due to individual variability. The calves of both
females were the same age and so theoretically they should have exhibited
similar concentrations of reproductive hormones.
High variability was observed in the concentrations of female reproductive
hormones in blow samples and this could be due to dilution from seawater. Every
effort was made to ensure that seawater did not contaminate saliva and blow
samples. However, when a dolphin surfaces to breathe, seawater sometimes
collects on top of the blowhole. Problems with dilution can be reduced by using
an endogenous standard to standardise sample concentrations. One possible
standard is creatinine which is used with urinary hormonal assays (Berman et al.
1980; Lasley and Kirkpatrick 1991). Creatinine is a by-product of endogenous
muscle metabolism and is excreted in urine in a constant fashion (Ruckebusch et
al. 1991). At this time, it is not known whether creatinine is present in the blow
samples of cetaceans. If creatinine proves to be an impractical internal standard,
then the concentration of specific trace minerals found in seawater but not in blow
could be used to estimate the degree of dilution. Further work is required to
determine a suitable comparative dilution compound for dolphin saliva and blow
samples to ensure that seawater dilution is not an issue.
117 Chapter 5 – Female Reproductive Hormones
5.5. Conclusion
A replicable and accurate method for determining pregnancy in bottlenose
dolphins has been developed using non-invasively collected saliva and blow
samples. It is currently unsuitable for daily monitoring of oestrous cycles or for
determining sexual maturity in dolphins. This is due to an inadequate limit of
detection for progestins and oestrogens in the acquired samples from a single
quadropole LC-MS. Recently, a comparative study between GC-MS and LC-MS
found that, due to the complexity of progestins and oestrogens, LC-MS-MS is the
method of choice for analysis of these compounds (Diaz-Cruz et al. 2003).
Unfortunately, there was no access to a LC-MS-MS for the present program.
Further development of the methods described here using LC-MS-MS may
improve the assay sensitivity so that dolphin oestrous cycles may be monitored
using saliva and blow samples.
118
CHAPTER 6
Progesterone Stability
6.1. Introduction
Methods of determining human salivary progesterone concentrations used to
describe the female reproductive cycle were developed over 25 years ago
(Walker et al. 1979; Connor et al. 1982; Metcalf et al. 1984). Since that time
salivary progesterone has been assayed frequently in humans (Wang and
Knyba 1985; Ellison et al. 1986; Meulenberg and Hofman 1989; Ellison 1993;
Lu et al. 1999; Lu et al. 1999; Gröschl et al. 2001; Ishikawa et al. 2002;
Stikkelbroeck et al. 2003; Schultheiss et al. 2004). Salivary progesterone
concentration has been determined also in animals such as false killer whales
(Atkinson et al. 1999); monk seals (Pietraszek and Atkinson 1994);
Californian sea lions (Iwata et al. 2003); and capuchin monkeys (DiGiano et
al. 1992).
Although salivary progesterone is used commonly there seems to be a lack of
consistency in the literature in reference to the stability of progesterone in
saliva samples. Early literature gives varying results for progesterone stability.
It has been considered stable in human saliva samples at room temperature
for 48 hours (Riad-Fahmy et al. 1982), for 72 hours (Banerjee et al. 1985), for
up to six weeks (Ellison et al. 1986), and for up to six months (Lipson and
Ellison 1989). Progesterone concentrations in saliva samples have been also
reported to decrease over a three week period at room temperature (Gröschl
et al. 2001).
119 Chapter 6 – Progesterone Stability
Some of this variability in stability of salivary progesterone concentration comes
from the use of different inhibitors to prevent degradation, such as sodium azide
(Lipson and Ellison 1989; Gröschl et al. 2001) or trifluoro-acetic acid (TFA)
(Gröschl et al. 2001). Different inhibitors stabilise progesterone for varying
lengths of time. Other variability may come from the variety of assay techniques
used in different laboratories. Hagen et al. (2003) showed that there was a large
amount of variability between different laboratories using the same
immunoassays. Inconsistent progesterone results were obtained from the same
saliva samples when sent to different laboratories (Hagen et al. 2003). In
addition, Banerjee et al. (1985) showed that storing saliva samples in
polyethylene, polypropylene or glass vials could result in differing progesterone
concentrations. Polyethylene vials appear to absorb approximately 30% of
progesterone within three days of being stored at room temperature (Banerjee et
al. 1985).
In light of these inconsistencies in the literature about the stability of human
salivary hormones, a comprehensive study was developed on progesterone
stability in bottlenose dolphin saliva and blow. In addition, the testosterone
stability experiments that were conducted using dolphin saliva and blow (Chapter
4) highlight the need to determine hormone stability with each new hormone in
different matrices.
The aim of the study presented in this chapter was to:
• determine the stability of progesterone in dolphin saliva and blow; and
• determine optimum storage conditions (including temperature and
inhibitors) for dolphin saliva and blow samples.
120 Chapter 6 – Progesterone Stability
6.2. Materials and Methods
6.2.1. Sample Preparation
Saliva and blow were collected from six female bottlenose dolphins at Sea World,
the Gold Coast, Queensland, Australia (Chapter 5). The saliva samples from
different individuals were pooled. The same was done for blow. Progesterone
stability experiments follow those described for testosterone (Chapter 4).
6.2.2. LC-MS assay
LC-MS was used to determine progesterone concentrations as described in
Chapter 5.
6.2.3. Inhibitors
Progesterone stability experiments were set up to determine which was the
superior inhibitor, either 100 µg/ml amoxycillin/potassium clavulanate
® (Augmentin ) or 100 mM MnCl 2. Experiments with no inhibitors were used as
controls.
6.2.4. Freeze-thaw experiments
Pooled saliva from different individuals and pooled blow from different individuals
were spiked with 50 ng/ml or 0.5 ng/ml progesterone (n = 5). The method of
drying off stock standard solutions to obtain these low concentrations of
progesterone has been described (Chapter 4). Samples were spiked and stored
at
-80 oC between sample runs. Each spiked sample was analysed after initial
spiking and then after each thaw cycle. A total of three freeze-thaw cycles were
conducted. Samples were not extracted but were injected directly onto the
column.
121 Chapter 6 – Progesterone Stability
6.2.5. Short-term storage
In order to assess stability of progesterone at 21 oC, saliva and blow samples
were spiked with 50 ng/ml or 0.5 ng/ml progesterone. Three different treatments
were set up: no inhibitors (NI), addition of 100 µg/ml Augmentin ®, or addition of
o 100 mM MnCl 2. Measurements at 21 C were made at time 0, 3, 6, 9, 12, 15 and
18 hours after initial spiking.
6.2.6. Long-term storage
® The stability of all three treatments (no inhibitors, MnCl 2, and Augmentin ) was
assessed at -20 oC and -80 oC. After initial spiking, samples were stored at -20 oC
and analysed at weeks 0, 1, 2, 3 and 4; and at weeks 0, 4, 6, 8, 10 and 12 when
stored at -80 oC. All spiked samples were analysed according to the time
schedules given until they exhibited a significant change relative to the initial
spiking concentration.
6.2.7. Statistical analysis
Significant differences in progesterone concentration in the freeze-thaw and
stability studies were determined by ANOVA. Mann-Whitney U or Kruskal-Wallis
tests were used if the data did not meet the assumptions of ANOVA.
122 Chapter 6 – Progesterone Stability
6.3. Results
6.3.1. Freeze-thaw experiments
When samples were spiked with 50 ng/ml progesterone, the progesterone
concentration in the saliva (ANOVA: F = 10.61, df = 3, p < 0.001) and in the blow
(ANOVA: F = 47.39, df = 3, p < 0.001) increased significantly after each cycle of
freeze-thawing (fig. 6.1a). Similarly, when samples were spiked with 0.5 ng/ml
progesterone, the progesterone concentration in the saliva (ANOVA: F = 35.90,
df = 3, p < 0.001) increased significantly after each freeze-thaw cycle.
Interestingly, blow samples spiked with 0.5 ng/ml progesterone increased slightly
over two freeze-thaw cycles and significantly decreased (ANOVA: F = 3.41, df =
3, p = 0.047) after the third freeze-thaw cycle (fig. 6.1b).
6.3.2. Short-term storage
6.3.2.1. Saliva at 21 oC
At 21 oC with no inhibitors added, progesterone concentrations in saliva
significantly decreased (table 6.1) within three hours when spiked with 50 ng/ml
progesterone. Yet when spiked with 0.5 ng/ml progesterone, concentrations of
progesterone increased significantly within one hour of initial spiking (table 6.1).
When MnCl 2 was added to the saliva significant increases in progesterone
concentration were observed after 12 hours with 50 ng/ml and after nine hours
with 0.5 ng/ml spiking (table 6.1). Progesterone concentrations increased by
98.1% for 50 ng/ml spiking over 18 hours (t 0 = 49.04 ± 4.74 ng/ml vs. t 18 = 97.13 ±
26.95 ng/ml) then started to decrease by 21 hours (t 21 = 77.38 ± 12.67 ng/ml).
123 Chapter 6 – Progesterone Stability
a) 700
600
500
400
300
200 Progesteroneconcentration (ng/ml)
100 Blow Saliva 0 Initial Thaw-1 Thaw-2 Thaw-3
b) 1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6 Progesteroneconcentration (ng/ml) 0.5
0.4 Blow Saliva 0.3 Initial Thaw-1 Thaw-2 Thaw-3
Figure 6.1 : a) Three cycles of freezing and thawing of saliva and blow with 50
ng/ml progesterone. b) Three cycles of freezing and thawing of saliva and blow
with 0.5 ng/ml progesterone. Mean ± SE.
