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Theriogenology xxx (2014) 1–12

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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
Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA
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
develop and validate immunologic methods to analyze relaxin in the blood of rodents and
pigs is initially discussed, which is followed by a summary of the development and vali-
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,

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

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

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

presence of a “third ovarian hormone” in addition to 2 major of ultrasonography, measurement of systemic concentrations
steroids (i.e., estrogen and progesterone) [3]. This cynicism of progesterone less than (nonpregnant) or greater than
resulted in a lull in research until the 1950s and early 1960s (pregnant) a baseline concentration was generally considered
when Steinetz et al. took advantage of advanced protein the conventional or “gold standard” for hormonal diagnosis of
isolation techniques and improved in vivo methods to purify pregnancy in certain companion and farm animal species
[4] and characterize relaxin [5]. At about the same time, [22–24] and still is considered preferable for many wild ani-
development of a more sensitive and precise in vitro tech- mal species [25–27].
nique such as radioimmunoassay (RIA) to quantify hormones Blood, urine, and, to some extent, feces are typically the
was developed [6]. With the validation of relaxin RIAs in a most common types of biological material for hormonal
few key species such as the pig, rat, and mouse [7–10] and analysis in wild animals to monitor general and reproductive
motivation for continued research, relaxin was established as health as well as for research purposes [27,28]. Commercial
a vital hormone during pregnancy in the late 1980s. enzyme immunoassay (EIA) kits are widely available and have
In most species studied, the placenta appears to be the been adapted and validated to measure progesterone or
greatest source of relaxin with a lesser extent produced by progesterone metabolites in some of these matrices. Although
other reproductive organs in females (e.g., CL, uterus) and progesterone is considered a “gestational hormone,” an
males (e.g., testes, prostate) as well as in nonreproductive or- elevation of concentrations on the basis of single-sample
gans (e.g., heart, kidneys) [7–10]. Discovery of the pregnancy- analysis is not necessarily pregnancy specific. Circulating
specific increase in circulating concentrations of relaxin concentrations comparable to pregnancy are also observed
initially in the pig, rat, and mouse provided the inspiration to during the luteal phase of the estrous cycle and during pro-
investigate the basic and applied aspects of relaxin associated longed luteal maintenance, an abnormal condition that may
with pregnancy in other laboratory, companion, domestic, and occur in nonpregnant or pseudopregnant animals [29]. Hence,
wild animal species that continue till date. to distinguish pregnancy from nonpregnancy on the basis of
The present review is not intended to be an exhaustive progesterone as the “gold standard,” it is necessary to collect a
or comprehensive review of the history, nature, or roles of repeat sample after a period from the first sample. If the
relaxin within and among various animal species of which concentration of progesterone is sustained or has increased in
there are many [7–10]. Instead, the review primarily fo- the repeat sample, then there is greater certainty that the
cuses on the seminal work of Steinetz et al. who first animal is pregnant. The collection of multiple repeat samples
characterized circulating concentrations of relaxin in dogs may be done in wild animals under managed care but is
[11] and, thereafter, patented [12] the first clinical appli- typically not done within the same reproductive cycle or
cation of the canine relaxin assay as a hormonal approach season in free-ranging animals, in part, because of logistical
to distinguish between pregnant and nonpregnant or reasons and state, federal, and international laws that regulate
pseudopregnant bitches [13]. Dr. Steinetz had envisioned research on wildlife.
that his initial research on relaxin in the dog could be In addition to progesterone, other pregnancy-associated
applied to other companion, farm, and wild animal species hormones have been used or investigated as potential diag-
and make a difference in the conservation of threatened or nostic aids to determine pregnancy status, including estro-
endangered wildlife. This review includes a historic over- gens in horses [30,31], chorionic gonadotropin in horses [32]
view of the nature of relaxin and early work to develop and and primates [33], a chorionic gonadotropin–like substance in
validate immunologic methods to analyze and characterize bottlenose dolphins [34], prolactin in elephants [35] and dogs
relaxin for hormonal diagnosis of pregnancy in the com- [36,37], FSH in dogs [38], and PGFM in domestic and wild cats
panion dog and cat. The initial sections are intended to [39]. Notably, these hormonal aids are only specific for certain
provide context for the latter sections of this review on the stages of pregnancy and may not be able to differentiate be-
latest research applied toward development and validation tween pseudopregnancy because of embryo and/or fetal loss.
