Zoo Biology - 2010 - Wheaton - The Use of Long Acting Subcutaneous Levonorgestrel LNG Gel Depot As An Effective

10982361, 2011, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/zoo.20354 by C/O Glucksman Library, Wiley Online Library on [02/11/2022].
See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Zoo Biology 30 : 498–522 (2011)
RESEARCH ARTICLE
The Use of Long Acting Subcutaneous

Levonorgestrel (LNG) Gel Depot as an
Effective Contraceptive Option for
Cotton-Top Tamarins (Saguinus oedipus)
C.J. Wheaton,1 A. Savage,1 A. Shukla,2 D. Neiffer,3 W. Qu,2 Y. Sun,2
and B.L. Lasley4
1
Disney’s Animal Kingdoms, Education and Science Department, Lake Buena Vista,
Florida
2
University of Tennessee, Health Science Center, Department of Pharmaceutical
Sciences, Memphis, Tennessee
3
Disney’s Animal Kingdom, Department of Animal Health, Lake Buena Vista, Florida
4
University of California Davis, Center for Health and Environment, Davis, California
Cotton-top tamarins (Saguinus oedipus) are a critically endangered species that

have been bred successfully in captivity for many years. For two decades, the
Cotton-top Tamarin SSPr has been challenged with a high rate of reproduction
combined with a history of contraceptive failures and nonrecommended births
using the current Depo Proveras (medroxyprogesterone acetate) injection
followed by MGA (melengestrol acetate) implant contraception combination.
To address these issues we have developed and tested the use of levonorgestrel
(LNG) as an effective contraception option for cotton-top tamarins. LNG was
delivered in an injectable, gel matrix consisting of polylactic-co-glycolic acid,
triethyl citrate and N-methylpyrrolidone. This gel matrix forms a biodegradable
depot at the subcutaneous injection site providing slow release of the active
ingredient. Gel matrix composition and LNG concentration were adjusted in
four gel formulations to maximize the duration of contraceptive efficacy
while minimizing immediate post-injection increases in fecal LNG concentration.
LNG treatment (68.4478.61 mg/kg) successfully eliminated ovarian cycles
(fecal pregnanediol-3-glucuronide (PdG) and estrone conjugates (E1C)) for
198.8770.3 days (formulation four; range 19–50 weeks). It was demonstrated
Correspondence to: C.J. Wheaton, Disney’s Animal Kingdom, PO Box 10,000, Lake Buena Vista,
FL 32830. E-mail: Catharine.J.Wheaton@disney.com
Received 8 March 2010; Revised 25 May 2010; Accepted 27 August 2010
DOI 10.1002/zoo.20354
Published online 11 October 2010 in Wiley Online Library (wileyonlinelibrary.com).
r 2010 Wiley Periodicals, Inc.

10982361, 2011, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/zoo.20354 by C/O Glucksman Library, Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
LNG Contraception in Cotton-Top Tamarins 499
that subcutaneous LNG depot injection was an effective, reversible contraceptive

option for the management of cotton-top tamarins in captivity. Zoo Biol
30:498–522, 2011. r 2010 Wiley Periodicals, Inc.
Keywords: contraception; biodegradable injectate; estrone conjugates; pregnanediol-