124 Chapter 6 – Progesterone Stability
Table 6.1: Changes in spiked progesterone concentration in bottlenose dolphin
o ® saliva and blow at 21 C with no inhibitors or inhibitors (Augmentin or MnCl 2)
added. Storage time indicates the time when changes were first observed.
SALIVA Hormone Inhibitor Storage Statistical test statistic df p concentration time (ng/ml) 50 No inhibitor 2 hours ANOVA: F = 2.9 2 NS 50 No inhibitor 3 hours ANOVA: F = 21.1 3 <0.001 0.5 No inhibitor 1 hour ANOVA: F = 10.7 1 0.008
50 MnCl 2 9 hours ANOVA: F = 3.3 2 NS
50 MnCl 2 12 hours ANOVA: F = 5.9 3 0.004
0.5 MnCl 2 6 hours ANOVA: F = 1.1 6 NS
0.5 MnCl 2 9 hours ANOVA: F = 1.5 2 0.002 50 Augmentin ® 6 hours ANOVA: F = 2.25 6 NS 50 Augmentin ® 9 hours ANOVA: F = 47.77 2 <0.001 0.5 Augmentin ® 6 hours ANOVA: F = 1.74 6 NS 0.5 Augmentin ® 9 hours ANOVA: F = 34.22 2 <0.001
BLOW Hormone Inhibitor Storage Statistical test statistic df p concentration time (ng/ml) 50 No inhibitor 3 hours ANOVA: F = 2.8 3 NS 50 No inhibitor 4 hours ANOVA: F = 3.1 4 0.0403 0.5 No inhibitor 1 hour ANOVA: F = 2.7 1 NS 0.5 No inhibitor 2 hours ANOVA: F = 9.4 2 0.0061
50 MnCl 2 18 hours ANOVA: F = 1.9 5 NS
0.5 MnCl 2 9 hours ANOVA: F = 5.8 2 NS
0.5 MnCl 2 12 hours ANOVA: F = 17.5 3 0.002 50 Augmentin ® 18 hours ANOVA: F = 1.23 6 NS 0.5 Augmentin ® 18 hours ANOVA: F = 1.3 6 NS
125 Chapter 6 – Progesterone Stability
In saliva spiked with 0.5 ng/ml progesterone concentrations increased by 104.0%
over 18 hours (t 0 = 0.54 ± 0.04 ng/ml vs. t 18 = 1.10 ± 0.24 ng/ml) and then began
to decrease by 21 hours (t 21 = 0.78 ± 0.14 ng/ml).
Similarly, when Augmentin ® was added to saliva significant increases in
progesterone concentrations occurred after nine hours for both 50 ng/ml and 0.5
ng/ml spiking (table 6.1). Progesterone concentrations increased 79% over 18
hours for 50 ng/ml (t 0 = 49.22 ± 3.31 ng/ml vs. t 18 = 87.87 ± 16.69 ng/ml) but after
21 hours concentrations in the the 50 ng/ml spiked samples began to decrease
(t 21 = 77.38 ± 5.66 ng/ml). For those samples spiked with 0.5 ng/ml, progesterone
concentrations increased by 73% over 18 hours (t 0 = 0.53 ± 0.02 ng/ml vs. t 18 =
0.91 ± 0.17 ng/ml). As with the higher spiking concentration, after 21 hours the
progesterone concentrations began to decrease for 0.5 ng/ml spiked samples (t 21
= 0.80 ± 0.06 ng/ml).
6.3.2.2. Blow at 21 oC
When blow samples were spiked with 50 ng/ml progesterone and no inhibitors
were added, significant changes in progesterone concentration occurred after
four hours at room temperature (table 6.1). Significant increases were observed
after two hours at room temperature when blow was spiked with 0.5 ng/ml
progesterone (table 6.1).
The addition of inhibitors greatly improved the stability of progesterone in blow
samples. When MnCl 2 was added to blow samples spiked with 50 ng/ml
progesterone, no significant changes in concentration were observed over 18
hours. However when blow was spiked with 0.5 ng/ml progesterone a significant
increase was observed after 12 hours (table 6.1). When Augmentin ® was added
126 Chapter 6 – Progesterone Stability
to blow samples that were spiked with either 50 ng/ml or 0.5 ng/ml progesterone,
there were no significant changes in progesterone concentration over 18 hours.
6.3.3. Long-term storage
6.3.3.1. Saliva at -20 oC
When saliva was stored at -20 oC, significant changes were observed in spiked
progesterone concentrations (table 6.2). However the time it took for changes to
be observed differed depending on whether or not an inhibitor had been added to
the sample. Progesterone concentrations decreased significantly after one week
for both 50 ng/ml and 0.5 ng/ml spiking (table 6.2) if no inhibitors were added.
When MnCl 2 was added significant decreases occurred in both 50 ng/ml and 0.5
ng/ml samples after one week (table 6.1). The addition of Augmentin ® improved
the stability of progesterone at -20 oC to more than three weeks for 50 ng/ml and
more than four weeks for 0.5 ng/ml progesterone (table 6.2).
6.3.3.2. Blow at -20 oC
Progesterone concentrations decreased significantly in blow samples spiked with
50 ng/ml or 0.5 ng/ml progesterone without any inhibitor after one week at -20 oC
(table 6.2). The addition of MnCl 2 did not improve the stability of progesterone in
blow. Significant decreases in progesterone concentration were seen after one
week for both 50 ng/ml and 0.5 ng/ml spiked samples (table 6.2). The addition of
Augmentin ® improved progesterone stability in blow at -20 oC. There were no
significant changes in progesterone concentration for both 50 ng/ml and 0.5 ng/ml
spiked samples after four weeks at -20 oC (table 6.2).
127 Chapter 6 – Progesterone Stability
Table 6.2: Changes in spiked progesterone concentration in bottlenose dolphin
o ® saliva and blow at -20 C with no inhibitors or inhibitors (Augmentin or MnCl 2)
added. Storage time indicates the time when changes were first observed.
SALIVA Hormone Inhibitor Storage Statistical test statistic df p concentration time (ng/ml) 50 No inhibitor 1 week ANOVA: F = 143.5 1 <0.001 0.5 No inhibitor 1 week ANOVA: F = 33.1 1 <0.001
50 MnCl 2 1 week Mann-Whitney: Z = 2.9 0.004
0.5 MnCl 2 1 week Mann-Whitney: Z = 2.9 0.004 50 Augmentin ® 3 weeks ANOVA: F = 2.0 3 NS 50 Augmentin ® 4 weeks ANOVA: F = 21.4 4 <0.001 0.5 Augmentin ® 4 weeks ANOVA: F = 1.6 4 0.207
BLOW Hormone Inhibitor Storage Statistical test statistic df p concentration time (ng/ml) 50 No inhibitor 1 week ANOVA: F = 13.533 1 0.008 0.5 No inhibitor 1 week ANOVA: F = 22.088 1 0.001
50 MnCl 2 1 week ANOVA: F = 460.2 3 <0.001
0.5 MnCl 2 1 week ANOVA: F = 14.1 1 0.007 50 Augmentin ® 4 weeks ANOVA: F = 3.1 1 NS 0.5 Augmentin ® 4 weeks ANOVA: F = 1.4 3 NS
128 Chapter 6 – Progesterone Stability
6.3.3.3. Saliva at -80 oC
When saliva was stored at -80 oC, significant changes in progesterone
concentration were observed (table 6.3). With no inhibitors added, progesterone
concentrations decreased after 4 weeks for both 50 ng/ml and 0.5ng/ml spiked
samples. The addition of MnCl 2 to the spiked saliva samples (50 ng/ml or 0.5
ng/ml) did not improve progesterone stability (table 6.3). However, the addition of
Augmentin ® improved salivary progesterone stability at -80 oC. Significant
increases in progesterone concentration were seen in 50 ng/ml spiked samples
after 12 weeks. There was no significant change in 0.5 ng/ml spiked samples with
Augmentin ® after 12 weeks at -80 oC (table 6.3).
6.3.3.4. Blow at -80 oC
When no inhibitors were added the concentration of progesterone significantly
decreased in both 50 ng/ml and 0.5 ng/ml spiked samples after four weeks (table
6.3). The addition of MnCl 2 to spiked blow samples did not improve the stability of
progesterone. Significant decreases in progesterone concentration were seen
after four weeks for both 50 ng/ml and 0.5 ng/ml spiked samples (table 6.3).
When Augmentin ® was used as an inhibitor, progesterone stability in blow
samples improved. Significant changes in progesterone concentration were
observed after eight weeks when spiked with 50 ng/ml and after six weeks when
spiked with 0.5 ng/ml progesterone (table 6.3).
129 Chapter 6 – Progesterone Stability
Table 6.3: Changes in spiked progesterone concentration in bottlenose dolphin
o ® saliva and blow at -80 C with no inhibitors or inhibitors (Augmentin or MnCl 2)
added. Storage time indicates the time when changes were first observed.