of immunologic assays to detect and evaluate relaxin in Besides hormones, pregnancy-associated factors have
wild terrestrial and marine mammal species for the pur- been indicated to have some diagnostic value, which include
pose of diagnosing pregnancy status. early conception factor [40], early pregnancy factor [41],
pregnancy-associated glycoproteins [42,43], fibrinogen, and
2. Hormonal diagnosis of pregnancy other acute-phase proteins [44]; C-peptide [38]; pregnancy-
specific protein B (www.biotracking.com); and cerulo-
Diagnosis of pregnancy is a hypothesis-based approach to plasmin [45]. For pregnancy diagnosis, pregnancy-associated
determine whether a female is nonpregnant or pregnant, glycoprotein has been reported in cattle [46,47]; pregnancy-
where the null hypothesis is the former and the alternative specific protein B in cattle, goat, sheep, bison, deer, and elk
the latter. The application of real-time ultrasonography is [48–52]; and ceruloplasmin in the giant panda [45]. Although
currently considered the most definitive tool for researchers there appears to be a variety of pregnancy-related hormones
and clinicians to test the hypothesis or diagnose pregnancy in or factors as potential diagnostic aids to determine pregnancy
many companion [14–16], farm [17–19], and wild [20,21] status, many are species specific and may not have broad
animal species. Although the technology is readily available, applicability across species.
it is relatively expensive to purchase and maintain, requires
the integration of technical and biological and/or anatomic 3. Relaxin
knowledge to capture and interpret images, and is generally
not applicable to wildlife species without physical or chemical The structure and heterogeneity of relaxin among spe-
restraint, especially under field conditions. Before the advent cies have been reviewed [7–10]. Genomically, relaxin is
D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12 3

encoded by 3 genes, RLN1, RLN2, and RLN3, in humans and returns to low concentrations during the postpartum period.
nonhuman primates; rodents have 2 genes (RLN1 and Regardless of the diversity in the pattern or fluctuation of
RLN3). In reference to humans, the 3 forms of relaxin are circulating concentrations of relaxin during pregnancy,
commonly referred to as H1-, H2-, and H3-relaxin [53]. The relatively higher concentrations preceding parturition fol-
most predominant form and that which is found in the lowed by lower concentrations postpartum appear to be
circulatory system is H2-relaxin. In general, relaxin is a 6- consistent among many species. In some species, increasing
kDa heterodimeric polypeptide with an A chain of 24 concentrations of relaxin before parturition occur concomi-
amino acids and B chain of 29 amino acids joined by 3 di- tantly with decreasing concentrations of progesterone. In
sulfide bonds with an intrachain disulfide loop in the A controlled studies that have examined the role of relaxin in
chain in a manner analogous to that of insulin. In regard to relation to progesterone in laboratory (e.g., rats, rabbits) [66]
the latter, identification of the primary structure of relaxin and farm (e.g., pig, sheep) [68] animals, it has been suggested
from different animal species suggests that relaxin is part of that the decrease in progesterone before parturition repre-
a larger family of relaxin-like factors that belong to the sents a necessary withdrawal of the ‘‘progesterone block’’
insulin superfamily [54]. In addition, although the size and that has maintained the gravid uterus in a quiescent state.
2-chain composition of relaxin are similar among species, Correspondingly, the increase in relaxin represents a mech-
there appears to be limited (<76%) evolutionary conser- anism to maintain myometrial quiescence and protect
vation of the amino acid sequence of relaxin among the against premature contractions and delivery.
approximately 25 species in which the sequence of relaxin
is known [7]. 4. Companion dog
Historically, the development of immunoassays for
relaxin has been hampered by the diversity of the primary The first canine relaxin RIA was developed and validated
structure of relaxin among species [7,55,56]. Initially, the lack for the companion dog with the use of antiporcine relaxin
of tyrosine and histidine in porcine relaxin made it impos- [11,58,59]. Table 1 provides a summary of the historic
sible to correctly radiolabel the hormone with 125iodine. progression and current use of relaxin immunoassays in
However, since the structural characterization of porcine the dog. Although modifications were done to improve the
relaxin [57], it has been correctly labeled by chemical addi- efficiency and effectiveness of subsequent canine RIAs
tion of 125iodine-labeled tyrosine [58]. Coupled with a [13,71,72], antiporcine relaxin was still the primary anti-
species-specific relaxin antibody, a porcine RIA was devel- body until development of synthetic canine relaxin and
oped and validated. Species-specific RIAs have since been production of anticanine relaxin [64,73]. Synthetic canine
developed for the rat [59–61], horse [62], human [63], and relaxin was also used to prepare radiolabeled ligand and
dog [64]. By adapting procedures and reagents originally reference standards, all of which improved reliability,
intended for one species, RIAs for relaxin have been devel- specificity, and sensitivity of the RIA [64].