3-glucuronide
INTRODUCTION
Cotton-top tamarins (Saguinus oedipus) have been bred successfully in captivity
for many years [Evans, 1983; Price and McGrew, 1990; Snowdon et al., 1985; Tardif
and Clapp, 1993; Tardif and Harrison, 1986]. In most captive colonies, social groups
consist of the adult breeding pair and their offspring from one or more litters. Unlike
their wild counterparts who exhibit reproductive seasonality with a single litter per
year [Savage et al., 1997, 2009], reproductive seasonality is not observed in captivity.
Adult female cotton-top tamarins give birth to twins on average every 29 weeks
[Snowdon et al., 1985; Ziegler et al., 1987a] with a post-partum ovulation on day 19
[French, 1983]. Juvenile females living in their natal social group are reproductively
suppressed until approximately two years of age [Savage et al., 1988; Ziegler et al.,
1987b]. Infant care is shared by all members of the social group with mothers, fathers
and siblings all participating in carrying the offspring [Cleveland and Snowdon, 1984;
Tardif et al., 1986, 1990, 1992; Washabaugh et al., 2002]. Early infant caretaking
experience has been found to be essential in providing sufficient experience to learn
parental care skills. Cotton-top tamarins denied early infant caretaking experiences
often kill or neglect their own offspring [Snowdon et al., 1985; Leong et al., 2004].
Thus, to insure a self-sustaining captive breeding colony, it is essential that all animals
born into social groups have the opportunity to care for offspring. Given the high
rate of reproduction, the need to provide animals the opportunity to assist in rearing
siblings and limited space in captive settings, techniques to improve chemical
contraception in this species are needed.
Synthetic progestins are commonly selected as contraception treatments which
function to raise circulating progestin concentrations to physiological levels that
mimic the actions of endogenous ovarian progesterone [Asa, 2005]. The increased
progestin concentrations may then act in one or more of a combination of ways to
effect contraception via inhibition of folliculogenesis, ovulation, thickening of the
cervical mucus or slowing of ovum transport interfering with successful fertilization
or implantation [Pérez-Palacios et al., 1981; Asa et al., 1996; Nagle et al., 2009; AZA
Contraceptive Advisory Group, 2010]. These contraceptive effects are often dose and
species specific. Low contraceptive doses may permit ovulation and even luteal
activity even though pregnancy does not occur [Filippi et al., 2004; Möhle et al.,
1999]. Higher treatment doses may be needed to produce cycle suppression; however,
follicle growth may persist and the estrogen produced may be sufficient to produce
estrous behavior [Hynes et al., 2007].
In New World primates, higher doses and shorter treatment intervals are
needed for successful contraception because of higher circulating concentrations of
progesterone [Coe et al., 1992; Porton and DeMatteo, 2005]. Initial steroid hormone
contraception studies in cotton-top tamarins revealed that 3 of 3 females continued
Zoo Biology
500 Wheaton et al.
to exhibit normal cycles when treated with 3 mg/kg, and 3 of 4 females with 10 mg/kg
medroxyprogesterone (Depo-Proveras ) [Lee-Parritz and Roberts, 1994]. Higher
dosage increased effectiveness. One female injected with 96 mg/kg Depo-Proveras
on day 10 post-partum conceived 11 weeks later [reported in Porton and DeMatteo,
2005]. Melengestrol acetate (MGA) implants (0.5 g) suppressed ovarian cycles in 5 of
5 females tested [Lee-Parritz and Roberts, 1994].
Under the auspices of the Cotton-top Tamarin SSPr and the Association of
Zoos and Aquariums (AZA) Contraceptive Advisory Group (CAG), recommenda-
tions to participating cotton-top Tamarin SSPr institutions in 2000 were that
cotton-top tamarin females receive an injection of 20 mg/kg Depo-Proveras
(medroxyprogesterone acetate; Pfizer, New York, NY) within ten days post partum,
followed by insertion of an MGA implant (melengestrol acetate; ZooPharm division
of Wildlife Pharmaceuticals, Fort Collins, CO) by week 14. During this post-partum
treatment of the adult female, daughters older than one year living in the social
group were recommended to receive MGA implants to prevent development of
ovarian activity while the primary adult female was suppressed [Porton and
DeMatteo, 2005]. These contraception recommendations were designed to minimize
behavioral and social disruption to the group prior to the weaning of the infants.
A 2005 survey [Portion and DeMatteo] reviewing results from institutions
following the Cotton-top Tamarin SSPr Depo Proveras/MGA contraceptive
recommendations revealed several concerns regarding the efficacy of this combina-
tion treatment. Originally estimated to be effective for up to 14 weeks, results
reported by Portion and DeMatteo [2005] indicated that the initial estimate of
duration of efficacy of the Depo Proveras was artificially high. Recommendations
were changed to 20 mg/kg injection of Depo Proveras at 35- to 40-day intervals for
New World monkeys [Porton and DeMatteo, 2005]. The most significant problem
reported for MGA implant contraception in cotton-top tamarins was implant loss
(17%) due to abscess or allogrooming and implant removal by cagemates. Reported
failure rates for MGA implants (defined as the MGA implant still present at the time
of conception) in callitrichids were very low (1.3%) [Porton and DeMatteo, 2005].
Small captive primates like tamarins are not routinely caught and handled on a
regular basis making implant loss difficult to detect. Loss of an active MGA implant
in cotton-top tamarins results in an almost immediate return of ovarian activity
[approximately 11 days, N 5 2, C. Wheaton and A. Savage, unpublished data].
The first ovarian cycle following parturition is fertile [French, 1983], and the
first ovulation following contraceptive, loss or failure is likely no exception. Cotton-
top Tamarin SSPr data compiled from January 2001 through December 2009
revealed that out of a total of 150 recorded births, only 56 of those young (37.3%)
came from recommended pairings. The remainder of the pregnancies resulted from
an undetermined or unreported combination of Depo Proveras contraceptive
failure prior to MGA implant insertion, MGA contraception failure, MGA loss
[Filippi et al., 2004; Porton and DeMatteo, 2005], vasectomy failure (N 5 2), and
nonrecommended pairings [R. Phillips, Cotton-top Tamarin SSPr Coordinator,
pers. comm.]. Few zoos routinely monitor hormone profiles in their female cotton-
top tamarins following Depo Proveras injection preventing accurate determination
of continued efficacy of the Depo Proveras treatment at the time of MGA implant
insertion. In addition, MGA implant loss was not routinely determined or reported
during annual surveys preventing accurate assessment of cause of contraception
Zoo Biology
failure [R. Phillips, Cotton-top Tamarin SSPr Coordinator, pers. comm.].