SALIVA Hormone Inhibitor Storage Statistical test statistic df p concentration time (ng/ml) 50 No inhibitor 4 weeks ANOVA: F = 194.0 1 <0.001 0.5 No inhibitor 4 weeks ANOVA: F = 25.2 1 <0.001
50 MnCl 2 4 weeks ANOVA: F = 150.7 1 <0.001
0.5 MnCl 2 4 weeks ANOVA: F = 22.0 1 <0.001 50 Augmentin ® 8 weeks K-W: Chi-square = 5.3 2 NS 50 Augmentin ® 12 weeks ANOVA: F = 122.3 3 <0.001 0.5 Augmentin ® 12 weeks ANOVA: F = 0.05 3 NS
BLOW Hormone Inhibitor Storage Statistical test statistic df p concentration time (ng/ml) 50 No inhibitor 4 weeks ANOVA: F = 149.449 1 <0.001 0.5 No inhibitor 4 weeks ANOVA: F = 14.75 1 0.005
50 MnCl 2 4 weeks ANOVA: 15.4 1 0.006
0.5 MnCl 2 4 weeks ANOVA: F = 13.6 1 0.008 50 Augmentin ® 6 weeks ANOVA: F = 0.5 2 NS 50 Augmentin ® 8 weeks ANOVA: F = 60.0 3 <0.001 0.5 Augmentin ® 4 weeks ANOVA: F = 0.9 1 NS 0.5 Augmentin ® 6 weeks K-W: Chi-Square = 6.9 2 0.0307
130 Chapter 6 – Progesterone Stability
6.4. Discussion
Progesterone was unstable in bottlenose dolphin saliva and blow samples. The
degree of stability varied depending on storage temperature and whether
inhibitors were used.
The freeze-thaw experiments showed increases in progesterone concentrations
with each freeze-thaw cycle except for 0.5ng/ml progesterone in blow which
decreased at the third freeze-thaw cycle. As discussed with testosterone stability,
repetitive thawing is known to cause error with steroid hormones (Chattoraj and
Watts 1987). The increase and/or decrease in concentration could be due to the
breaking of chemical bonds during thawing. Every effort should be made to
ensure that freezing and thawing is kept to a minimum when storing and shipping
biological samples for hormone analysis.
When inhibitors were not added, progesterone concentrations changed rapidly in
both saliva and blow samples. As with testosterone the addition of either MnCl 2 or
Augmentin ® improved the stability of progesterone in saliva and blow at room
temperature. Although MnCl 2 was a slightly better inhibitor for saliva at room
temperature, Augmentin ® proved to be the superior inhibitor overall. At lower
temperatures, MnCl 2 did not stabilise progesterone as there was no difference
between samples with or without MnCl 2. This indicates that bacteria may have
influenced progesterone stability at lower temperatures, as stability improved with
the addition of a broad spectrum antibiotic.
Interestingly, salivary progesterone concentrations at room temperature
increased over an 18 hour period. This trend was seen in saliva samples
regardless of the presence or absence of the inhibitor. Yet when the same
131 Chapter 6 – Progesterone Stability
samples were assayed at 21 hours after spiking the progesterone concentrations
had begun to decrease. There is some progesterone metabolism in human saliva
(Laine and Ojanotko 1999) but this did not affect salivary progesterone
concentrations significantly. Progesterone metabolism in human saliva was
attributed to the cellular content of saliva. Metabolic activity was greater in whole
saliva than in centrifuged saliva (Laine and Ojanotko 1999). As steroid
metabolism has not been determined in dolphin saliva, the rate of metabolic
activity may be higher than in humans. In addition the stability experiments
conducted in this study used whole saliva.
There were no significant changes in progesterone concentration when
Augmentin ® was used with blow samples. This indicates that changes in blow
progesterone concentrations are more than likely due to bacterial changes rather
than metabolic ones. Cetaceans are known to have a number of infectious and
non-infectious diseases in their respiratory system (Dunn et al. 2001; Gulland et
al. 2001). Microbiological studies of cetacean blow will allow for a greater
understanding of the different types of bacteria found in blow.
MnCl 2 was tested as an inhibitor in this program as it was an effective inhibitor for
testosterone. However, it does not appear to have an affect on progesterone
stability at low temperatures. There may be more bacterial activity occurring at
lower temperatures and MnCl 2 has no affect on bacteria. Alternatively, there may
be metabolic activity occurring at lower temperatures. Although MnCl 2 is known to
influence testosterone steroidogenesis (Cheng et al. 2003) it may have no effect
on progesterone metabolism because the androgen/oestrogen metabolic
pathway is separate to the progesterone metabolic pathway (fig. 1.5).
Progesterone stability studies in dolphin saliva and blow that assess both
132 Chapter 6 – Progesterone Stability
progesterone and its metabolites will clarify this issue. Alternatively, it may be
possible to use a progesterone steroidogenesis inhibitor.
133 Chapter 6 – Progesterone Stability
6.5 Conclusion
From this study it has been determined that dolphin saliva can be stored for six
hours at 21 oC, four weeks at -20 oC and eight weeks at -80 oC with the addition of
100 µg/ml Augmentin ® before changes in progesterone concentrations occur.
Dolphin blow can be stored with 100 µg/ml Augmentin® added for 18 hours at
21 oC, four weeks at -20 oC and four weeks at -80 oC before changes in
progesterone concentrations occur. In order to minimise any changes in
endogenous hormone concentration samples should be extracted as soon as
possible. It is also advised that samples are shipped on dry ice or in liquid
nitrogen to prevent any freeze-thawing during transit.
Many biological programs are conducted under the premise that samples are
stable at -20 oC for a number of months or years without inhibitors added. Stability
studies of progesterone in non-invasively collected samples, such as faeces
(Schlenker et al. 1999; Lynch et al. 2003), urine (Kesner et al. 1995) and saliva
(Lipson and Ellison 1989; Gröschl et al. 2001), have revealed changes in
progesterone concentration over varying lengths of time and storage
temperature. Similar results were found in this study. If stability is not addressed
then observed changes in hormone concentrations may be an artefact of storage
time and temperature, rather than an indication of an animal’s biological activity.
134
CHAPTER 7
Whale Blow Collection: Is It Feasible?
"They [whales] are blowing on every point of the compass, and frequently taint the whole atmosphere with the utmost disagreeable effluvia that can be conceived." Charles Clerke, Lieutenant of the Resolution in his journal, 30 Dec 1774, off Tiera del Fuego.
7.1. Introduction
Anatomical and physiological studies of whales were carried out on whaling
ships or in whaling stations (Matthews 1937; Chittleborough 1955;
Chittleborough 1965). Since commercial whaling ceased in the 1980s,
reproductive studies on large whale species have been limited post whaling.
It is problematic to determine the reproductive hormone cycles of great
whales (Chapter 1). So our current understanding of their breeding behaviour
is from histological analysis from the whaling days and more recent photo-
identification studies. Behavioural studies have led to a greater understanding
of breeding biology in some species, such as humpback whales, but many
questions remain unanswered. Most whale populations around the world
appear to be recovering, while others, such as the blue whales in the Gulf of
St. Lawrence and the North Atlantic right whale, have low reproductive rates
(Caswell et al. 1999). Why the reproductive rates of some populations are so
low is unknown. It is impossible to determine if these low reproductive rates
are a result of reproductive dysfunction or due to other external factors.
135 Chapter 7 – Whale Blow Collection
7.1.1. Humpback whale reproduction
Humpbacks (Cetacea: Balaenopteridae) whales migrate annually from their polar
and temperate feeding grounds to their tropical breeding areas (Matthews 1937;
Dawbin 1966).
Age is determined in this species by the number of growth layers that are present
in the animal’s ear wax plugs (Chittleborough 1959; Lockyer 1984).
Chittleborough (1959) believed that there were four growth layers per year and so
age of sexual maturity was between five and six years. However, Lockyer (1984)
estimated that there were only two growth layers per year, indicating that the age
of sexual maturity was from ten to twelve years. Ongoing photo-identification
studies of different humpback whale populations have indicated that the age of
sexual maturity is between five and seven years with some animals reaching
maturity as late as ten years (Clapham and Mayo 1987; Clapham et al. 1993).
These estimates were obtained from the presence or absence of calves with
known aged females. Using genetic analysis it is possible to know which males
are siring calves (Clapham and Palsbøll 1997) and so age of sexual maturity may
also be estimated for known males.
Males have a seasonal cycle indicated by the reduced testes weight in male
humpback whales in the feeding grounds compared with testes weight in the
breeding areas (Matthews 1937; Chittleborough 1955; Chittleborough 1955a).
Due to the high variability in body length of male humpback whales during
puberty it is difficult to determine sexual maturity on body length alone
(Chittleborough 1955a). Testes weight increases rapidly before puberty and
continues to rise sharply for some time after sexual maturity has been reached
(Chittleborough 1955a). Without a method to determine testosterone
136 Chapter 7 – Whale Blow Collection
concentrations in humpback whales it is difficult to determine the proportion of
mature whales that have the capacity to sire calves. Reproductive decline in a
population can be a result of reproductive dysfunction in males or females or a
combination of the two.
