Theriogenology xxx (2014) 1–12

Contents lists available at ScienceDirect

Theriogenology
journal homepage: www.theriojournal.com

Review article

Relaxin: A hormonal aid to diagnose pregnancy status in

wild mammalian species
Don R. Bergfelt a, *, Augustine T. Peter b, Mohd A. Beg c
a
Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies
b
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA
c
King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: In the beginning of 1960s, seminal studies characterizing circulating concentrations of
Received 25 April 2014 immunoreactive relaxin in companion dogs and evaluating the differences in concentra-
Received in revised form 26 July 2014 tions among pregnant, nonpregnant, and pseudopregnant bitches indicated the potential
Accepted 27 July 2014
for relaxin to be applied clinically as a diagnostic aid to detect pregnancy status in wild
animal species. A brief historical overview of the nature of relaxin and early work to
Keywords:
develop and validate immunologic methods to analyze relaxin in the blood of rodents and
Conservation
pigs is initially discussed, which is followed by a summary of the development and vali-
Immunoassay
Pregnancy diagnosis dation of relaxin immunoassays to diagnose pregnancy in companion dogs and cats.
Relaxin Thereafter, observation of the pregnancy-specific increase in circulating concentrations of
Wild mammal relaxin in laboratory, companion, and farm animal species leads to discussion on the
application of radioimmunoassays, enzyme immunoassays, and a rapid immunomigration
assay to diagnose pregnancy in wild terrestrial (e.g., wolves, lions, elephants, rhinoceros,
panda) and marine (e.g., seals, dolphins) mammal species. A reference table is included
with a comprehensive list of numerous species and essential reagents that have been used
in various in-house and commercial immunoassays to successfully analyze relaxin quan-
titatively and qualitatively in blood (serum or plasma) and to some extent in urine.
Although the detection of relaxin concentrations has the potential to aid in the diagnosis of
pregnancy in many wild animal species, there are challenges in other species. Future ef-
forts should focus on validation of nonradiolabeled relaxin immunoassays for broader
application among species and improving techniques (e.g., extraction, purification) to
analyze relaxin in samples other than blood (e.g., urine, feces, saliva, blow, skin, blubber)
that can be collected in a less-invasive or -stressful manner and processed accordingly for
basic and applied purposes, especially with application toward conservation of threatened
or endangered species.
Published by Elsevier Inc.

1. Introduction Subsequently, in a classical endocrine-related experiment,

The hormone relaxin was discovered approximately
90 years ago by Hisaw [1] when he found that adminis-
tration of serum from pregnant guinea pigs or rabbits into
virgin guinea pigs induced relaxation of the pubic ligament.
* Corresponding author. Tel.: þ1703 731 6775; fax: þ1 703 349 6453.
E-mail address: drbergfelt.don@hotmail.com (D.R. Bergfelt).
administration of an aqueous extract of porcine CL into
ovariectomized guinea pigs relaxed the pubic ligament.
Thus, the physiological basis for the hormone “relaxin” was
first established [2].
Most of the early research to isolate and purify relaxin
was done with excised pig ovaries because they are a rich
source of relaxin, especially during pregnancy. In the early
1930s, it was difficult to establish relaxin as an independent
hormone because many researchers were skeptical of the

0093-691X/$ – see front matter Published by Elsevier Inc.