Currently, the 2010 AZA Contraception Advisory Group guidelines for New World
monkeys [2010] note that Depo Proveras injection is effective for approximately 30
days requiring MGA implant insertion by the fourth week post-partum.
More importantly, several reports have raised concerns about potential health
effects associated with MGA. Detrimental effects such as endometrial hyperplasia
and decidualization have been reported in Goeldi’s monkeys (Callimico goeldii),
squirrel monkeys (Saimiri sciureus) [Murnane et al, 1996] and common marmosets
(Callithrix jacchus) [Möhle et al., 1999] [see Nettles, 1997; Munson et al., 2005; Gray
and Cameron, 2010 for a complete review]. Although few adverse effects have been
reported yet in callitrichids [Munson et al., 2005], the high proportion of Depo
Proveras/MGA implant combination contraceptive failures and MGA implant loss
observed in cotton-top tamarins have spurred investigation of alternative, more
reliable contraceptive options and methods [Asa, 1993, 2005].
Levonorgestrel (LNG; a synthetic progestin contraceptive) has been success-
fully used in women and has provided a model for a safe and effective means of
contraception for domestic and wildlife species as an alternative to MGA. Looper
et al. [2001] and Tell et al. [1999] have developed a method that can be applied to
captive primates. They have tested LNG efficacy as a contraceptive when delivered
as a cesium-irradiated, slow-release, injectable matrix to domestic cats and quail. The
biodegradable polymer-based matrix formulations form a biodegradable solid at the
injection site and were designed for slow release of LNG. Given that the LNG-
loaded biodegradable formulations can be designed to release LNG over an
extended period and is unlikely to be removed by cagemates, this contraceptive
method has a high likelihood of effectiveness in primates.
This study is the first to investigate the efficacy of LNG gel depot as a means to
contracept cotton-top tamarins. Here we report results from physiological and
enzyme immunoassay (EIA) validations (LNG Challenge) for the use of the LNG
EIA [Munro et al., 1996] for noninvasive monitoring of LNG contraception in
cotton-top tamarins. Fecal LNG elimination profiles were used to direct changes in
the gel matrix component concentrations (effecting gel viscosity and LNG loading
dose) to maximize the duration of contraceptive efficacy while minimizing
physiological LNG peaks observed immediately postinjection. In addition, fecal
samples collected from study females were measured for ovarian hormone
metabolites pregnanediol-3-glucuronide (PdG) and estrone conjugates (E1C) content
to assess reproductive activity through ovarian cycles. Fecal PdG and E1C are
known to be the predominant fecal steroid hormone metabolites and indicate the
luteal stage following ovulation in an ovarian cycle in cotton-top tamarins
[Heistermann et al., 1993; Ziegler et al., 1989]. Effectiveness of LNG contraception
was assessed by measurement of the latency to and duration of suppression of
ovarian cycles.
Behavior is an important component of social cohesion in cotton-top tamarin
family groups [Savage et al., 1998]. During LNG gel depot contraceptive testing,
behavioral observations were conducted to determine whether there were any
changes in behavior related to LNG treatment or ovarian cycle suppression.
Observations focused on species-specific behaviors that have been associated
with pair-bonding and ovarian cyclicity, specifically, estrogen excretion [Savage
et al., 1988].
Zoo Biology
502 Wheaton et al.
Reversibility of LNG contraceptive treatment was assessed through monitoring