In female humpback whales puberty (first ovulation) and sexual maturity (when
first pregnancy occurs) are not reached at the same time. Typically pregnancy
does not occur in the first but rather in the female’s second ovulatory season
(Chittleborough 1955b). From histological examination of ovaries and distribution
of near-term foetuses, the gestation of humpback whales is estimated to be 12
months (Chittleborough 1958). Females are thought to be impregnated in the
calving areas or along the migration to and from these areas (Chittleborough
1958; Dawbin 1966). When females return to their breeding sites the following
season they give birth to a single calf (Clapham and Mayo 1990). Ongoing photo-
identification studies of known individuals from different populations indicate that
females are on a two to three year breeding cycle. Some animals calve annually
while others have longer calving intervals (Chittleborough 1955b; Chittleborough
1958; Chittleborough 1965; Glockner-Ferrari and Venus 1983; Clapham and
Mayo 1987; Glockner-Ferrari and Ferrari 1990; Hogg 1998).
Pod composition varies throughout the migration but generally consists of two to
three whales (Paterson and Paterson 1984). Larger aggregations are found in the
breeding areas (Herman and Antinoja 1977; Tyack and Whitehead 1983) and
feeding grounds (Baker et al. 1987; Clapham and Mayo 1987).
Males in the breeding areas are competing for mates and so aggressive
behaviours are often observed (Tyack and Whitehead 1983). Competition pods
are seen frequently and are associated with courtship behaviours. These pods
137 Chapter 7 – Whale Blow Collection
consist of a number of males jostling for the primary escort position behind a
female, although some pods have been observed without a female present
(Herman and Antinoja 1977; Tyack and Whitehead 1983). It is believed that when
a female comes into oestrus she will mate with the male closest to her (Tyack
and Whitehead 1983).
Females with last year’s calf are generally the first to arrive in the breeding areas,
followed by juveniles, adults and pregnant females (Dawbin 1966). It is thought
that males associate with sexually mature females at the beginning of the
breeding season. As these females depart the breeding areas males then
associate with females and calves (Tyack and Whitehead 1983). Catch data of
females with calves showed that post-partum ovulation occurs and pregnancy
may result (Matthews 1937; van Lennep and van Utrecht 1953; Chittleborough
1958). So males may escort females with calves waiting for the opportunity for
mating to arise (Glockner-Ferrari and Ferrari 1984). This lends itself to a number
of interesting hormonal questions.
− Is there a group of males that are more successful breeders in any
population and do they have higher testosterone concentrations?
− Are these animals dominating the primary escort position in competition
pods?
− When do females come into oestrus and how long is their cycle?
− Do subordinate males escort female-calf pods on the migration as this leads
to a greater opportunity to breed?
− Do all females have post-partum ovulation?
− How can the concentrations of reproductive hormones in free-swimming
humpback whales be determined?
138 Chapter 7 – Whale Blow Collection
By using captive bottlenose dolphins a method for determining the presence of
reproductive hormones in cetacean blow was developed and validated (Chapter
3 and 5). The aim of the study presented in this chapter was to ascertain whether
this method could be used to determine the concentration of reproductive
hormones in free-swimming great whales by:
• determining the feasibility of collecting blow from free-swimming
humpback whales, and
• determining if testosterone or progesterone could be found and then
quantified in samples of humpback whale blow using LC-MS.
7.2. Materials and Methods
7.2.1. Sample collection materials
Three different sample collection materials were trialled. The first was cotton
gauze (Smith-Neilsen, Beecham, Australia), the second was Millipore filtration net
(Millipore, Sydney, Australia), which is inert and has a small micropore mesh, and
the third was Koltex nylon knee high stockings (Koltex, Australia).
Experiments were conducted to determine which collection material was the most
inert for whale blow collection. The collection material (either gauze, net or
stocking) was placed in the collection tube with 5 ml of inhibitor mix (100 mM
o MnCl 2 and 100 µg/ml amoxicillin/potassium clavulanate) and frozen at -80 C
overnight. The materials were then extracted as per the extraction method
described in Chapter 3. Positive and negative scans of all extracts were
conducted on a 0-100% mobile phase B gradient over 50 mins. Both mobile
phases were used for the scans: scan 1 - mobile phase A = 0.5% acetic acid,
139 Chapter 7 – Whale Blow Collection
mobile phase B = 0.5% acetic acid, 90% acetonitrile; and scan 2 - mobile phase
A = 0.5% formic acid, mobile phase B = 0.5% formic acid, 90% acetonitrile.
To ensure that nylon stocking was inert, two different methods of cleaning were
examined. In the first the nylon stocking was sonicated for 15 mins with 0.5%
methanol (MeOH) and then sonicated for a further 15 mins with Milli-Q water,
changing the water every 5 mins. In the second the stocking was sonicated for 15
mins in 100% acetonitrile and then for a further 15 mins with Milli-Q water,
changing the water every 5 mins.
7.2.2. Blow sample collection
Blow samples were collected from southward migrating humpback whales at
Peregian Beach, Queensland, Australia (fig. 7.1) in October 2003 and 2004. The
samples were collected using a 13-metre carbon fibre pole (Composite
Engineering, Concord, Mass., USA) that was bracket mounted to the bow of a 5-
metre aluminium vessel (fig. 7.2) (Moore et al. 2001). The blow collection device
was a 5-inch bamboo double ring with a collection material secured over it (fig.
7.3). The blow collection device was mounted on the top of the pole (fig. 7.4) and
the blow was collected as the whale surfaced.
140 Chapter 7 – Whale Blow Collection
Brisbane
Sydney
Figure 7.1: Study site at Peregian Beach, Queensland, Australia
141 Chapter 7 – Whale Blow Collection
Figure 7.2: Thirteen metre carbon fibre pole used to collect blow samples from
whales. The pole was bracket-mounted to the vessel (a) and steered by a crew
member (b).
142 Chapter 7 – Whale Blow Collection
a)
b)
143 Chapter 7 – Whale Blow Collection
Figure 7.3: The blow collection device was a 5-inch bamboo double ring with
nylon stocking that had been sonicated with 100% acetonitrile stretched across it.
144 Chapter 7 – Whale Blow Collection
a)
Blow collection device
Whale blow b) passing over collection device at
the end of the pole
13-m carbon fibre pole
Humpback whale
Figure 7.4: a) The blow collection device was mounted to the top of the pole and
samples were collected when approaching the whale. b) This picture documents
the collection of a blow that was graded as a medium quality sample.
145 Chapter 7 – Whale Blow Collection
Blow samples were graded based on the amount of blow that was seen to pass
over the blow collection device during sampling. Samples were rated as 3 if the
end of the pole was within 5 metres of the whale’s blowholes when it surfaced.
However the sample was rated as 2 if there were high winds during collection or if
the sample was collected when the pole approached within 5 - 10 metres of the
whale. Any sample collection that was further than 10 metres from the whale was
rated as the poorest quality, 1.
Once the blow had been collected, the collection material was removed from the
ring and placed in a 50 ml polypropylene collection tube with 5 ml inhibitor mix.
Samples were kept on ice in a shady area of the boat until arrival on-shore where
they were stored at -20 oC for two to three weeks. Samples were shipped on dry
ice and stored in -80 oC for three weeks until extraction.
Seawater samples were collected after each blow collection and extracted and
assayed using the same method for blow.
7.2.3. Blow sample analysis
Whale blow samples were extracted as per Chapter 3 and the dry extracts from
the SPE were stored at -80 oC for one to four weeks prior to analysis. To minimise
any changes to hormone concentrations in the samples, only four blow samples
were extracted at a time to ensure that extraction time remained less than nine
hours.
Extracted blow samples were reconstituted in 60 µl 60% acetonitrile and 5 µl
injections used for all analyses. Samples were run using both the 55% isocratic
testosterone method (Chapter 3), referred to as the “testosterone method” here
146 Chapter 7 – Whale Blow Collection
and the 50% isocratic female hormone method (Chapter 5), referred to as the
“female method”. Samples were scanned using both positive and negative scan
mode on a 0 - 100% mobile phase B gradient over 50 mins. Two scan runs were
done, one with mobile Phase A = 0.5% acetic acid and mobile Phase B = 0.5%
acetic acid, 90% acetonitrile; and the other with mobile Phase A = 0.5% formic
acid and mobile phase B = 0.5% formic acid, 90% acetonitrile. The scanning
protocol was: 0 - 100% phase B for 30 mins; 100% Phase B for 5 mins; 100 - 0%
phase B for 5 minutes; and 0% phase B for 10 mins (to allow for equilibration of
the column). Blow samples from an individual whale were analysed with all
methods (testosterone, female and scanning gradients) on the same day.
Different individual whales were analysed on different days.
Seawater samples were analysed from multiple locations in the Peregian Beach
region using the testosterone and female methods and the scanning gradients.
Seawater results were compared to whale blow. To ensure that results obtained
were from the whale and not from any other factors, the inhibitor mix was frozen
at -80 oC with collection material and then extracted. This extract was analysed
with the same methods as the samples and results compared with whale blow.
During the 2003 season, nine blow samples were collected from nine individual
humpback whales. All animals were adults and the sex was assumed for three
whales, due to the presence of a calf in the case of females (n = 1) or due to the
animal singing in the case of males (n = 2) (Table 7.1a). Of the nine samples,
three were graded as high quality (3), two were graded as medium quality (2) and
four were rated as poor quality (1).
147 Chapter 7 – Whale Blow Collection
During the 2004 season, a total of 26 blow samples were collected (Table 7.1b).