http://dx.doi.org/10.1016/j.theriogenology.2014.07.030
2 D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12
presence of a “third ovarian hormone” in addition to 2 major
steroids (i.e., estrogen and progesterone) [3]. This cynicism
resulted in a lull in research until the 1950s and early 1960s
when Steinetz et al. took advantage of advanced protein
isolation techniques and improved in vivo methods to purify
[4] and characterize relaxin [5]. At about the same time,
development of a more sensitive and precise in vitro tech-
nique such as radioimmunoassay (RIA) to quantify hormones
was developed [6]. With the validation of relaxin RIAs in a
few key species such as the pig, rat, and mouse [7–10] and
motivation for continued research, relaxin was established as
a vital hormone during pregnancy in the late 1980s.
In most species studied, the placenta appears to be the
greatest source of relaxin with a lesser extent produced by
other reproductive organs in females (e.g., CL, uterus) and
males (e.g., testes, prostate) as well as in nonreproductive or-
gans (e.g., heart, kidneys) [7–10]. Discovery of the pregnancy-
specific increase in circulating concentrations of relaxin
initially in the pig, rat, and mouse provided the inspiration to
investigate the basic and applied aspects of relaxin associated
with pregnancy in other laboratory, companion, domestic, and
wild animal species that continue till date.
The present review is not intended to be an exhaustive
or comprehensive review of the history, nature, or roles of
relaxin within and among various animal species of which
there are many [7–10]. Instead, the review primarily fo-
cuses on the seminal work of Steinetz et al. who first
characterized circulating concentrations of relaxin in dogs
[11] and, thereafter, patented [12] the first clinical appli-
cation of the canine relaxin assay as a hormonal approach
to distinguish between pregnant and nonpregnant or
pseudopregnant bitches [13]. Dr. Steinetz had envisioned
that his initial research on relaxin in the dog could be
applied to other companion, farm, and wild animal species
and make a difference in the conservation of threatened or
endangered wildlife. This review includes a historic over-
view of the nature of relaxin and early work to develop and
validate immunologic methods to analyze and characterize
relaxin for hormonal diagnosis of pregnancy in the com-
panion dog and cat. The initial sections are intended to
provide context for the latter sections of this review on the
latest research applied toward development and validation
of immunologic assays to detect and evaluate relaxin in
wild terrestrial and marine mammal species for the pur-
pose of diagnosing pregnancy status.
2. Hormonal diagnosis of pregnancy
Diagnosis of pregnancy is a hypothesis-based approach to
determine whether a female is nonpregnant or pregnant,
where the null hypothesis is the former and the alternative
the latter. The application of real-time ultrasonography is
currently considered the most definitive tool for researchers
and clinicians to test the hypothesis or diagnose pregnancy in
many companion [14–16], farm [17–19], and wild [20,21]
animal species. Although the technology is readily available,
it is relatively expensive to purchase and maintain, requires
the integration of technical and biological and/or anatomic
knowledge to capture and interpret images, and is generally
not applicable to wildlife species without physical or chemical
restraint, especially under field conditions. Before the advent
of ultrasonography, measurement of systemic concentrations
of progesterone less than (nonpregnant) or greater than
(pregnant) a baseline concentration was generally considered
the conventional or “gold standard” for hormonal diagnosis of
pregnancy in certain companion and farm animal species
[22–24] and still is considered preferable for many wild ani-
mal species [25–27].
Blood, urine, and, to some extent, feces are typically the
most common types of biological material for hormonal
analysis in wild animals to monitor general and reproductive
health as well as for research purposes [27,28]. Commercial
enzyme immunoassay (EIA) kits are widely available and have
been adapted and validated to measure progesterone or
progesterone metabolites in some of these matrices. Although
progesterone is considered a “gestational hormone,” an
elevation of concentrations on the basis of single-sample
analysis is not necessarily pregnancy specific. Circulating
concentrations comparable to pregnancy are also observed
during the luteal phase of the estrous cycle and during pro-
longed luteal maintenance, an abnormal condition that may
occur in nonpregnant or pseudopregnant animals [29]. Hence,
to distinguish pregnancy from nonpregnancy on the basis of
progesterone as the “gold standard,” it is necessary to collect a
repeat sample after a period from the first sample. If the
concentration of progesterone is sustained or has increased in
the repeat sample, then there is greater certainty that the
animal is pregnant. The collection of multiple repeat samples
may be done in wild animals under managed care but is
typically not done within the same reproductive cycle or
season in free-ranging animals, in part, because of logistical
reasons and state, federal, and international laws that regulate
research on wildlife.
In addition to progesterone, other pregnancy-associated
hormones have been used or investigated as potential diag-
nostic aids to determine pregnancy status, including estro-
gens in horses [30,31], chorionic gonadotropin in horses [32]
and primates [33], a chorionic gonadotropin–like substance in
bottlenose dolphins [34], prolactin in elephants [35] and dogs
[36,37], FSH in dogs [38], and PGFM in domestic and wild cats
[39]. Notably, these hormonal aids are only specific for certain
stages of pregnancy and may not be able to differentiate be-
tween pseudopregnancy because of embryo and/or fetal loss.
Besides hormones, pregnancy-associated factors have
been indicated to have some diagnostic value, which include
early conception factor [40], early pregnancy factor [41],
pregnancy-associated glycoproteins [42,43], fibrinogen, and
other acute-phase proteins [44]; C-peptide [38]; pregnancy-
specific protein B (www.biotracking.com); and cerulo-
plasmin [45]. For pregnancy diagnosis, pregnancy-associated
glycoprotein has been reported in cattle [46,47]; pregnancy-
specific protein B in cattle, goat, sheep, bison, deer, and elk
[48–52]; and ceruloplasmin in the giant panda [45]. Although
there appears to be a variety of pregnancy-related hormones
or factors as potential diagnostic aids to determine pregnancy
status, many are species specific and may not have broad
applicability across species.
3. Relaxin
The structure and heterogeneity of relaxin among spe-
cies have been reviewed [7–10]. Genomically, relaxin is