the resumption of ovarian cycles following clearance of LNG after each injection.
Finally, reversibility was assessed in one female through an SSPr permitted
pregnancy who was allowed to cycle, breed and conceive following clearance of LNG
treatment.
METHODS
Animals
Five multiparous (Al, Eu, Ge, Ro, Te), one primiparous (Pr) and two
nulliparous (Da, Me) female cotton-top tamarins housed at Disney’s Animal
Kingdoms (DAK), Lake Buena Vista, FL (Al, Eu, Ge, Ro, Pr, Da, Me) and Central
Florida Zoo (CFZ), Sanford, FL (Te) were used in this study (age range 4–12.5 years
at the beginning of this study). Females Ge and Eu remained contracepted with
MGA implants during the initial validation for noninvasive monitoring of LNG
during the LNG challenge. For paired females (Al, Ge, Eu, Pr), males (No, Ce, Sh,
Ho) were vasectomized. Males No, Ce and Ho were used to test gel injectability
(viscosity), optimal injection volume, method of insertion and placement using gel
formulation 1 (vehicle only) prior to the start of LNG gel depot injection testing in
the females (F1; Table 1).
MGA implants were removed from females Ge, Eu, Ro and Al 1.5–2 years
prior to LNG gel depot contraceptive treatment. Fecal steroid analysis showed that
ovarian cycle activity resumed within ten days of MGA implant removal. One female
(Ro) was housed with her (0.2) young offspring (Da, Me). During the course of the
study Da began cycling and was subsequently used as a subject in the study. Female
Me remained contracepted with MGA subdermal implants until her transfer to
another institution in July 2005. Female (Te) was housed with an adult, intact male
and 2.1 offspring at CFZ.
Housing
Paired females Al, Ge, Pr and female Ro with daughters Da and Me (DAK)
were housed in large outdoor enclosures (2.92 m 3.05 m 2.08 m) that contained a
heated nestbox (0.38 m 0.48 m 0.31 m), a complex array of natural vegetation and
branches, and were on public display. One pair of cotton-tops (Eu) was housed
indoors during the LNG Challenge validation in an enclosure measuring
(0.18 m 3.31 m 1.68 m) that contained a nestbox and a complex array of ropes
and branches. The pair was then moved to an outdoor enclosure measuring
(3.05 m 3.35 m 2.44 m) that contained a heated nestbox (0.38 m 0.48 m 0.31 m)
and an array of vegetation and branches.
Female Te (CFZ) was housed with her intact male and offspring in an
outdoor enclosure (3.96 m 2.13 m 2.74 m) that contained two nestboxes
(0.14 m 0.14 m 0.81 m) and a complex array of natural vegetation and branches.
This female had a history of contraceptive failures while following the recommended
Cotton-top tamarin SSPr contraception protocol (two pregnancies were confirmed
as Depo Proveras contraceptive injection failures after eight weeks, and one
pregnancy following MGA implant contraception after 13.8 months) and was used
in the study to test the efficacy and reversibility of LNG (Table 1).
Zoo Biology
TABLE 1. Study Design Outlining the Development, Validation and Test Phases of Efficacy and Reversibility of LNG Contraception
Gel depot injection test condition, formulation no. and % LNG dose
Validation 0% 4% 6% Observations reversibilitya
Contraception LNG Contraception Ovarian Pregnancy