Twenty were females with calves and one was an adult male (an escort to a
female-calf pod). The sex was unknown for three samples as the identity of the
individual (female or escort) was unable to be determined when the blow was
collected. Two samples were collected from the same individual. Of the 26
samples collected, eight samples were graded as high quality (3), 15 were
graded as medium quality (2) and three were graded as poor quality (1).
7.2.4. 0-100% gradient analysis
Analysis of individual m/z of the scanning gradients was achieved by dividing the
chromatograms into 1 minute time periods. For 2004 whale blow samples,
seawater samples and cleaned stocking extracts, m/ zs with a greater than 50%
relative intensity were recorded in any given time period. The m/z s that were due
to seawater and stocking/inhibitors were then excluded from the whale blow
samples. Any m/z s which may be attributed to precursors or metabolites of
progesterone and testosterone were noted. The m/ z for these substances was
determined by adding or subtracting one to the molecular weight (mw) of the
compound:
i.e. [M+H] + or [M+H] -
where: M = the molecular mass
H = the addition or subtraction of a proton during mass
spectrometric analysis (Chapter 2).
148 Chapter 7 – Whale Blow Collection
Table 7.1: a) Humpback whale blow samples collected from Peregian Beach in
2003.
2003 Pod composition Blow code Date Time Animal blow Quality Adults Sub-adults Calves collected from Mn04-01 05-Oct-03 09:20 2 0 0 Unknown 2 Mn04-02 05-Oct-03 N/A 1 0 0 Unknown 3 Mn04-03 07-Oct-03 N/A 2 0 1 Unknown 2 Mn04-04 09-Oct-03 10:22 3 0 0 Unknown 1 Mn04-05 10-Oct-03 09:36 1 0 0 Unknown 3 Mn04-06 10-Oct-03 09:36 1 0 0 Unknown 1 Mn04-07 11-Oct-03 07:58 1 0 1 Female 1 Mn04-08 11-Oct-03 09:00 2 0 1 Escort 3 Mn04-09 11-Oct-03 14:30 2 0 1 Escort 1
149 Chapter 7 – Whale Blow Collection
Table 7.1: b) Humpback whale blow samples collected from Peregian Beach in
2004.
2004 Pod composition Animal blow Blow code Date Time Sub- Quality Adults Calves collected adults from Mn04-01 03-Oct-04 09:14 1 0 1 Female 3 Mn04-02 04-Oct-04 09:43 1 0 1 Female 3 Mn04-03 05-Oct-04 10:44 2 0 1 Female 3 Mn04-04 06-Oct-04 11:58 1 0 1 Female 3 Mn04-05 07-Oct-04 08:59 2 0 1 Female 3 Mn04-06 09-Oct-04 10:35 1 0 1 Female 3 Mn04-07 13-Oct-04 09:00 2 0 1 Female 3 Mn04-08 14-Oct-04 11:30 1 0 1 Female 3 Mn04-09 02-Oct-04 10:04 2 0 1 Female 2 Mn04-10 03-Oct-04 07:01 1 0 1 Female 2 Mn04-11 04-Oct-04 09:43 1 0 1 Female 2 Mn04-12 05-Oct-04 09:03 1 0 1 Female 2 Mn04-13 05-Oct-04 10:44 2 0 1 Unknown 2 Mn04-14 06-Oct-04 11:06 1 0 1 Female 2 Mn04-15 07-Oct-04 08:59 2 0 1 Female 2 Mn04-16 09-Oct-04 09:27 2 0 1 Unknown 2 Mn04-17 11-Oct-04 11:18 1 0 1 Female 2 Mn04-18 11-Oct-04 12:41 2 0 1 Unknwon 2 Mn04-19 12-Oct-04 09:03 2 0 1 Female 2 Mn04-20 12-Oct-04 09:25 2 0 1 Escort 2 Mn04-21 12-Oct-04 10:36 1 0 1 Female 2 Mn04-22 13-Oct-04 07:09 1 0 1 Female 2 Mn04-23 15-Oct-04 07:20 1 0 1 Female 2 Mn04-24 30-Sep-04 09:45 2 0 1 Female 1 Mn04-25 30-Sep-04 08:27 1 0 1 Female 1 Mn04-26 06-Oct-04 09:54 1 0 1 Female 1
150 Chapter 7 – Whale Blow Collection
The precursor of testosterone was androst-4-ene-3,17-dione (mw = 286.7) and
the metabolites were 5 α-dihydrotestosterone (mw = 290.44) and 19-hydroxy-
testosterone (mw = 304.42). The precursor for progesterone was pregnenolone
(mw = 316.49) and the metabolites were 11-deoxycorticosterone (mw = 330.47)
and hydroxyprogesterone (mw = 330.46).
7. 3. Results
7.3.1. Sample collection materials
Scans showed nylon stocking and Millipore net were more inert than cotton
gauze (fig. 7.5). Millipore net did not absorb liquid in the same manner as nylon
stocking. Untreated nylon stocking showed some interfering peaks, i.e. not
completely inert (fig. 7.5c). Scans of 0.5% methanol stocking (fig. 7.5d) showed
improvements but there were still interfering peaks. The 100% acetonitrile
cleaning method yielded optimal results (fig. 7.5e) and was the method used for
whale blow sample collection.
7.3.2. Blow sample analysis
Of the nine 2003 whales, two were found to have testosterone present (fig. 7.6),
Mn03-07 and Mn03-09. The blow samples from both Mn03-07 and Mn03-09 were
graded as high quality samples. Mn03-09 was a female-calf-escort pod and the
sample was collected from the escort. Mn03-07 was a single adult whale of
unknown sex. Progesterone was not detected in any of the samples.
In 2004, 24 whale blow samples were collected. Two blow samples were found to
have testosterone present, Mn04-13 and Mn04-16. The single sample from the
adult escort (Mn04-20) did not have testosterone present. Seven blow samples
151 Chapter 7 – Whale Blow Collection
from the 2004 whales had progesterone present. Six of these samples had
quantifiable peaks, i.e. signal-to-noise ratio greater than 5 to 1 (fig. 7.7). Fourteen
blow samples from known females did not have any progesterone present.
7.33. 0-100% gradient analysis
There were 341 different m/zs found in whale blow (Appendix 2). Each of the
scanning gradients using acetic acid phase and formic acid phase were
compared against the control chromatograms for stocking and the inhibitors (fig.
7.5e) as well as the sea water samples.
The positive and negative m/z s for the precursors and metabolites of testosterone
and progesterone are given in Table 7.2. An m/z representative of
dihydrotestosterone was observed in two whales. The m/z representative of
hydroxy-testosterone was observed in five individuals. Six whales had the m/z s
representative of pregnenolone.
152 Chapter 7 – Whale Blow Collection
Figure 7.5: MS chromatograms of scans of different collection materials. All
scans were carried out using both formic acid and acetic acid in the mobile
phases. However, only those chromatograms using mobile phase A: 0.5% formic
acid and mobile phase B: 0.5% formic acid, 90% acetonitrile, are shown here.
a) 0-100% mobile phase B gradient over 50 mins of cotton gauze;
b) 0-100% mobile phase B gradient over 50 mins of Millipore net;
c) 0-100% mobile phase B gradient over 50 mins of stocking;
d) 0-100% mobile phase B gradient over 50 mins of stocking washed in 0.5%
MeOH;
e) 0-100% mobile phase B gradient over 50 mins of stocking washed in 100%
acetonitrile.
153 Chapter 7 – Whale Blow Collection
a) 0-100% mobile phase B gradient over 50 mins of cotton gauze
Int. 1:TIC(1.00) 2:TIC(1.00)
50e6
40e6
30e6
20e6
10e6
0 5 10 15 20 25 30 35 40 45 min
b) 0-100% mobile phase B gradient over 50 mins of Millipore net
Int. 1:TIC(1.00) 2:TIC(1.00)
250e6
200e6
150e6
100e6
50e6
0 5 10 15 20 25 30 35 40 45 min
c) 0-100% mobile phase B gradient over 50 mins of stocking
Int. 1:TIC(1.00) 2:TIC(1.00) 40.0e6
35.0e6
30.0e6
25.0e6
20.0e6
15.0e6
10.0e6
5.0e6
0 5 10 15 20 25 30 35 40 45 min
154 Chapter 7 – Whale Blow Collection
d) 0-100% mobile phase B gradient over 50 mins of stocking washed in 0.5%
MeOH
Int. 1:TIC(1.00) 2:TIC(1.00)
35.0e6
30.0e6
25.0e6
20.0e6
15.0e6
10.0e6
5.0e6
0 5 10 15 20 25 30 35 40 45 min
e) 0-100% mobile phase B gradient over 50 mins of stocking washed in 100%
acetonitrile
Int. 1:TIC(1.00) 2:TIC(1.00)
100e6
75.0e6
50.0e6
25.0e6
0 5 10 15 20 25 30 35 40 45 min
155 Chapter 7 – Whale Blow Collection
Int. 1:289.20(1.00) 2:330.25(1.00)
200000
175000 Testosterone 150000
125000
100000
75000
50000
25000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 min
Figure 7.6: MS chromatogram showing presence of testosterone in humpback
whale (Mn03-07) blow collected in 2003 season. 55% isocratic mobile phase B
method using mobile phase A: 0.5% acetic acid, mobile phase B: 0.5% acetic
acid, 90% acetonitrile over 15 mins.