 D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12
encoded by 3 genes, RLN1, RLN2, and RLN3, in humans and
nonhuman primates; rodents have 2 genes (RLN1 and
RLN3). In reference to humans, the 3 forms of relaxin are
commonly referred to as H1-, H2-, and H3-relaxin [53]. The
most predominant form and that which is found in the
circulatory system is H2-relaxin. In general, relaxin is a 6-
kDa heterodimeric polypeptide with an A chain of 24
amino acids and B chain of 29 amino acids joined by 3 di-
sulfide bonds with an intrachain disulfide loop in the A
chain in a manner analogous to that of insulin. In regard to
the latter, identification of the primary structure of relaxin
from different animal species suggests that relaxin is part of
a larger family of relaxin-like factors that belong to the
insulin superfamily [54]. In addition, although the size and
2-chain composition of relaxin are similar among species,
there appears to be limited (<76%) evolutionary conser-
vation of the amino acid sequence of relaxin among the
approximately 25 species in which the sequence of relaxin
is known [7].
Historically, the development of immunoassays for
relaxin has been hampered by the diversity of the primary
structure of relaxin among species [7,55,56]. Initially, the lack
of tyrosine and histidine in porcine relaxin made it impos-
sible to correctly radiolabel the hormone with 125iodine.
However, since the structural characterization of porcine
relaxin [57], it has been correctly labeled by chemical addi-
tion of 125iodine-labeled tyrosine [58]. Coupled with a
species-specific relaxin antibody, a porcine RIA was devel-
oped and validated. Species-specific RIAs have since been
developed for the rat [59–61], horse [62], human [63], and
dog [64]. By adapting procedures and reagents originally
intended for one species, RIAs for relaxin have been devel-
oped and validated for other species. Notably, rabbit anti-
porcine relaxin (R6) was found to cross-react with relaxins of
many species because of its affinity for a conserved receptor-
binding domain [59]. Hence, the R6 antiserum has been used
to develop and validate RIAs for numerous wild animal
species as summarized in Table 1.
Relaxin is produced by multiple tissues as a preprorelaxin
precursor, which is processed to the mature bioactive
hormone [65]. Relaxin is recognized as a multifunctional
endocrine and paracrine hormone and/or factor with many
roles in female and male reproduction, as a neuropeptide in
the central nervous system, as a vasodilator and cardiac
stimulant in the cardiovascular system, and as an antifibrotic
agent [7–10]. Although relaxin is produced in highest con-
centrations by female reproductive tract tissues (e.g.,
placenta, CL, uterus) during pregnancy, the primary source
varies among species [7–10]. A major source of relaxin is the
placenta in the golden hamster, rabbit, dog, and horse; CL
in the mouse, rat, pig, and humans; and uterus in the
guinea pig.
Temporal changes in circulating concentrations of
immunoreactive relaxin during pregnancy and the peri-
partum period appear to be species specific. Generally,
among laboratory (e.g., rats, rabbits) [66], farm (e.g., pigs,
sheep) [67,68], and wild (e.g., elephants, rhinoceros, leop-
ards) [69,70] animals, serum or plasma concentrations of
relaxin are relatively low, intermediate, or high from early- to
mid-pregnancy with a gradual or marked surge-like increase
during late pregnancy, before parturition. Thereafter, relaxin
3
returns to low concentrations during the postpartum period.
Regardless of the diversity in the pattern or fluctuation of
circulating concentrations of relaxin during pregnancy,
relatively higher concentrations preceding parturition fol-
lowed by lower concentrations postpartum appear to be
consistent among many species. In some species, increasing
concentrations of relaxin before parturition occur concomi-
tantly with decreasing concentrations of progesterone. In
controlled studies that have examined the role of relaxin in
relation to progesterone in laboratory (e.g., rats, rabbits) [66]
and farm (e.g., pig, sheep) [68] animals, it has been suggested
that the decrease in progesterone before parturition repre-
sents a necessary withdrawal of the ‘‘progesterone block’’
that has maintained the gravid uterus in a quiescent state.
Correspondingly, the increase in relaxin represents a mech-
anism to maintain myometrial quiescence and protect
against premature contractions and delivery.
4. Companion dog
The first canine relaxin RIA was developed and validated
for the companion dog with the use of antiporcine relaxin
[11,58,59]. Table 1 provides a summary of the historic
progression and current use of relaxin immunoassays in
the dog. Although modifications were done to improve the
efficiency and effectiveness of subsequent canine RIAs
[13,71,72], antiporcine relaxin was still the primary anti-
body until development of synthetic canine relaxin and
production of anticanine relaxin [64,73]. Synthetic canine
relaxin was also used to prepare radiolabeled ligand and
reference standards, all of which improved reliability,
specificity, and sensitivity of the RIA [64].
In pregnant bitches [11,71], relaxin was first detected in
daily plasma samples as an increase in concentrations around
3 to 4 weeks after mating with maximum concentrations
around 5 to 7 weeks. Thereafter, concentrations either
remained elevated or decreased before parturition (gestation
length approximately 9 weeks). In nonpregnant and pseu-
dopregnant bitches [11], relaxin concentrations were below
detectable limits of the RIA throughout the 9 weeks of
observation. Notably, diurnal changes associated with circu-
lating concentrations of relaxin were not observed [74].
On the basis of the detectable increase in relaxin during
pregnancy in the bitch, it is thought that relaxin is pro-
duced in association with implantation of the embryo and
development of the fetal-placenta unit [74]. In this regard,
it has been shown that the placental syncytiotrophoblast
is a major source of relaxin in dogs [13,72]. Discovery of
preprorelaxin localization in the canine placenta and
determination of its nucleic acid sequence [75] confirmed
earlier observations that the placenta is likely the major
source of relaxin in the bitch. Relaxin is also produced to a
lesser extent by the ovary [13] as evidenced by a decrease in
serum relaxin in ovariectomized pregnant bitches [13]. An
ovarian source of relaxin is further supported by the
detection of relaxin and corresponding receptors in luteal
tissue collected during pregnancy [76]. Because the embryo
and/or fetoplacental unit appears to be a major source of
relaxin in bitches, it has been suggested that a decrease in
circulating concentrations of relaxin can also be used to
detect pregnancy loss in dogs [77]. In this regard, serum
Table 1