Zoob Name Sex conditionc challenged conditione Vehiclef F1 F2 F3 F4 Behaviorg cycles and birth
DAK Ge F MGA x MGA removed x x x x x x

Ce M Intact Vasectomy x x
Eu F MGA x MGA removed x x x x

Sh M Intact Vasectomy
Al F MGA MGA removed x x x x x x

No M Intact Vasectomy x x
Pr F n/a None x x x x x x
Ho M Intact Vasectomy x x
Ro F MGA MGA removed x x x x x x

Da F None None x x x x
Me F MGA MGA x
CFZ Te F MGA MGA removed x x x

M M Intact None (Intact)
a
Reversibility of LNG contraceptive treatment was assessed through monitoring of resumption of ovarian cycles following clearance of LNG after each injection (minimum of 1–3
cycles) and one (SSPr permitted) pregnancy in CFZ female Te who was allowed to cycle, breed and conceive following clearance of LNG treatment.
b
DAK (Disney’s Animal Kingdoms, Lake Buena Vista, FL); CFZ (Central Florida Zoo, Sanford, FL).
c
Females Ge and Eu were maintained under MGA subdermal implant contraception for the LNG Challenge validation test. Female Pr (daughter of Al, No) was separated from her
family group to be paired with vasectomized male Ho and allowed to begin normal ovarian cycles prior to LNG gel depot contraception treatment. Ro was housed with juvenile
and sub-adult daughters Da (not contracepted) and Me (MGA implant).
d
LNG Challenge: i.m. injection of 1 mg/kg purified LNG (d( )-Norgestrel, dissolved in corn oil (5 mg/ml)). Serum, fecal and urine samples were collected for up to one week
following injection to analyze relative patterns of circulating and excreted LNG to determine optimal noninvasive sample collection (urine or feces) for the remainder of the study.
e
Adult males were vasectomized and MGA implants were removed from females 1.5 to 2 years prior to first LNG gel depot injection (DAK females). For CFZ female Te, the MGA implant
was removed on the day of LNG gel depot injection and she remained housed with an adult, intact male and her 2.1 offspring.
f
Biodegradable gel depot injection volume and location was tested in males Ce, No and Ho using blank gel formulation one (F1; vehicle only) to determine optimum injection protocol for
LNG loaded gel formulations.
g
Zoo Biology
Behavioral observations were conducted to assess possible changes in species specific affiliative or pair-bonding behaviors related to LNG contraception treatment or ovarian cycle suppression.
504 Wheaton et al.
Fecal Sample Collection and Extraction