156 Chapter 7 – Whale Blow Collection
Int. 1:315.30(1.00) 2:356.25(1.00) 30000
27500
25000 22500 20000
17500 Progesterone 15000
12500
10000
7500
5000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 min
Figure 7.7: MS chromatogram showing presence of progesterone in humpback
whale (Mn04-07) blow collected in 2004 season. 50% isocratic mobile phase B
method using mobile phase A: 0.5% formic acid, mobile phase B: 0.5% formic
acid, 90% acetonitrile over 12 mins.
157 Chapter 7 – Whale Blow Collection
Table 7.2: Positive ([M+H] +) and negative ([M+H] -) m/z for some of the precursors
and metabolites of testosterone and progesterone (mw = molecular weight, M =
molecular mass and H = proton).
Hormone name mw [M+H] + [M+H] - Type
Testosterone 288.2 289.2 287.2 androst-4-ene-3,17-dione 286.7 287 285.7 precursor/metabolite 5α-dihydrotestosterone 290.44 291.5 289.44 metabolite 5β-dihydrotestosterone 290.44 291.5 289.44 metabolite 19-hydroxy-testosterone 304.42 305.5 303.42 metabolite Progesterone 314.3 315.3 313.3 Pregnenolone 316.49 317.5 315.49 precursor/metabolite 11-deoxycorticosterone 330.47 331.47 329.47 metabolite 21 α-hydroxyprogesterone 330.46 331.46 329.46 metabolite 11 α-hydroxyprogesterone 330.46 331.46 329.46 metabolite 11 β-hydroxyprogesterone 330.46 331.46 329.46 metabolite
158 Chapter 7 – Whale Blow Collection
7.4. Discussion
7.4.1 Sample collection materials
Analysis of different collection materials showed that cotton gauze was unsuitable
for whale blow collection. The same brand, but a smaller size, did not have
interfering results (Chapter 3). Due to this inconsistency, and that cotton gauze is
known to effect immunoassay results (Shirtcliff et al. 2001), the use of cotton
gauze for steroid hormone analysis is inadvisable. Although inert, Millipore net
was unsuitable as it did not absorb enough liquid. There were concerns that if the
pole bounced whilst at sea that any blow on the net would be dislodged. Nylon
stocking absorbed liquid easily but interfering compounds were noted if the
stocking was not cleaned. Sonication with 100% acetonitrile proved an affective
cleaning method. These results support the need for analysis of different
collection materials prior to sample collection. Different materials may cause
interference with hormonal results depending on the chemical analysis used.
7.4.2. Blow sample analysis
Three of the blow samples with testosterone present were collected from whales
in female-calf-escort pods. In humpback whales it is believed that escorts of
female-calf pods are typically males waiting for the opportunity to mate (Glockner-
Ferrari and Ferrari 1984). The presence of testosterone in these samples
supports this hypothesis. No testosterone was found in any of the high quality
samples from known females. The fourth sample with testosterone present was
collected from an adult of unknown sex. There are no distinguishing features that
can be used from the surface to determine sex in humpback whales unless the
genital area is observed (Glockner-Ferrari and Ferrari 1990). However, this
animal may have been male due to the presence of testosterone in the sample.
Future collection of blow samples from individuals of known sex or in conjunction
159 Chapter 7 – Whale Blow Collection
with genetic sampling will determine the practicality of sex determination from
blow sampling. High testosterone concentrations in blow samples is likely to
indicate sexually mature males due to the marked difference in testes size and
weight between pre-pubescent and pubescent whales (Chittleborough 1955a).
All 2004 female blow samples were collected from known lactating females (due
to the presence of a calf). Humpback whales nurse their young until they return to
the breeding area the following season (Chittleborough 1958). The presence of
progesterone in seven samples, and not in the other fourteen, may be attributed
to a number of different factors. Chittleborough (1958) showed that humpback
whales experience post-partum ovulation. If this is the case, progesterone in
some samples and not in others may be due to the different stages of the
oestrous cycle or that ovulation has resulted in pregnancy. Although the resight
histories of East Australian female humpback whales show calves in consecutive
years (Hogg 1998) it is not commonplace. If there is post-partum ovulation it does
not always result in pregnancy. The lack of progesterone in some samples may
be also due to the whales lactating. Suckling stimuli suppresses gonadotropin
release (particularly luteinising hormone) preventing normal follicular maturation
and growth (McNeilly 1988). Or alternatively the lack of progesterone in the
samples may be due to the quality of the blow samples themselves.
Progesterone could not be quantified in the blow of cycling female bottlenose
dolphins (Chapter 5). Using the same LC-MS method progesterone could be
quantified in 30% (6 of 20) of the humpback whales sampled. In humpback
whales, where successful pregnancy from post-partum ovulation is not common,
perhaps progesterone concentrations in the blow of cycling females is higher
than in bottlenose dolphins. Recently, Rolland et al. (2005) reported the need to
160 Chapter 7 – Whale Blow Collection
dilute North Atlantic right whale faecal samples when assaying for reproductive
hormones due to higher than normal concentrations (for mammals) of
reproductive hormones in the samples. As progesterone concentrations in cycling
and pregnant female humpback whales are unknown, it is difficult to ascertain
why progesterone was determined in some blow samples and not in others.
7.4.3. 0-100% gradient analysis
Humpback whale blow contains a plethora of different compounds. The gradient
analyses showed a number of different substances, some of small molecular
weight and others which make up larger compounds, such as proteins. At this
stage it is unclear whether these compounds are specific fluid from the lung
mucosa, are part of the oil found in whale blow or whether they have passed
across the capillary wall of the lungs from the bloodstream.
Steroid hormones can be excreted in conjugated or unconjugated forms. In
addition they can be metabolised in certain body fluids such as saliva (Whitten et
al. 1998; Laine and Ojanotko 1999). The form in which hormones are secreted is
species dependent (Whitten et al. 1998). Preliminary assessment of the m/z
values indicates that the precursors and metabolites of testosterone and
progesterone may be present in the samples of whale blow. Whether these
compounds are from the bloodstream or whether they have been produced
through metabolism/catabolism in the lungs is undetermined. By using more
sensitive technologies like MS-MS it may be possible to better understand which
form of the reproductive hormones are present in whale blow. However the
gradient analyses showed that a variety of different compounds were present.
With further analytical development and enhanced collection techniques, whale
blow has the potential to provide answers to a range of physiological questions.
161 Chapter 7 – Whale Blow Collection
7.5. Conclusion
It is feasible to collect blow from a free-swimming whale. Testosterone and
progesterone can be quantified in samples of whale blow using the LC-MS
methods developed here. The collection of blow samples from humpback whales
in conjunction with life history and genetic data will further our understanding of
their breeding cycle. Development of more sensitive analysis and collection
techniques will enable blow sampling to be used to determine reproductive
function in great whales in both their breeding areas and feeding grounds.
Moreover these techniques make possible the assessment of reproductive
dysfunction in declining or threatened populations.
162
CHAPTER 8
General Discussion
Reproductive physiology plays a vital role in population growth and vitality.
Understanding how an animal reproduces both physiologically and behaviourally
is important in population conservation. The aim of this study was to develop a
non-invasive technique that could be used to determine reproductive hormones in
cetaceans. Although a number of non-invasive methods exist for captive
cetaceans none of these were feasible to use with great whales. The new method
developed and validated in this thesis proposed the use of blow samples to
determine (and eventually monitor) reproductive hormones in cetaceans.
8.1. Analytical chemistry
Traditional methods of hormonal analysis, such as immunoassays, were not
practical for this program as they required approximately 200 µl of sample. The
blow samples were typically 50 µl in volume so an alternative method was
needed. LC-MS was found to offer great promise. Reproducible and accurate LC-
MS methods were developed for the determination of testosterone in male
dolphins (Chapter 3) and progesterone in pregnant female dolphins (Chapter 5).
Interestingly the concentration of hormones was greater in blow samples than in
saliva samples. At this time the reasons for this are unknown. Future comparative
analysis of blow and saliva samples with plasma samples may provide further
insight.
Although promising, there are still limitations to the LC-MS methods at this stage.
The limit of detection of the oestradiol and oestrone methods was not low enough
163 Chapter 8 – General Discussion
for their concentrations to be assessed in cycling dolphins. The progesterone
method allowed for the detection of progesterone but was not sensitive enough to
quantify concentrations in cycling female dolphins. Due to the complexity of
progestins and oestrogens, LC-MS-MS may be required to analyse these
compounds (Diaz-Cruz et al. 2003). The use of MS-MS or time-of-flight mass
spectrometry (TOF) may permit further development of this method as this
technology decreases the limits of detection for reproductive hormones. In
addition these methods will allow for the determination of the molecular
breakdown of the compounds found in both the saliva and blow of cetaceans.
Due to the aquatic nature of cetaceans it is difficult to ensure that there is no
seawater contamination of the samples. To combat problems with seawater
dilution a comparative dilution factor is required. Creatinine is an endogenous by-
product of muscle metabolism and is excreted in urine in a constant fashion
(Ruckebusch et al. 1991). It is used widely as a dilution factor to determine
hormone concentrations in urine. Creatinine is a small molecule (mw = 149.59)
and so could easily pass into saliva and lung mucosa. However, it is unknown at
this time if creatinine is present in cetacean saliva and blow. Alternatively other
substances which are excreted at a constant rate could be used to determine
dilution. The next step for this program would be to find a compound to identify
dilution rates so concentrations can be compared across animals.