oped and validated for other species. Notably, rabbit anti- In pregnant bitches [11,71], relaxin was first detected in
porcine relaxin (R6) was found to cross-react with relaxins of daily plasma samples as an increase in concentrations around
many species because of its affinity for a conserved receptor- 3 to 4 weeks after mating with maximum concentrations
binding domain [59]. Hence, the R6 antiserum has been used around 5 to 7 weeks. Thereafter, concentrations either
to develop and validate RIAs for numerous wild animal remained elevated or decreased before parturition (gestation
species as summarized in Table 1. length approximately 9 weeks). In nonpregnant and pseu-
Relaxin is produced by multiple tissues as a preprorelaxin dopregnant bitches [11], relaxin concentrations were below
precursor, which is processed to the mature bioactive detectable limits of the RIA throughout the 9 weeks of
hormone [65]. Relaxin is recognized as a multifunctional observation. Notably, diurnal changes associated with circu-
endocrine and paracrine hormone and/or factor with many lating concentrations of relaxin were not observed [74].
roles in female and male reproduction, as a neuropeptide in On the basis of the detectable increase in relaxin during
the central nervous system, as a vasodilator and cardiac pregnancy in the bitch, it is thought that relaxin is pro-
stimulant in the cardiovascular system, and as an antifibrotic duced in association with implantation of the embryo and
agent [7–10]. Although relaxin is produced in highest con- development of the fetal-placenta unit [74]. In this regard,
centrations by female reproductive tract tissues (e.g., it has been shown that the placental syncytiotrophoblast
placenta, CL, uterus) during pregnancy, the primary source is a major source of relaxin in dogs [13,72]. Discovery of
varies among species [7–10]. A major source of relaxin is the preprorelaxin localization in the canine placenta and
placenta in the golden hamster, rabbit, dog, and horse; CL determination of its nucleic acid sequence [75] confirmed
in the mouse, rat, pig, and humans; and uterus in the earlier observations that the placenta is likely the major
guinea pig. source of relaxin in the bitch. Relaxin is also produced to a
Temporal changes in circulating concentrations of lesser extent by the ovary [13] as evidenced by a decrease in
immunoreactive relaxin during pregnancy and the peri- serum relaxin in ovariectomized pregnant bitches [13]. An
partum period appear to be species specific. Generally, ovarian source of relaxin is further supported by the
among laboratory (e.g., rats, rabbits) [66], farm (e.g., pigs, detection of relaxin and corresponding receptors in luteal
sheep) [67,68], and wild (e.g., elephants, rhinoceros, leop- tissue collected during pregnancy [76]. Because the embryo
ards) [69,70] animals, serum or plasma concentrations of and/or fetoplacental unit appears to be a major source of
relaxin are relatively low, intermediate, or high from early- to relaxin in bitches, it has been suggested that a decrease in
mid-pregnancy with a gradual or marked surge-like increase circulating concentrations of relaxin can also be used to
during late pregnancy, before parturition. Thereafter, relaxin detect pregnancy loss in dogs [77]. In this regard, serum
Table 1

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]
EIAb None Rabbit anticanine and None Plasma Pos [109]
anticanine conjugated to
Cat (Felis silvestris catus) RIA Porcine Rabbit antiporcine (5858, Porcine Plasma Pos [80,90]
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]
Coyote (Canis latrans) EIA None Rabbit anticanine and None Plasma Pos [89]
anticanine conjugated to
Gray wolf (Canis lupus) EIAb None Rabbit anticanine and None Plasma Pos [84,88]
anticanine conjugated to
Mexican gray wolf (Canis EIAb None Rabbit anticanine and None Plasma Pos [88]
lupus baileyi) anticanine conjugated to
Red wolf (Canis rufus) EIAb None Rabbit anticanine and None Plasma Pos [88]
anticanine conjugated to
Fennec fox (Vulpes zerda) EIAb None Rabbit anticanine and None Plasma Pos [88]
anticanine conjugated to
Island fox (Urocyon EIAb None Rabbit anticanine and None Plasma Pos [88]
littoralis) anticanine conjugated to
African wild dog (Lycaon EIAb None Rabbit anticanine and None Plasma Pos [88]
pictus) anticanine conjugated to
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]
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]

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]
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]
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]
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]
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]
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.