First morning void fecal samples were collected 3 times per week for all females
for a minimum of 90 days prior to treatment until return of ovarian cycles and
clearance of LNG following their last injection. A total of 5,205 fecal samples were
collected during the course of the study between 9/17/02 and 7/15/08. Samples were
collected by holding a clean plastic bucket (Nalgene Nunc International, Rochester,
NY) underneath the female while defecating. The fecal sample was placed in 5 ml
Sarstedt tubes (Sarstedt, Inc., Newton, NC) and stored at 501C until extraction. The
fecal extraction protocol used was validated for a number of species by Graham et al.
[2001]. In brief, fecal samples were thawed at room temperature and 0.5 g (70.02 g) was
weighed out and placed in an 8 ml glass sample vial (National Scientific, Duluth, GA).
Five milliliters of 80% HPLC grade methanol in dH20 was added to extract the steroid
metabolites. Samples were shaken overnight (10–12 hr), then centrifuged (Centrifuge
5804 R, Eppendorf; Hamburg, Germany) at 2,400 rpm, at 51C for 30 min. The
supernatant was decanted into an 8 ml vial, labeled and stored at 501C until assayed.
Enzyme Immunoassays
The PdG and E1C EIAs used in this study have been described previously in
Phillips and Wheaton [2008]. The protocol was modified from Savage et al. [1997].
Pregnanediol-3-glucuronide
Fecal PdG was analyzed using antiserum P70 (diluted to 1:15,000) and R13904
(1:30,000) prepared in rabbit. The PdG horseradish peroxidase (HRP) conjugate was
diluted 1:40,000 to 1:75,000 depending on lot number date and titration. Standards
were prepared using pregnanediol-3-glucuronide (5b-pregnane-3a, 20a-diol glucur-
onide, Sigma P3635) at a range of 0.078–40 ng/50 ml. The inter- and intra-assay
coefficients of variation were 19.25 and 13.57% (low control, 0.31 ng/50 ml at 74.39%
binding), and 13.08% and 10.21% (high control, 2.14 ng/50 ml at 28.44% binding) in
209 assays. Sensitivity of the PdG assay was 0.11 ng/50 ml per well.
Estrone conjugates
Fecal E1C was analyzed using antiserum R522 (diluted 1:4,000) and R522-2
(lot 7/7/05, diluted 1:14,000) prepared in rabbit. For the E1C R522 antiserum (156
plates assayed between March 2004 and December 2005) the conjugate (E1C HRP,
lot 8/6/01) was diluted to 1:20,000 and used a low control of 4 pg/50 ml and a high
control of 40 pg/50 ml. Using the replacement E1C antiserum R522-2 (63 plates
assayed between January 2006 to October 2009), the new conjugate (E1C HRP, lot
3/14/05) was titrated to 1:150,000. This antiserum and conjugate reagent combination
increased the slope of the standard curve from 1.17 to 2.13, reducing the mean
maximum readable range of the assay from 43.54 pg/50 ml to 27.72 pg/50 ml at (20%
B/Bo; standards prepared from estrone 3-b-glucuronide sodium salt (Sigma E1752)).
This required a change in the high control and standard set for the second reagent kit
(standards ranged from 1.162 to 200 pg/50 ml). The inter- and intra-assay coefficients
of variation were 15.22 and 14.82% (low control, 4.00 pg/50 ml at 82.22% binding,
N 5 219 assays), 7.85 and 9.32% (high control, Lot 1; 40.03 pg/50 ml at 21.34%
binding, N 5 153 plates) and 5.48 and 6.49% (high control, Lot 2; 24.76 pg/50 ml at
21.34% binding, N 5 63 plates). Sensitivity of the E1C assay was 2.76 pg per well.
Zoo Biology
Levonorgestrel
The LNG enzyme immunoassay, developed and validated for use in humans
and nonhuman primates [Munro et al., 1996], was modified using the same assay
protocol as described above for the E1C and PdG EIAs. The R5864 polyclonal
antisera to 3-O-carboxymethyl oxime (CMO) derivative of LNG was prepared in
rabbit and diluted to 1:12,000 for coating plates. The LNG HRP conjugate was used
at a working dilution 1:80,000. Standards were prepared using purified Levonorges-
trel (d( )-Norgestrel, N2260 Sigma-Aldrich, St. Louis, MO). Standard curves
generated from LNG standards (3.9–500 pg/50 ml) were parallel with serially diluted
fecal sample extracts from 1:2 through 1:2,000. No substantial cross-reactivities were
found for the R5864 antiserum with the exception of Norethindrone (a synthetic
progesterone birth control). Cross-reactivity with progesterone and estradiol-17b
was less than 0.001% [Munro et al., 1996]. In-house tests for cross-reactivities for
progestagen metabolites and PdG found no substantial cross-reactions (o1%).
Spike recovery tests of two negative and one positive pooled sample (fecal extracts)
with 350 pg LNG run in serial dilutions against the standard curve resulted in
101.171.5% recovery at 50% binding. Sample dilutions ranged from 1:2,000 to
1:4,000 for post injection LNG peaks through to a 1:3 dilution when LNG was
effectively eliminated from treated cotton-top tamarins. The inter- and intra-assay
coefficients of variation for the LNG EIA were 13.34 and 17.01% (low control,
9.93 pg/50 ml at 73.87% binding) and 9.41 and 10.35% (high control, 99.60 pg/50 ml
at 18.81% binding) in 197 plates. Sensitivity of the LNG assay was 4.48 pg per well.
Clearance of the LNG treatment was considered complete when sample extracts
assayed at a 1:3 dilution read at 480% binding on the standard curve.
For all steroid assay systems, fecal sample extracts were assayed in duplicate at
a minimum dilution of 1:3 to minimize interference from the solvent. The
conservative range of the assay was determined to be between 80 and 20% B/Bo.
Samples with CV values greater than 20% or that fell outside the range were
re-diluted and re-assayed until they fell within the readable range of the standard curve.
Ovarian Cycle Monitoring