8.2. Stability
One major research area that was investigated in this study was the stability of
testosterone (Chapter 4) and progesterone (Chapter 6). There is a lack of
consistency in the literature in reference to testosterone and progesterone
164 Chapter 8 – General Discussion
stability. A number of recent studies have shown that steroid hormones are not
stable and that each compound reacts differently depending on the species and
type of biological sample (Gröschl et al. 2001; Khan et al. 2002; Millspaugh et al.
2002; Lynch et al. 2003). No uniform method has been used to determine stability
in biological samples. Some studies analyse samples frequently (daily or weekly)
at room temperature and in the refrigerator (4 oC) (Granger et al. 2004) but only
analyse samples at six-monthly intervals when they have been stored at lower
temperatures (-20 oC and -80 oC). But as was shown in this study, hormone
concentrations can change over hours at room temperature and over weeks,
rather than months, at lower temperatures (-20 oC and -80 oC).
The addition of inhibitors improved the stability of testosterone and progesterone
in saliva and blow which has been shown with other biological samples (Gröschl
et al. 2001; Hunt and Wasser 2003; Lynch et al. 2003). Sodium azide is
commonly used as an inhibitor (Lipson and Ellison 1989; Gröschl et al. 2001) but
due to its toxicity it was not considered for this study. MnCl 2 proved to be the best
inhibitor for testosterone as it stops testosterone steroidgenesis (Cheng et al.
2003). As testosterone and progesterone belong to different metabolic pathways
(fig. 1.5), it was not surprising that MnCl 2 was not effective as an inhibitor for
progesterone. However, amoxycillin/potassium clavulanate (Augmentin ®) proved
to be an effective inhibitor for progesterone. The different stabilising effects of
® MnCl 2 and Augmentin with testosterone and progesterone indicated that there
are a number of different factors affecting stability in saliva and blow. Further
analysis of the precursors and metabolites of testosterone and progesterone will
indicate if metabolism is occurring in the samples. The use of a broad spectrum
antibiotic is useful as bacteria are present in both saliva and blow.
165 Chapter 8 – General Discussion
Samples used in the stability experiments were spiked with a known
concentration of either testosterone or progesterone. Using endogenous
concentrations of hormones to assess stability can be problematic. This is
because the endogenous concentration of these hormones may have already
undergone changes prior to the commencement of the stability experiment. By
spiking samples with known concentrations of hormone this problem is resolved.
It is important also to spike samples with high and low hormone concentrations as
small fluctuations in hormone concentrations were seen in these experiments.
Fluctuations did not affect the stability of the high concentration samples but gave
significant changes when lower concentrations were present.
Why testosterone and progesterone concentrations increase and then decrease
over time in saliva and blow samples of bottlenose dolphins is unknown. There is
very little published information on the composition of cetacean saliva and blow.
Lipid micelles in faeces breakdown over time releasing steroid hormones that are
bound to them (Yalkowsky 1999) and so increasing hormone concentrations
(Hunt and Wasser 2003). Cetaceans have large quantities of fat in their bodies.
Blow is oily, and perhaps lipids are binding to the steroid hormones which are
released as the lipids degrade, so increasing hormone concentrations in the blow
samples.
Alternatively, the instability of testosterone and progesterone in saliva and blow
may be due to the presence of cells in the samples. Saliva samples contain
epithelial cells, as the samples originate from the roof of the animal’s mouth.
Progesterone metabolism is known to occur in human saliva due to the presence
of cells (Laine and Ojanotko 1999). Cetacean blow exits the blowholes at
enormous speeds. It is possible that epithelial cells that line the trachea are
166 Chapter 8 – General Discussion
expelled with the blow. Cytological examination of both saliva and blow samples
would determine if this is the case.
Non-invasively collected samples are becoming more popular in the study of
hormone fluctuations of wildlife (Monfort 2003). The different sample matrices
that are used by wildlife biologists would contain different bacteria, proteins, and
enzymes which may be influencing hormone concentrations. It is imperative that
stability studies of each steroid hormone are conducted when using different
matrices for different species. The stability of the steroid hormone studied needs
to be addressed prior to using non-invasively collected samples as part of long-
term biological studies. This will ensure that changes seen in hormone
concentration are due to biological activity rather than an artefact of storage.
8.3. Whale blow collection
Australian humpback whales are known to be most reproductively receptive
between June and September when they are off the coasts of Australia
(Chittleborough 1954). Unlike dolphins these animals are not polyoestrous.
Histological examination of the ovaries has shown that humpback whales ovulate
only once during their ovulatory period, with a few ovulating twice (Chittleborough
1954). A method has been developed to determine reproductive hormones in
faecal samples of northern right whales (Rolland et al. 2005) but, as there is no
feeding off the Australian coast, faecal sampling is not a possibility when
determining hormone concentrations of humpback whales. An alternative to
faecal sampling is required to determine hormone concentrations in breeding
areas.
167 Chapter 8 – General Discussion
A method was developed to determine the concentration of reproductive
hormones present in the blow of free-swimming humpback whales (Chapter 7).
Although a number of analytical variables remain, such as dilution and stability,
this method could lead to a greater understanding of great whale reproduction.
Further development of the analytical method using MS-MS should allow for the
determination of concentrations of progesterone as well as oestradiol. A
technique for sexing adult whales from blow samples becomes possible by
determining an oestradiol/testosterone ratio (Gross et al. 1995). Sexing animals
by their blow will take some time as it will need to be done in conjunction with
studies that identify the sex of animals either by genetic sampling or photo-
identification.
To determine ranges for reproductive hormones humpback whales could be
sampled on their northward and southward migration over a number of years.
Although there is the possibility of sampling the same individuals within a season,
it is more likely that the same individuals would be sampled across seasons.
Biological ranges for a variety of reproductive hormones can be determined for
different sexes and ages by using blow samples in conjunction with ongoing
photo-identification studies and life history information.
It is anticipated that baseline information on reproductive hormones of great
whale populations will lead to a better understanding of how their populations are
interacting:
− Is it only a proportion of females that are breeding and, if so, is it because
the other females are not cycling?
− Do primary male escorts have higher testosterone concentrations than
other males?
168 Chapter 8 – General Discussion
− Do subordinate males find alternative mating strategies, such as escorting
female-calf pods?
These are the types of questions that can be answered with further development
of blow sample collection.
8.4. Conclusion
This study developed analytical methods to assess reproductive hormones in
cetacean blow samples with the view to develop methods which could be useful
to study free-swimming great whales. Reproducible and validated LC-MS
methods now exist for determining testosterone and progesterone in saliva and
blow. This study highlighted the need to assess steroid hormone stability in the
biological matrices being used. Further development using the latest mass
spectrometric technology will enhance the sensitivity and accuracy of these
methods. Longitudinal studies using saliva and blow will provide baseline values
for both male and female captive dolphins. Non-invasive sampling of cetaceans
can be used on a daily basis with minimal stress to the animal. In addition, further
sampling of humpback whales will provide reference ranges for reproductive
hormone concentrations on a vulnerable species in an area where alternative
non-invasive sampling is not possible.
Any signs of population decline or issues with reproductive efficiency may be due
to changes in internal physiology and/or external environmental factors. Sampling
and analytical methods which incorporate multiple hormone analysis from one
sample would be useful due to the difficulty and expense of collecting samples
from free-swimming whales. Developing a technique that could determine steroid
169 Chapter 8 – General Discussion
hormones associated with reproduction and stress would be beneficial as there is
not just one factor that affects an animal or population.
Of all vertebrate species in the world, reproductive behaviour has been described
for only 200 (Wildt and Wemmer 1999). Part of conservation management is a
comprehensive understanding of population growth. Baseline data on
reproductive physiology and a comprehensive knowledge of breeding biology is
essential. The analytical methods and techniques developed in this study provide
a solid foundation for future researchers and conservation managers in relation to
the reproductive physiology of cetaceans.
170
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Appendix 1
Peer-reviewed journal publication from Chapter 3.
190
Appendix 2
All mass-to-charge ratios ( m/ z) found in humpback whale blow. Those m/z ’s
that were only found in one animal were excluded. Data is presented in the
one minute time period in which the m/z is found dependent on which phase
was used, either formic acid or acetic acid.