Positive or negative for the immunoassay of relaxin in respective matrices on the basis of results in corresponding citations.
Commercial EIA (ReproCheK) or RIM (Witness) assay kits for relaxin are available from Synbiotics Corp, San Diego, CA, USA.

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

relaxin decreased in association with pregnancy loss maternal and embryo and/or fetal development for the
related to uterine pathology [78,79]. successful delivery of offspring and neonatal care thereafter.
In 1992, a patent was awarded to Steinetz et al. [12] on the However, the methods for diagnosis and evaluation of
basis of the clinical application that a pregnancy-specific pregnancy in wild animals are only practical if they can be
increase in circulating concentrations of relaxin can be applied in a manner that avoids or minimizes stress-related
used as a diagnostic aid for early pregnancy detection in responses, which can potentially disrupt ovulation or jeop-
companion dogs and cats. As a result, commercial EIA and ardize pregnancy [87]. The collection of blood samples usu-
rapid immunomigration (RIM) assay kits (ReproCheK and ally require some form of physical or chemical restraint,
Witness, respectively; Synbiotics Corp, San Diego, CA, USA) whereas the collection of urine, feces, saliva, hair, skin, or
have been developed, validated, and adapted for use in blubber samples can typically be done without interfering
numerous animal species (Table 1) to give a fast qualitative with the animal or remotely (e.g., biopsy darting) without
indication of whether concentrations of relaxin have capture, restraint, and release. Thus, although noninvasive
increased greater than a baseline concentration indicative of hormone monitoring of wild animals under managed care or
pregnancy. free-ranging conditions is an important tool for evaluating
an animal’s reproductive status [27], there are many chal-
5. Companion cat lenges associated with collecting samples in a safe manner
while minimizing animal stress and sample contamination,
In the companion cat (Felis silvestris catus), a porcine processing or preserving samples for short- or long-term
relaxin RIA has been used to characterize serum [80,81] storage or transportation, and interpreting assay results
and urine [81] concentrations of relaxin (Table 1). Sam- that may be confounded by metabolites or matrix effects.
ples were collected daily or every 3 or 4 times per week Immunoassay of relaxin has mostly been done using
[80] or once weekly [81] during pregnancy. In both serum blood (serum or plasma) from various species; however,
and urine, an increase in relaxin was detected 21 to 25 days analysis has also been done to some extent with urine, milk,
after mating. Thereafter, concentrations peaked 35 to and saliva in various domestic and wild animals (e.g., dog,
49 days before declining about 2 weeks before parturition cat, wolf, leopard, Pallas cat, sand cat, cheetah, lion, lynx,
(gestation length approximately 63 days or 9 weeks). Re- panda, alpaca, and dolphin). The use of serum, plasma, and,
sults suggested that urinary relaxin in the cat is pregnancy to some extent, urine to develop and validate immunoassays
specific because only pregnant queens excreted detectable with different combinations of relaxin and antirelaxin from
concentrations of relaxin in contrast to undetectable con- human, dog, pig, and horse to diagnose pregnancy status in
centrations in mated nonpregnant queens. The urinary wild mammalian species are discussed in the following
profiles of relaxin concentrations on the basis of RIA anal- sections in concordance with Table 1.
ysis paralleled that of serum, which further justified the use
of urine as a reliable noninvasive approach for pregnancy 6.1. Wild terrestrial mammals
diagnosis in companion cats.