4
Reference list of primary reagents used in RIA, EIA, and RIM assays for analysis of relaxin in companion dogs and cats and various wild terrestrial and marine mammal species.

Species Assay Relaxin-labeled Relaxin primary Relaxin reference Biological matrices Matrix suitabilitya Citation
format antigen antibody standard
Companion dogs and cats
Dog (Canis familiaris) RIA Porcine Rabbit antiporcine (R6) Porcine Plasma, serum Pos (all) [11,13]
RIA Canine Rabbit anticanine (79888) Canine Serum Pos [64]
RIA Porcine Rabbit antiporcine (5859) Canine Plasma Pos [71]
RIA Canine Rabbit antiporcine (SMA-1) Canine Plasma Pos [72]
RIA Canine Rabbit antiporcine (SMA-1) Canine Serum Pos [73]
EIA Porcine Rabbit antiporcine (258) Human recombinant H2, Serum Pos [78]
porcine
EIAb None Rabbit anticanine and None Plasma Pos [109]
anticanine conjugated to
peroxidase
Cat (Felis silvestris catus) RIA Porcine Rabbit antiporcine (5858, Porcine Plasma Pos [80,90]
R6)
RIA Canine Rabbit anticanine (79888) Canine Urine Pos [81,83]

D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12

RIMb None Rabbit anticanine bound to None Plasma, serum, urine Pos (all) [81,83,110]
colloidal gold
Wild terrestrial mammals
Spotted hyena (Crocuta RIA Porcine Rabbit antiporcine (R6) Porcine (B29) Serum, ovarian extracts, Pos (all) [92]
crocuta) placental tissue extract
Maned wolf (Chrysocyon RIA Canine Rabbit anticanine (79888) Canine Serum, urine Pos (all) [70]
brachyurus)
b
Coyote (Canis latrans) EIA None Rabbit anticanine and None Plasma Pos [89]
anticanine conjugated to
peroxidase
Gray wolf (Canis lupus) EIAb None Rabbit anticanine and None Plasma Pos [84,88]
anticanine conjugated to
peroxidase
Mexican gray wolf (Canis EIAb None Rabbit anticanine and None Plasma Pos [88]
lupus baileyi) anticanine conjugated to
peroxidase
Red wolf (Canis rufus) EIAb None Rabbit anticanine and None Plasma Pos [88]
anticanine conjugated to
peroxidase
Fennec fox (Vulpes zerda) EIAb None Rabbit anticanine and None Plasma Pos [88]
anticanine conjugated to
peroxidase
Island fox (Urocyon EIAb None Rabbit anticanine and None Plasma Pos [88]
littoralis) anticanine conjugated to
peroxidase
African wild dog (Lycaon EIAb None Rabbit anticanine and None Plasma Pos [88]
pictus) anticanine conjugated to
peroxidase
Leopard (Panthera RIA Canine Rabbit anticanine (79888) Canine Urine Pos [81]
pardus) RIMb None Rabbit anticanine bound to None Urine Neg [83]
colloidal gold
Pallas cat (Otocolobus RIA Canine Rabbit anticanine (79888) Canine Urine Pos [81]
manul) RIMb None Rabbit anticanine bound to None Urine Pos [83]
colloidal gold
 Sand cat (Felis margarita) RIA Canine Rabbit anticanine (79888) Canine Urine Pos [81]
RIMb None Rabbit anticanine bound to None Urine Neg [83]
colloidal gold
Cheetah (Acinonyx RIA Canine Rabbit anticanine (79888) Canine Urine Pos [81]
jubatus) RIMb None Rabbit anticanine bound to None Urine Neg [83]
colloidal gold
Lion (Panthera leo) RIA Canine Rabbit anticanine (79888) Canine Urine Pos [81]
RIMb None Rabbit anticanine bound to None Urine Neg [83]
colloidal gold
Iberian lynx (Lynx RIMb None Rabbit anticanine bound to None Serum, urine Pos (serum), Neg (urine) [85]
pardinus) colloidal gold
Asian elephant (Elephas RIA Equine Rabbit antiequine (3150) Equine Plasma Pos [35,69,93]
maximus)
African elephant RIA Equine Rabbit antiequine (3150) Equine Serum Pos [35,69]
(Loxodonta africana)
Sumataran rhinoceros RIA Porcine Rabbit antiporcine (R6) Porcine Serum Pos [69]
(Dicerorhinus
sumatrensis)

D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12

Giant panda (Ailuropoda RIA Porcine Rabbit antiporcine (R6) Porcine Serum, urine Pos (serum), Neg (urine) [69]
melanoleuca)
Alpaca (Vecugna pacos) EIA Porcine Rabbit antiporcine (258) Porcine Plasma, saliva, milk, urine Pos (plasma) [87]
Neg (saliva)
Neg (milk)
Neg (urine)
Rhesus monkey (Macaca RIA Porcine Rabbit antiporcine (R6) Porcine Serum Pos [95]
mulatta)
Baboon (Papio RIA Porcine Rabbit antiporcine (R6) Porcine Plasma, amniotic fluid, Pos (all) [96]
cynocephalus) decidua extracts, placental
tissue extracts
Chimpanzee (Pan RIA Porcine Rabbit antiporcine (R6) Porcine Serum Pos [97]
troglodytes)
Marmoset monkey RIA Porcine Rabbit antiporcine (R6) Porcine Serum Pos [98]
(Callithrix jacchus)
Marmoset monkey EIA Porcine Rabbit antiporcine (258) Porcine Serum Pos [99]
(Callithrix jacchus)
Wild marine mammals
Northern fur seal RIA Synthetic canine Rabbit anticanine (80037) Synthetic canine Serum, placental tissue Pos (all) [103]
(Callorhinus ursinus) extract
Steller sea lion RIA Synthetic canine Rabbit anticanine (80037) Synthetic canine Placental tissue extract Pos [103]
(Eumetopias jubatus)
Harbor seal (Phoca RIA Synthetic canine Rabbit anticanine (80037) Synthetic canine Placental tissue extract Pos [103]
vitulina)
Bottlenose dolphin RIA Human recombinant H2 Rabbit antiporcine (R6) Synthetic canine Serum, placental tissue Pos (all) [106]
(Tursiops truncatus) extract
Antarctic minke whale RIA Porcine Sheep antiporcine (S540) Porcine Luteal tissue extract Pos [105]
(Balaenoptera
acutorostrata)
Bryde’s whale RIA Porcine Sheep antiporcine (S540) Porcine Luteal tissue extract Pos [105]
(Balaenoptera edeni)

Sources and availability of reagents can be found in respective references that have been cited.
Abbreviations: EIA, enzyme immunoassay; RIA, radioimmunoassays; RIM, rapid immunomigration.
a
Positive or negative for the immunoassay of relaxin in respective matrices on the basis of results in corresponding citations.
b
Commercial EIA (ReproCheK) or RIM (Witness) assay kits for relaxin are available from Synbiotics Corp, San Diego, CA, USA.