199 Formic acid Acetic acid Formic acid Acetic acid phase phase phase phase Time Time Time Time m/z m/z m/z m/z period period period period
13,14 178.95 13,14 178.95 23,24 316.4 18,19 184.05 09,10 321 19,20 203.05 19,20 203.05 19,20 322.15 15,16 206 15,16 205.95 08,9 323.1 08,9 323.1 08,9 207.95 08,9 208 12,13 327.1 12,13 327.1 11,12 208.95 11,12 328.15 14,15 210.95 14,15 210.95 28,29 333.3 20,21 212.05 16,17 336.1 26,27 212.05 08,9 340.1 08,9 340.1 12,13 213 12,13 213 19,20 340.3 19,20 340.15 21,22 220.3 22,23 342.2 22,23 342.2 13,14 223.7 23,24 342.25 05,6 224.05 26,27 343.3 16,17 224.1 28,29 344.1 28,29 344.1 10,11 227 10,11 226.95 17,18 348.6 17,18 348.35 14,15 227.05 11,12 348.9 17,18 227.2 17,18 227.25 15,16 348.95 10,11 228.95 10,11 228.95 28,29 350.15 28,29 350.15 14,15 229 15,16 354.15 09,10 236.05 24,25 356.25 24,25 356.2 17,18 236.05 29,30 356.35 14,15 238.05 14,15 238.05 14,15 359 09,10 239.1 09,10 239.1 11,12 360.15 22,23 241.25 22,23 241.25 14,15 362.2 48,49 241.2 18,19 362.3 18,19 362.3 28,29 242.3 09,10 364.1 14,15 252.05 14,15 252.05 11,12 365.95 09,10 256.05 09,10 256.05 21,22 368.25 23,24 261.8 23,24 261.8 19,20 369.95 19,20 369.95 28,29 261.05 18,19 370.75 18,19 266.45 13,14 371.15 13,14 371.15 11,12 267.05 11,12 267.05 18,19 373.15 10,11 268.1 16,17 375.05 16,17 375.05 24,25 269.75 28,29 377.2 28,29 377.15 28,29 271.05 12,13 378 12,13 378 05,6 274.05 18,19 379.1 18,19 379.2 19,20 277.05 19,20 277.05 28,29 380.3 22,23 282.4 16,17 383.05 11,12 283.1 11,12 283.1 08,9 384 18,19 283.85 18,19 384.2 07,8 289 14,15 385.35 28,29 289.1 18,19 392.4 18,19 392.15 13,14 295.05 13,14 295.05 14,15 395 28,29 300.2 28,29 300.2 28,29 397.05
200 Formic acid Acetic acid Formic acid Acetic acid phase phase phase phase Time Time Time Time m/z m/z m/z m/z period period period period
18,19 300.3 18,19 401.25 18,19 401.25 28,29 304.25 13,14 404.15 09,10 313.95 18,19 406.35 16,17 316.05 16,17 316.05 08,9 407.1 08,9 407.05 21,22 316.3 28,29 407.15 28,29 407.15 14,15 409 17,18 475.35 23,24 412.25 15,16 476.3 15,16 476.3 13,14 413.1 13,14 413.05 16,17 476.3 18,19 414.95 18,19 414.95 16,17 478.1 14,15 415.2 19,20 479.15 16,17 416 18,19 480.45 18,19 480.25 26,28 416.2 20,21 480.3 12,13 419.15 22,23 481.35 22,23 481.45 18,19 423.35 18,19 423.35 28,29 482.45 28,29 424.25 28,29 424.25 33,34 482.5 14,15 425.2 15,16 485.05 18,19 428.25 21,22 486.55 15,16 430.1 15,16 430.1 12,13 487 12,13 487 17,18 431.3 17,18 487 23,24 432.1 19,20 487.9 19,20 487.9 16,17 433.95 18,19 489.2 18,19 489.4 11,12 434.1 11,12 434.1 14,15 491.05 14,15 491.35 14,15 434.2 14,15 434.1 09,10 493.3 12,13 434.15 19,20 494 19,20 493.8 14,15 435.15 10,11 496.15 10,11 496.15 19,20 436.4 27,28 496.35 27,28 496.3 18,19 437.1 18,19 437.1 17,18 497.4 11,12 439 18,19 500.15 15,16 439.1 15,16 439 10,11 501.15 21,22 439.2 21,22 439.4 19,20 502.35 19,20 502.35 26,28 441.15 21,22 502.85 12,13 442.1 18,19 503.05 18,19 503.55 19,20 443.75 19,20 443.6 16,17 503.35 18,19 445.25 17,18 504.115 17,18 504.25 14,15 448.25 16,17 505.15 16,17 505.15 19,20 449.8 32,33 505.5 26,27 450.4 18,19 506.1 18,19 506.1 17,18 453.3 17,18 453.3 19,20 506.25 15,16 454.2 09,10 507.4 17,18 455 21,22 508.5 12,13 456.05 19,20 511.55 15,16 456.1 21,22 512.3 23,24 456.4 28,29 512.4 18,19 458.5 18,19 458.4 21,22 516.25 21,22 516.25
201 Formic acid Acetic acid Formic acid Acetic acid phase phase phase phase Time Time Time Time m/z m/z m/z m/z period period period period
21,22 458.75 19,20 518.35 16,17 459.25 18,19 519.2 18,19 519.25 17,18 464.25 17,18 464.5 16,17 520.3 09,10 466.3 09,10 466.3 19,20 520.2 19,20 520.25 28,29 467.35 21,22 520.15 19,20 467.35 14,15 522.15 09,10 471.1 09,10 471.05 21,22 523.3 19,20 472.6 19,20 472 14,15 524.2 19,20 474.5 18,19 524.8 18,19 524.7 17,18 475.35 17,18 475.2 18,19 526.1 18,19 526.1 13,14 529.2 13,14 529.2 18,19 592.55 18,19 592.55 24,25 529.4 16,17 594.15 16,17 594.15 26,27 529.4 26,27 529.35 13,14 597.1 21,22 531.85 21,22 531.95 21,22 597.1 19,20 532.15 28,29 600.4 19,20 533.1 19,20 533.1 14,15 605.4 16,17 533.7 17,18 608.35 18,19 535.85 20,21 610.2 20,21 610.2 32,33 535.4 14,15 612.35 14,15 612.35 15,16 536.35 19,20 612.55 19,20 612.45 21,22 537.7 14,15 613.35 19,20 538.2 19,20 538.2 13,14 614.4 23,24 545.25 19,20 614.45 27,28 545.35 27,28 545.35 28,29 614.5 28,29 614.5 11,12 546.2 11,12 546.2 18,19 624.45 19,20 546.25 19,20 546.25 19,20 634.65 19,20 634.55 20,21 547.05 20,21 546.95 19,20 637.15 18,19 548.25 19,20 646.8 19,20 548.45 17,18 652.5 21,22 551 21,22 551 22,23 655.15 17,18 551.35 21,22 656.65 21,22 656.65 19,20 555.65 19,20 555.65 28,29 658.55 06,07 555.85 19,20 659.6 19,20 659.6 18,19 555.9 18,19 555.9 21,22 678.3 21,22 556.2 19,20 679.05 28,29 556.4 19,20 701.4 19,20 557.25 17,18 705.3 18,19 558.2 17,18 722.15 19,20 561.3 19,20 561.3 19,20 739.7 19,20 562.25 19,20 563.25 17,18 564.35 18,19 566.35 19,20 568.05 19,20 568.35
202 Formic acid Acetic acid Formic acid Acetic acid phase phase phase phase Time Time Time Time m/z m/z m/z m/z period period period period
28,29 570.35 17,18 574.15 13,14 575.2 19,20 576.35 19,20 576.2 23,24 577.4 23,24 577.45 16,17 580.35 23,24 581.25 19,20 581.85 19,20 582 28,29 583.8 18,19 588.45 17,18 588.45 19,20 588.45 18,19 589.4 19,20 590.55 17,18 591.4 21,22 591
203 Formic acid Acetic acid Formic acid Acetic acid phase phase phase phase Time Time Time Time m/z m/z m/z m/z period period period period
All m/z on this page were observed using negative scan mode. 19,20 187.1 - 15,16 454.2 - 10,11 223 - 10,11 222.9 - 15,16 456.15 - 16,17 225.05 - 13,14 475.1 - 12,13 235.05 - 15,16 476.3 - 18,19 236.05 - 18,19 487.35 - 17,18 236.1 - 19,20 492.4 - 19,20 239.05 - 10,11 493.3 - 22,23 242.2 - 18,19 497.4 - 18,19 497.4 - 09,10 243.15 - 19,20 504.1 - 18,19 252 - 09,10 507.3 - 18,19 261.25 - 18,19 511.5 - 19,20 277.05 - 20,21 512.4 - 23,24 277.15 - 23,24 277.15 - 19,20 518.25 - 19,20 518.25- 25,26 291.25 - 16,17 521 - 13,14 296.95 - 10,11 523.35 - 10,11 523.35 - 24,25 296.95 - 19,20 533.75 - 25,26 311.15 - 10,11 537.35 - 28,29 311.25 - 17,18 551.35 - 17,18 551.25 - 09,10 320 - 17,18 560.2 - 27,28 325.15 - 27,28 325.25 - 20,21 608.25 - 20,21 608.35 - 06,07 338.2 - 19,20 609.2 - 28,29 339.1 - 18,19 610.5 - 28,29 357.1 - 19,20 624.45 - 09,10 364.05 - 18,19 665.55 - 12,13 364.15 - 17,18 703.35 - 28,29 371.15 - 19,20 729.3 - 12,13 373.2 - 19,20 754.2 - 14,15 374.25 - 17,18 560.2 - 12,13 376 - 20,21 608.25 - 20,21 608.35 - 14,15 384.3 - 19,20 609.2 - 28,29 385.3 - 18,19 610.5 - 14,15 398.25 - 19,20 624.45 - 15,16 409.1 - 18,19 665.55 - 09,10 410.15 - 17,18 703.35 - 13,14 425.25 - 13,14 425.1 - 19,20 729.3 - 13,14 434.1 - 13,14 434.1 - 19,20 754.2 - 14,15 435.2 - 12,13 440.15 - 14,15 449.25 - 19,20 451.4 -
204