In addition to RIA analysis, a relaxin commercial RIM 6.1.1. Canids
assay kit (Witness) has been used successfully with urine to In female gray wolves (Canis lupus) under managed care,
detect pregnancy in the companion cat (Table 1) [82,83]. use of the commercial EIA ReproCheK kit (Table 1) accu-
Although the RIM assay was originally designed for use rately distinguished pregnancy from nonpregnancy in all
with serum or plasma as a qualitative test to diagnose animals [84]. Use of the ReproCheK kit for pregnancy
pregnancy in dogs and cats, the Witness kit was shown to diagnosis was further evaluated in gray wolves, Mexican
be reliable with cat urine after diluting samples with equal gray wolves (Canis lupus baileyi), red wolves (Canis rufus),
parts of nonpregnant cat serum [83]. All known pregnant fennec foxes (Vulpes zerda), and African wild dogs (Lycaon
queens tested positive, whereas nonpregnant queens pictus) under managed care and free-ranging island foxes
tested negative. Application of nonradiolabeled commer- (Urocyon littoralis) [88]. Corresponding to those animals
cial EIA and RIM kits in companion dogs and cats has been confirmed pregnant with ultrasound or observed with
shown to be valid for relaxin and pregnancy diagnosis pups, the relaxin EIA was positive for gray wolves, fennec
within the same family of canidae [84] and felidae [85] for foxes, and island foxes greater than 25 days after mating.
conservation purposes as listed in Table 1 and discussed in Application of the ReproCheK kit less than 25 days resulted
the next section. in an approximately 8% false-negative rate in island foxes
on the basis of a positive ultrasound examination or
6. Wildlife observation of pups. The relaxin EIA was negative for red
wolves and wild dogs, which was supported by no obser-
For the conservation and survival of wild animal species vation of pups. The false-positive rate was nil on the basis of
that are threatened or endangered and at the threshold of negative test results in known nonmated Mexican gray
extinction, zoos, aquariums, and other managed care habi- wolves, red wolves, and island foxes.
tats are essential for gaining fundamental knowledge of the In coyotes (Canis latarans) [89], results of the relaxin EIA
reproductive biology of these species [86]. A comprehensive ReproCheK kit (Table 1) with plasma samples indicated all
understanding of the physiology and endocrinology of control animals (i.e., males and females that were not
reproduction is a prerequisite for a sustainable and produc- mated or were with castrated males) tested negative for
tive conservation program. The capability to diagnose and relaxin, whereas all mated animals with pups tested posi-
evaluate pregnancy status is a basic requirement to manage tive approximately 28 days after mating. In mated coyotes
D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12 7

without pups, 87% tested negative for relaxin and 13% collected from pregnant lynx and companion cat, immu-
tested positive. The basis for the 13% that tested positive nohistochemical and mRNA expression of the relaxin
could not be clarified because pregnancy or pregnancy loss ligand and receptor was stronger in the placenta compared
could not be confirmed. Nonetheless, the combined results with the ovary and uterus. Thus, the combined results
indicated that a commercial EIA for canine relaxin can be provide evidence that the placenta is a major source of
used with serum or plasma as a reliable method for relaxin during pregnancy in some felids.
detection of pregnancy in wild canid species beginning
greater than 25 days after mating. 6.1.3. Other carnivores
The potential for using urine from wild canids was In female spotted hyenas (Crocuta crocuta) under
initially indicated by the detection of a relaxin-like sub- managed care, a porcine relaxin RIA (Table 1) was used to
stance in the urine of a pregnant maned wolf (Chrysocyon characterize circulating concentrations of relaxin in serum
brachyurus) using a canine relaxin RIA (Table 1) [70]. Future samples collected at various times during pregnancy
studies are required to confirm and broaden the application (gestation length approximately 110 days) [92]. Relative to
of immunoassay of relaxin in urine in this and other canid parturition (Day 0), concentrations were near or below the
species. detectable limits of the assay during early pregnancy (103
to 87 days). During midpregnancy (55 to 42 days),
6.1.2. Felids there was an increase in relaxin that progressively rose to
In leopards (Panthera pardus) [81], a canine relaxin RIA peak concentrations near parturition (19 to 0 days).
(Table 1) run with weekly urine samples was found to be a Relaxin was not detected in prepubertal, adult, nonpreg-
reliable method for pregnancy diagnosis on the basis of nant female or male hyenas; results were below the
detection of relaxin 25 to 28 days after mating with peak detectable limits of the RIA.
concentrations at 60 to 64 days of pregnancy. Thereafter, In extracts of placental and ovarian tissue from a preg-
concentrations declined before parturition (gestation length nant hyena, relaxin concentrations were higher in the
approximately 101 days). Correspondingly, the canine placenta compared with those of the ovary [92]; thus, the
relaxin RIA run with urine samples differentiated nonpreg- placenta appears to be a major source of relaxin during
nant from pregnant sand cats (Felis margarita), Pallas cats pregnancy in this species.