5
6 D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12
relaxin decreased in association with pregnancy loss
related to uterine pathology [78,79].
In 1992, a patent was awarded to Steinetz et al. [12] on the
basis of the clinical application that a pregnancy-specific
increase in circulating concentrations of relaxin can be
used as a diagnostic aid for early pregnancy detection in
companion dogs and cats. As a result, commercial EIA and
rapid immunomigration (RIM) assay kits (ReproCheK and
Witness, respectively; Synbiotics Corp, San Diego, CA, USA)
have been developed, validated, and adapted for use in
numerous animal species (Table 1) to give a fast qualitative
indication of whether concentrations of relaxin have
increased greater than a baseline concentration indicative of
pregnancy.
5. Companion cat
In the companion cat (Felis silvestris catus), a porcine
relaxin RIA has been used to characterize serum [80,81]
and urine [81] concentrations of relaxin (Table 1). Sam-
ples were collected daily or every 3 or 4 times per week
[80] or once weekly [81] during pregnancy. In both serum
and urine, an increase in relaxin was detected 21 to 25 days
after mating. Thereafter, concentrations peaked 35 to
49 days before declining about 2 weeks before parturition
(gestation length approximately 63 days or 9 weeks). Re-
sults suggested that urinary relaxin in the cat is pregnancy
specific because only pregnant queens excreted detectable
concentrations of relaxin in contrast to undetectable con-
centrations in mated nonpregnant queens. The urinary
profiles of relaxin concentrations on the basis of RIA anal-
ysis paralleled that of serum, which further justified the use
of urine as a reliable noninvasive approach for pregnancy
diagnosis in companion cats.
In addition to RIA analysis, a relaxin commercial RIM
assay kit (Witness) has been used successfully with urine to
detect pregnancy in the companion cat (Table 1) [82,83].
Although the RIM assay was originally designed for use
with serum or plasma as a qualitative test to diagnose
pregnancy in dogs and cats, the Witness kit was shown to
be reliable with cat urine after diluting samples with equal
parts of nonpregnant cat serum [83]. All known pregnant
queens tested positive, whereas nonpregnant queens
tested negative. Application of nonradiolabeled commer-
cial EIA and RIM kits in companion dogs and cats has been
shown to be valid for relaxin and pregnancy diagnosis
within the same family of canidae [84] and felidae [85] for
conservation purposes as listed in Table 1 and discussed in
the next section.
6. Wildlife
For the conservation and survival of wild animal species
that are threatened or endangered and at the threshold of
extinction, zoos, aquariums, and other managed care habi-
tats are essential for gaining fundamental knowledge of the
reproductive biology of these species [86]. A comprehensive
understanding of the physiology and endocrinology of
reproduction is a prerequisite for a sustainable and produc-
tive conservation program. The capability to diagnose and
evaluate pregnancy status is a basic requirement to manage
maternal and embryo and/or fetal development for the
successful delivery of offspring and neonatal care thereafter.
However, the methods for diagnosis and evaluation of
pregnancy in wild animals are only practical if they can be
applied in a manner that avoids or minimizes stress-related
responses, which can potentially disrupt ovulation or jeop-
ardize pregnancy [87]. The collection of blood samples usu-
ally require some form of physical or chemical restraint,
whereas the collection of urine, feces, saliva, hair, skin, or
blubber samples can typically be done without interfering
with the animal or remotely (e.g., biopsy darting) without
capture, restraint, and release. Thus, although noninvasive
hormone monitoring of wild animals under managed care or
free-ranging conditions is an important tool for evaluating
an animal’s reproductive status [27], there are many chal-
lenges associated with collecting samples in a safe manner
while minimizing animal stress and sample contamination,
processing or preserving samples for short- or long-term
storage or transportation, and interpreting assay results
that may be confounded by metabolites or matrix effects.
Immunoassay of relaxin has mostly been done using
blood (serum or plasma) from various species; however,
analysis has also been done to some extent with urine, milk,
and saliva in various domestic and wild animals (e.g., dog,
cat, wolf, leopard, Pallas cat, sand cat, cheetah, lion, lynx,
panda, alpaca, and dolphin). The use of serum, plasma, and,
to some extent, urine to develop and validate immunoassays
with different combinations of relaxin and antirelaxin from
human, dog, pig, and horse to diagnose pregnancy status in
wild mammalian species are discussed in the following
sections in concordance with Table 1.
6.1. Wild terrestrial mammals
6.1.1. Canids
In female gray wolves (Canis lupus) under managed care,
use of the commercial EIA ReproCheK kit (Table 1) accu-
rately distinguished pregnancy from nonpregnancy in all
animals [84]. Use of the ReproCheK kit for pregnancy
diagnosis was further evaluated in gray wolves, Mexican
gray wolves (Canis lupus baileyi), red wolves (Canis rufus),
fennec foxes (Vulpes zerda), and African wild dogs (Lycaon
pictus) under managed care and free-ranging island foxes
(Urocyon littoralis) [88]. Corresponding to those animals
confirmed pregnant with ultrasound or observed with
pups, the relaxin EIA was positive for gray wolves, fennec
foxes, and island foxes greater than 25 days after mating.
Application of the ReproCheK kit less than 25 days resulted
in an approximately 8% false-negative rate in island foxes
on the basis of a positive ultrasound examination or
observation of pups. The relaxin EIA was negative for red
wolves and wild dogs, which was supported by no obser-
vation of pups. The false-positive rate was nil on the basis of
negative test results in known nonmated Mexican gray
wolves, red wolves, and island foxes.
In coyotes (Canis latarans) [89], results of the relaxin EIA
ReproCheK kit (Table 1) with plasma samples indicated all
control animals (i.e., males and females that were not
mated or were with castrated males) tested negative for
relaxin, whereas all mated animals with pups tested posi-
tive approximately 28 days after mating. In mated coyotes
 D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12
without pups, 87% tested negative for relaxin and 13%
tested positive. The basis for the 13% that tested positive
could not be clarified because pregnancy or pregnancy loss
could not be confirmed. Nonetheless, the combined results
indicated that a commercial EIA for canine relaxin can be
used with serum or plasma as a reliable method for
detection of pregnancy in wild canid species beginning
greater than 25 days after mating.
The potential for using urine from wild canids was
initially indicated by the detection of a relaxin-like sub-
stance in the urine of a pregnant maned wolf (Chrysocyon
brachyurus) using a canine relaxin RIA (Table 1) [70]. Future
studies are required to confirm and broaden the application
of immunoassay of relaxin in urine in this and other canid
species.
6.1.2. Felids
In leopards (Panthera pardus) [81], a canine relaxin RIA
(Table 1) run with weekly urine samples was found to be a
reliable method for pregnancy diagnosis on the basis of
detection of relaxin 25 to 28 days after mating with peak
concentrations at 60 to 64 days of pregnancy. Thereafter,
concentrations declined before parturition (gestation length
approximately 101 days). Correspondingly, the canine
relaxin RIA run with urine samples differentiated nonpreg-
nant from pregnant sand cats (Felis margarita), Pallas cats
(Octocolobus manul), cheetahs (Actinonyx jubatus), and lions
(Panthera leo) [83]. However, in comparison to peak con-
centrations in companion cats (range, 14–30 ng/mL) at
midpregnancy, concentrations were relatively greater in
pregnant sand and Pallas cats (range, 21–64 ng/mL), com-
parable in pregnant cheetahs (range, 5–17 ng/mL), and lower
in pregnant lions (range, 6–10 ng/mL). In contrast, applica-
tion of the commercial RIM Witness kit did not detect relaxin
in unadulterated urine from a pregnant leopard or in urine
diluted with nonpregnant leopard serum (personal com-
munication cited in Harris et al. [83]). Correspondingly, the
RIM assay was used with urine from sand cats, Pallas cats,
cheetahs, and lions but was not successful in differentiating
pregnant from nonpregnant animals [83].
In Iberian lynx (Lynx pardinus), application of the RIM
Witness kit (Table 1) was successful using serum in which
relaxin was detected in all pregnant cats between 32 and
56 days after mating [85]. In contrast, the RIM assay kit did
not detect relaxin in freshly collected or frozen samples of
lynx urine at the same stage of pregnancy. However, by
concentrating the pregnant and nonpregnant urine sam-
ples about 50-fold using ultrafiltration, the RIM assay
detected relaxin as a weak reaction for pregnancies greater
than or equal to 29 days but not those less than 29 days or
in nonpregnant lynx. Apparently, development of processes
to filter and concentrate urine need to be explored to
improve the suitability of urine for immunoassay of relaxin
using the RIM Witness assay kit in this and other felids.
Temporally, the increase in serum, plasma, and urine
concentrations of relaxin using RIA corresponds with the
placental production of relaxin beginning approximately
20 days of pregnancy in the companion cat [90]. More
recently [91], DNA and protein sequences of relaxin from
placental tissue of an Iberian lynx were determined and
used to generate a felid-specific relaxin antibody. In tissues
7
collected from pregnant lynx and companion cat, immu-
nohistochemical and mRNA expression of the relaxin
ligand and receptor was stronger in the placenta compared
with the ovary and uterus. Thus, the combined results
provide evidence that the placenta is a major source of
relaxin during pregnancy in some felids.
6.1.3. Other carnivores
In female spotted hyenas (Crocuta crocuta) under
managed care, a porcine relaxin RIA (Table 1) was used to
characterize circulating concentrations of relaxin in serum
samples collected at various times during pregnancy
(gestation length approximately 110 days) [92]. Relative to
parturition (Day 0), concentrations were near or below the
detectable limits of the assay during early pregnancy (103
to 87 days). During midpregnancy (55 to 42 days),
there was an increase in relaxin that progressively rose to
peak concentrations near parturition (19 to 0 days).
Relaxin was not detected in prepubertal, adult, nonpreg-
nant female or male hyenas; results were below the
detectable limits of the RIA.
In extracts of placental and ovarian tissue from a preg-
nant hyena, relaxin concentrations were higher in the
placenta compared with those of the ovary [92]; thus, the
placenta appears to be a major source of relaxin during
pregnancy in this species.
In Giant pandas (Ailuropoda melanoleuca) under managed
care, use of a porcine relaxin RIA (Table 1) detected serum
concentrations of relaxin during the last 3 weeks of preg-
nancy (gestation length approximately 19 weeks) but not
during nonpregnancy or in males [69]. In contrast, analysis of
urine resulted in inconsistencies in the relative changes
during pregnancy compared with that of serum. The basis for
the inconsistent changes in concentrations between serum
and urine is not known but may be related, in part, to
metabolized or degraded forms of relaxin in the urine at
different stages of pregnancy or that other urinary compo-
nents interfered with the immunoanalysis. Perhaps, as in
some felids [85], filtering and concentrating the urine would
result in more consistent results. Nonetheless, future studies
are needed to clarify the use of urine for analysis of relaxin in
pandas.
6.1.4. Elephants, rhinoceros, and alpacas
In pregnant Asian elephants (Elephas maximus) and Af-
rican elephants (Loxodonta africana) under managed care,
use of an equine relaxin RIA (Table 1) indicated higher serum
concentrations of relaxin approximately 20 weeks of preg-