(Octocolobus manul), cheetahs (Actinonyx jubatus), and lions In Giant pandas (Ailuropoda melanoleuca) under managed
(Panthera leo) [83]. However, in comparison to peak con- care, use of a porcine relaxin RIA (Table 1) detected serum
centrations in companion cats (range, 14–30 ng/mL) at concentrations of relaxin during the last 3 weeks of preg-
midpregnancy, concentrations were relatively greater in nancy (gestation length approximately 19 weeks) but not
pregnant sand and Pallas cats (range, 21–64 ng/mL), com- during nonpregnancy or in males [69]. In contrast, analysis of
parable in pregnant cheetahs (range, 5–17 ng/mL), and lower urine resulted in inconsistencies in the relative changes
in pregnant lions (range, 6–10 ng/mL). In contrast, applica- during pregnancy compared with that of serum. The basis for
tion of the commercial RIM Witness kit did not detect relaxin the inconsistent changes in concentrations between serum
in unadulterated urine from a pregnant leopard or in urine and urine is not known but may be related, in part, to
diluted with nonpregnant leopard serum (personal com- metabolized or degraded forms of relaxin in the urine at
munication cited in Harris et al. [83]). Correspondingly, the different stages of pregnancy or that other urinary compo-
RIM assay was used with urine from sand cats, Pallas cats, nents interfered with the immunoanalysis. Perhaps, as in
cheetahs, and lions but was not successful in differentiating some felids [85], filtering and concentrating the urine would
pregnant from nonpregnant animals [83]. result in more consistent results. Nonetheless, future studies
In Iberian lynx (Lynx pardinus), application of the RIM are needed to clarify the use of urine for analysis of relaxin in
Witness kit (Table 1) was successful using serum in which pandas.
relaxin was detected in all pregnant cats between 32 and
56 days after mating [85]. In contrast, the RIM assay kit did 6.1.4. Elephants, rhinoceros, and alpacas
not detect relaxin in freshly collected or frozen samples of In pregnant Asian elephants (Elephas maximus) and Af-
lynx urine at the same stage of pregnancy. However, by rican elephants (Loxodonta africana) under managed care,
concentrating the pregnant and nonpregnant urine sam- use of an equine relaxin RIA (Table 1) indicated higher serum
ples about 50-fold using ultrafiltration, the RIM assay concentrations of relaxin approximately 20 weeks of preg-
detected relaxin as a weak reaction for pregnancies greater nancy that remained elevated until 26 weeks [35,69].
than or equal to 29 days but not those less than 29 days or Thereafter, concentrations decreased until a second surge of
in nonpregnant lynx. Apparently, development of processes relaxin occurred as parturition approached (gestation length
to filter and concentrate urine need to be explored to approximately 92 weeks). In male Asian elephants, plasma
improve the suitability of urine for immunoassay of relaxin concentrations of relaxin were not detected; however, rela-
using the RIM Witness assay kit in this and other felids. tively low concentrations were detected in nonpregnant fe-
Temporally, the increase in serum, plasma, and urine males during the estrous cycle [93]. In an Asian elephant
concentrations of relaxin using RIA corresponds with the with pregnancy loss, a precipitous decrease in relaxin was
placental production of relaxin beginning approximately observed approximately 1 week before spontaneous abor-
20 days of pregnancy in the companion cat [90]. More tion [93]. In this regard, relaxin may also be used to detect
recently [91], DNA and protein sequences of relaxin from pregnancy loss in elephants as suggested in the dog [77–79].
placental tissue of an Iberian lynx were determined and In Sumatran rhinoceroses (Dicerorhinus sumatrensis)
used to generate a felid-specific relaxin antibody. In tissues 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 dog and seal provided a reasonable basis to evaluate the
6 months of pregnancy [69]. Thereafter, concentrations potential of a previously validated canine relaxin RIA using
increased by 8 months and reached highest concentrations anticanine relaxin [64] to detect and characterize a relaxin-
2 weeks before parturition (gestation length approximately like substance in pinnipeds (Table 1).