nancy that remained elevated until 26 weeks [35,69].
Thereafter, concentrations decreased until a second surge of
relaxin occurred as parturition approached (gestation length
approximately 92 weeks). In male Asian elephants, plasma
concentrations of relaxin were not detected; however, rela-
tively low concentrations were detected in nonpregnant fe-
males during the estrous cycle [93]. In an Asian elephant
with pregnancy loss, a precipitous decrease in relaxin was
observed approximately 1 week before spontaneous abor-
tion [93]. In this regard, relaxin may also be used to detect
pregnancy loss in elephants as suggested in the dog [77–79].
In Sumatran rhinoceroses (Dicerorhinus sumatrensis)
under managed care, use of a porcine relaxin RIA (Table 1)
8 D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12
detected serum concentrations of relaxin by approximately
6 months of pregnancy [69]. Thereafter, concentrations
increased by 8 months and reached highest concentrations
2 weeks before parturition (gestation length approximately
62 weeks). Serum relaxin concentrations remained
elevated 4 days after parturition and were also detected in
maternal milk samples.
In pregnant alpacas (Vicugna pacos), use of a porcine
relaxin RIA (Table 1) indicated serum concentrations of
relaxin increased by 2 months of pregnancy but declined by
5 to 8 months [94]. Thereafter, concentrations increased until
parturition (gestation length approximately 12 months).
Relaxin was also examined in nonpregnant and pregnant
alpacas using a modified EIA (Table 1) [99] to determine the
potential to detect concentrations in milk, urine, and saliva
[87], which can be collected in a less-stressful manner than
blood. Plasma concentrations of relaxin increased after the
second month of pregnancy, whereas there were no differ-
ences in saliva and milk concentrations between nonpreg-
nant and pregnant alpacas. Relaxin EIA results with urine
were below the detectable limits of the assay.
6.1.5. Nonhuman primates
A porcine relaxin RIA (Table 1) was used to analyze
relaxin in nonhuman primates. In rhesus monkeys (Macaca
mulatta), plasma concentrations of relaxin were detected
from 3 weeks of pregnancy to parturition (gestation length
approximately 23 weeks) but not during nonpregnancy or
in males [95]. In baboons (Papio cynocephalus), high plasma
concentrations of relaxin were detected during pregnancy
[96]. In chimpanzees (Pan troglodytes), higher relaxin con-
centrations were detected during the first trimester of
pregnancy compared with lower concentrations during the
late luteal phase of the menstrual cycle [97]. In marmosets
(Callithrix jacchus), a porcine RIA [98] and EIA [99] (Table 1)
detected serum relaxin concentrations during early preg-
nancy (2–3 weeks) which continued to increase to peak
concentrations during midpregnancy and, thereafter,
gradually decreased as parturition (gestation length ap-
proximately 20 weeks) approached. In nonpregnant mar-
mosets, relaxin was not detected during the reproductive
cycle [98].
In pregnant ovariectomized rhesus monkeys and ba-
boons, removal of the ovaries or the CL-bearing ovary
resulted in nondetectable serum concentrations of relaxin
[95,96]. Thus, the CL seems to be the major source of relaxin
during pregnancy in some primates.
6.2. Marine mammals
6.2.1. Pinnipeds
In pinnipeds, studies documenting the detection of
immunoreactive relaxin could not be found in the scientific
literature. Therefore, to develop and validate a relaxin RIA
for use in seals, it was first considered how this species of
carnivore might be related to other carnivores, in which
relaxin has been detected. Anatomically, it was found that
the chorioallantoic placenta of pinnipeds is comparable
with many other carnivores [100]. In this regard, canids and
pinnipeds have a zonary endothelial–chorial placentation
[101,102]. Thus, the evolutionary proximity between the
dog and seal provided a reasonable basis to evaluate the
potential of a previously validated canine relaxin RIA using
anticanine relaxin [64] to detect and characterize a relaxin-
like substance in pinnipeds (Table 1).
Development and validation of a canine relaxin RIA for
use in managed care and wild Northern fur seals (Cal-
lorhinus ursinus), Steller sea lions (Eumetopias jubatus), and
harbor seals (Phoca vitulina) [103] were adapted from a
canine relaxin RIA [64]. In fur seals under managed care,
relaxin concentrations were highest in pools of serum from
pregnant seals compared with nonpregnant and post-
partum female and male seals. Higher concentrations of
relaxin in pregnant compared with nonpregnant and
postpartum fur seals are supportive of the pregnancy-
specific nature of relaxin in this species. Temporally, con-
centrations of relaxin were relatively low during Months 4
to 5 of pregnancy and high during Months 7 to 12 as
parturition approached (gestation length approximately
12 months). Notably, low concentrations during early-to
mid-pregnancy (Months 4–5) correspond with the end of
embryonic diapause, a period of delayed implantation that
reportedly ranges from 3 to 5 months in Northern fur seals
[104].
In extracts of placental tissue from a wild Northern fur
seal, Steller sea lion, and harbor seal, relaxin was readily
detected [103]. Because it is not known if other reproduc-
tive tissues (e.g., CL, uterus) produce relaxin in high
amounts, it remains to be clarified if the placenta is the
major source of relaxin in these species.
6.2.2. Cetaceans
In cetaceans, relaxin immunoreactivity was first re-
ported in extracts of luteal tissue from pregnant Antarctic
minke (Balaenoptera acutorostrata) and Bryde’s (Balae-
noptera edeni) whales [105] using a porcine relaxin RIA
with sheep antiporcine relaxin (Table 1). Relaxin bioactivity
from the 2 whales appeared similar to porcine relaxin as
determined by a mouse pubic symphysis relaxin assay [5].
Structurally, among whales, relaxin differed by about 3
residues, whereas, among species, the relaxin of the Bryde’s
whale differed at only one position from that of pig relaxin.
Thus, the results in whales provided a basis to evaluate the
potential of using antiporcine relaxin to develop a relaxin
RIA to detect and characterize a relaxin-like substance in
managed care and wild bottlenose dolphins.
Development and validation of a relaxin RIA for use in
bottlenose dolphins (Tursiops truncatus) consisted of H2
human relaxin as radiolabeled hormone, rabbit antiporcine
relaxin as the primary antibody, and synthetic canine
relaxin as reference standard [106] (Table 1). Temporally,
changes in circulating concentrations of relaxin were
characterized throughout 12 months of pregnancy in dol-
phins under managed care, which included dolphins with
live births and perinatal loss (i.e., late-term abortion or
stillbirth). Regardless of pregnancy outcomes, serum con-
centrations of relaxin progressively increased from rela-
tively low concentrations during early pregnancy (Months
1–4), intermediate concentrations during midpregnancy
(Months 5–8), and high concentrations during late preg-
nancy (Months 9–12). Subsequent to parturition, relaxin
returned to low concentrations during the postpartum
 D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12
period. Hence, relatively high concentrations of relaxin
during the prepartum period versus postpartum period are
supportive of the pregnancy-specific nature of relaxin in
this species. In pregnancies resulting in late-term abortion
or stillbirth, mean relaxin concentrations were 42%, 29%,
and 34% lower at early, mid, and late pregnancy, respec-
tively, compared with pregnancies resulting in live births.
In wild dolphins, there is a degree of uncertainty to di-
agnose pregnancy on the basis of single-sample analysis of
serum progesterone because a relatively high concentra-
tion in a sole sample is not necessarily pregnancy specific.
To increase the hormonal diagnostic certainty of preg-
nancy, a study was conducted to analyze relaxin and pro-
gesterone in the same serum samples collected from
populations of free-ranging dolphins [107]. Hormone
pregnancy diagnosis was based on concentrations of
relaxin and progesterone above respective baseline con-
centrations. Observed pregnancy diagnosis was based on
photoidentification of cow-calf pairs subsequent to sample
collections. Concordance between hormone and observed
diagnosed pregnancies was 100% for progesterone and 78%
for relaxin. The basis for lower predictability with relaxin
was attributed to samples collected during the first
trimester of pregnancy at which time concentrations are
relatively low [106]. Although the diagnostic value of
relaxin alone may be limited during early pregnancy
(Months 1–4), the combination of relatively high concen-
trations of relaxin and progesterone in a single-sample
analysis thereafter has the potential to provide greater
confidence to diagnose pregnancy than if either hormone
was used alone.
A preliminary analysis of relaxin in urine from dolphins
under managed care was conducted with the expectation
that the less-invasive nature of collecting urine compared
with blood would allow for more frequent serial collection
of hormonal data (personal communication, Bergfelt).
Concentrations of relaxin in increasing volumes of pools of
unextracted and acid-acetone–extracted [4] urine from
pregnant, nonpregnant, and male dolphins paralleled the
relaxin standard reference curve, thus indicating the suit-
ability to analyze relaxin in unextracted urine samples.
However, relative changes of a relaxin-like substance in
unextracted urine from individual dolphins during preg-
nancy were not consistent with the relative changes
observed in serum during pregnancy [106]. Additional
studies are needed to determine if acid-acetone–extracted
urine samples from individual dolphins during pregnancy
would be more consistent or if other matrices (e.g., blow)
may be more useful [108] for frequent noninvasive collec-
tion and analysis of relaxin in dolphins and other cetaceans.
Relaxin was readily detected in extracts of placental
tissue from multiple dolphins [106]. However, because
relaxin has also been detected in extracts of luteal tissue
from pregnant whales [105], the major source of relaxin
during pregnancy remains to be clarified in cetaceans.
7. Conclusions
Currently, immunologic methods (RIA, EIA, and RIM) to
successfully analyze relaxin quantitatively and qualitatively
in wild animal species have been adapted using different
9
combinations of relaxin and antirelaxin from human,
companion, and farm animal species. The potential for
immunoreactive relaxin to serve as an aid in the diagnosis
of pregnancy status is on the basis of detection primarily in
blood and to some extent in urine of which concentrations
are higher in pregnant than nonpregnant (postpartum)
animals. Although the increase in circulating concentra-
tions of relaxin appears to be pregnancy specific among the
various terrestrial and marine mammals evaluated thus far,
the primary source of production (e.g., placenta, CL, uterus)
and timing of the increase during pregnancy are different
among species. In some species, the increase in relaxin
seems to coincide with implantation of the embryo and
development of the fetal-placenta unit. Future efforts
should focus on validation of nonradiolabeled relaxin im-
munoassays for broader application among species and
improving techniques (e.g., extraction, purification) to
analyze relaxin in samples other than blood (e.g., urine,
feces, saliva, blow, skin, blubber) that can be collected in a
less-invasive or -stressful manner and processed accord-
ingly for basic and applied purposes, especially with
application toward conservation of threatened or endan-
gered species.
Acknowledgments
The authors would like to pay tribute to Dr. Bernard G.
Steinetz for his pioneering work to characterize the phys-
iological role of relaxin and his vision to realize the appli-
cation of relaxin as a clinical aid to initially differentiate
between pregnant and nonpregnant dogs and cats and,
thereafter, as a conservation tool to diagnose pregnancy
status in numerous wild animal species, especially those
that may be threatened or endangered.
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