62 weeks). Serum relaxin concentrations remained Development and validation of a canine relaxin RIA for
elevated 4 days after parturition and were also detected in use in managed care and wild Northern fur seals (Cal-
maternal milk samples. lorhinus ursinus), Steller sea lions (Eumetopias jubatus), and
In pregnant alpacas (Vicugna pacos), use of a porcine harbor seals (Phoca vitulina) [103] were adapted from a
relaxin RIA (Table 1) indicated serum concentrations of canine relaxin RIA [64]. In fur seals under managed care,
relaxin increased by 2 months of pregnancy but declined by relaxin concentrations were highest in pools of serum from
5 to 8 months [94]. Thereafter, concentrations increased until pregnant seals compared with nonpregnant and post-
parturition (gestation length approximately 12 months). partum female and male seals. Higher concentrations of
Relaxin was also examined in nonpregnant and pregnant relaxin in pregnant compared with nonpregnant and
alpacas using a modified EIA (Table 1) [99] to determine the postpartum fur seals are supportive of the pregnancy-
potential to detect concentrations in milk, urine, and saliva specific nature of relaxin in this species. Temporally, con-
[87], which can be collected in a less-stressful manner than centrations of relaxin were relatively low during Months 4
blood. Plasma concentrations of relaxin increased after the to 5 of pregnancy and high during Months 7 to 12 as
second month of pregnancy, whereas there were no differ- parturition approached (gestation length approximately
ences in saliva and milk concentrations between nonpreg- 12 months). Notably, low concentrations during early-to
nant and pregnant alpacas. Relaxin EIA results with urine mid-pregnancy (Months 4–5) correspond with the end of
were below the detectable limits of the assay. embryonic diapause, a period of delayed implantation that
reportedly ranges from 3 to 5 months in Northern fur seals
6.1.5. Nonhuman primates [104].
A porcine relaxin RIA (Table 1) was used to analyze In extracts of placental tissue from a wild Northern fur
relaxin in nonhuman primates. In rhesus monkeys (Macaca seal, Steller sea lion, and harbor seal, relaxin was readily
mulatta), plasma concentrations of relaxin were detected detected [103]. Because it is not known if other reproduc-
from 3 weeks of pregnancy to parturition (gestation length tive tissues (e.g., CL, uterus) produce relaxin in high
approximately 23 weeks) but not during nonpregnancy or amounts, it remains to be clarified if the placenta is the
in males [95]. In baboons (Papio cynocephalus), high plasma major source of relaxin in these species.
concentrations of relaxin were detected during pregnancy
[96]. In chimpanzees (Pan troglodytes), higher relaxin con- 6.2.2. Cetaceans
centrations were detected during the first trimester of In cetaceans, relaxin immunoreactivity was first re-
pregnancy compared with lower concentrations during the ported in extracts of luteal tissue from pregnant Antarctic
late luteal phase of the menstrual cycle [97]. In marmosets minke (Balaenoptera acutorostrata) and Bryde’s (Balae-
(Callithrix jacchus), a porcine RIA [98] and EIA [99] (Table 1) noptera edeni) whales [105] using a porcine relaxin RIA
detected serum relaxin concentrations during early preg- with sheep antiporcine relaxin (Table 1). Relaxin bioactivity
nancy (2–3 weeks) which continued to increase to peak from the 2 whales appeared similar to porcine relaxin as
concentrations during midpregnancy and, thereafter, determined by a mouse pubic symphysis relaxin assay [5].
gradually decreased as parturition (gestation length ap- Structurally, among whales, relaxin differed by about 3
proximately 20 weeks) approached. In nonpregnant mar- residues, whereas, among species, the relaxin of the Bryde’s
mosets, relaxin was not detected during the reproductive whale differed at only one position from that of pig relaxin.
cycle [98]. Thus, the results in whales provided a basis to evaluate the
In pregnant ovariectomized rhesus monkeys and ba- potential of using antiporcine relaxin to develop a relaxin
boons, removal of the ovaries or the CL-bearing ovary RIA to detect and characterize a relaxin-like substance in
resulted in nondetectable serum concentrations of relaxin managed care and wild bottlenose dolphins.
[95,96]. Thus, the CL seems to be the major source of relaxin Development and validation of a relaxin RIA for use in
during pregnancy in some primates. bottlenose dolphins (Tursiops truncatus) consisted of H2
human relaxin as radiolabeled hormone, rabbit antiporcine
6.2. Marine mammals relaxin as the primary antibody, and synthetic canine
relaxin as reference standard [106] (Table 1). Temporally,
6.2.1. Pinnipeds changes in circulating concentrations of relaxin were
In pinnipeds, studies documenting the detection of characterized throughout 12 months of pregnancy in dol-
immunoreactive relaxin could not be found in the scientific phins under managed care, which included dolphins with
literature. Therefore, to develop and validate a relaxin RIA live births and perinatal loss (i.e., late-term abortion or
for use in seals, it was first considered how this species of stillbirth). Regardless of pregnancy outcomes, serum con-
carnivore might be related to other carnivores, in which centrations of relaxin progressively increased from rela-
relaxin has been detected. Anatomically, it was found that tively low concentrations during early pregnancy (Months
the chorioallantoic placenta of pinnipeds is comparable 1–4), intermediate concentrations during midpregnancy
with many other carnivores [100]. In this regard, canids and (Months 5–8), and high concentrations during late preg-
pinnipeds have a zonary endothelial–chorial placentation nancy (Months 9–12). Subsequent to parturition, relaxin
[101,102]. Thus, the evolutionary proximity between the returned to low concentrations during the postpartum
D.R. Bergfelt et al. / Theriogenology xxx (2014) 1–12 9

period. Hence, relatively high concentrations of relaxin combinations of relaxin and antirelaxin from human,
during the prepartum period versus postpartum period are companion, and farm animal species. The potential for
supportive of the pregnancy-specific nature of relaxin in immunoreactive relaxin to serve as an aid in the diagnosis
this species. In pregnancies resulting in late-term abortion of pregnancy status is on the basis of detection primarily in
or stillbirth, mean relaxin concentrations were 42%, 29%, blood and to some extent in urine of which concentrations
and 34% lower at early, mid, and late pregnancy, respec- are higher in pregnant than nonpregnant (postpartum)
tively, compared with pregnancies resulting in live births. animals. Although the increase in circulating concentra-
In wild dolphins, there is a degree of uncertainty to di- tions of relaxin appears to be pregnancy specific among the
agnose pregnancy on the basis of single-sample analysis of various terrestrial and marine mammals evaluated thus far,
serum progesterone because a relatively high concentra- the primary source of production (e.g., placenta, CL, uterus)
tion in a sole sample is not necessarily pregnancy specific. and timing of the increase during pregnancy are different
To increase the hormonal diagnostic certainty of preg- among species. In some species, the increase in relaxin
nancy, a study was conducted to analyze relaxin and pro- seems to coincide with implantation of the embryo and
gesterone in the same serum samples collected from development of the fetal-placenta unit. Future efforts
populations of free-ranging dolphins [107]. Hormone should focus on validation of nonradiolabeled relaxin im-
pregnancy diagnosis was based on concentrations of munoassays for broader application among species and
relaxin and progesterone above respective baseline con- improving techniques (e.g., extraction, purification) to
centrations. Observed pregnancy diagnosis was based on analyze relaxin in samples other than blood (e.g., urine,
photoidentification of cow-calf pairs subsequent to sample feces, saliva, blow, skin, blubber) that can be collected in a
collections. Concordance between hormone and observed less-invasive or -stressful manner and processed accord-
diagnosed pregnancies was 100% for progesterone and 78% ingly for basic and applied purposes, especially with
for relaxin. The basis for lower predictability with relaxin application toward conservation of threatened or endan-
was attributed to samples collected during the first gered species.
trimester of pregnancy at which time concentrations are
relatively low [106]. Although the diagnostic value of
relaxin alone may be limited during early pregnancy Acknowledgments
(Months 1–4), the combination of relatively high concen-
trations of relaxin and progesterone in a single-sample The authors would like to pay tribute to Dr. Bernard G.
analysis thereafter has the potential to provide greater Steinetz for his pioneering work to characterize the phys-
confidence to diagnose pregnancy than if either hormone iological role of relaxin and his vision to realize the appli-
was used alone. cation of relaxin as a clinical aid to initially differentiate
A preliminary analysis of relaxin in urine from dolphins between pregnant and nonpregnant dogs and cats and,
under managed care was conducted with the expectation thereafter, as a conservation tool to diagnose pregnancy
that the less-invasive nature of collecting urine compared status in numerous wild animal species, especially those
with blood would allow for more frequent serial collection that may be threatened or endangered.
of hormonal data (personal communication, Bergfelt).
Concentrations of relaxin in increasing volumes of pools of
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