Results from fecal E1C and PdG EIA analysis were used to detect ovarian
cycles in study females. Suppression of E1C and PdG cycle profiles was used to
monitor the duration of efficacy of LNG treatment contraception. Paired females
were allowed one to five ovarian cycles between LNG injection treatments. Female
Ro (housed with daughters Da and Me) was limited to one cycle between injections
to minimize chance of aggression within the group [A. Savage, personal communication].
Affiliative Behavior
Focal behavior observations were conducted from August 2002 through May
2008. Animals were observed for 20 min between 0800 and 1700 hr three times per
week. A total of 2,549 observations were conducted on females Al, Ge, Pr (housed
with an adult male) and female Ro (housed with her two daughters) (see Table 1).
Behavior observations started 90 days prior to each females’ first LNG injection and
ended on the last day of ovarian cycle suppression. Inter-observer reliability for all
behaviors was 90%. Observers recorded frequency and duration of contact behaviors
(grooming and proximity) between family members (mothers and daughters and in
Zoo Biology
506 Wheaton et al.
male–female pairs). In paired animals, copulatory activity (including mounts and

attempted mounts), anogenital and suprapubic scent marking were recorded.
LNG Challenge
The LNG challenge validation was designed to verify the route of LNG
metabolism to determine the appropriate noninvasive sample (feces or urine) for
collection in the study. Females Ge and Eu were injected intramuscularly with a bolus
of LNG dissolved in oil (see Table 1). Subsequent serum, urine and fecal samples
were collected, extracted, and assayed in the LNG EIA. Fecal and urine sample LNG
EIA results were compared with serum LNG results to validate that eliminated
samples accurately reflected circulating LNG concentrations. Serial dilution
validations in the LNG EIA were used to confirm relative proportion of LNG
eliminated to feces and urine and compared to the standard curve for validation of
use in the LNG EIA. Results were used to determine sample collection choice for
noninvasive monitoring of LNG injection profiles and elementary pharmacokinetics
in LNG gel depot injected cotton-top tamarins in the remainder of the study.
Contraceptive efficacy was not intended to be assessed in this test. Accordingly,
females Ge and Eu remained contracepted with MGA implants to prevent unwanted
pregnancy while housed in their respective social groups with their (intact) adult
male (Ce and Sh; see Table 1).
Injectate preparation
Purified LNG (d( )-Norgestrel, N2260 Sigma-Aldrich) was dissolved in corn
oil (5 mg/ml). Approximately 100 ml was administered intramuscularly (rear leg) in
females Ge and Eu for a final dose of 1 mg/kg body weight.
Sample collection
Where possible, blood samples were collected at 0, 24, 48 and 72 hr following
LNG administration, and all urine and fecal samples were collected for 168 hr
(one week) following LNG administration for validation of the LNG EIA. Samples
were collected by holding a clean plastic bucket (Nalgene Nunc International)
underneath the female while defecating or urinating. Care was taken to avoid cross-
contamination of urine and fecal samples.
Sample preparation
Blood samples were centrifuged (2,500 rpm, 10 min) and the collected serum
was stored frozen ( 501F). Prior to LNG analysis, 250 ml of each serum sample was
extracted with 2 ml petroleum ether, the extract evaporated and re-suspended in
0.5 ml of assay buffer for LNG analysis.
Validation
Serum, urine and fecal extracts were serially diluted for comparison tests of
parallelism against the standard curve. Dilutions reflecting 50% B/Bo of the
standard curve were chosen for EIA analysis of samples collected following the LNG
injection. Urine and fecal LNG profiles were compared with serum to determine
optimal sample collection choice for the analysis of the LNG elimination profiles
following subcutaneous LNG gel depot injection in the remainder of LNG gel
formulation development testing in the study.
Zoo Biology
LNG Gel Injectate Formulation Development and Dosage

Injectate preparation
The LNG injectate used was a biodegradable gel consisting of a 24% weight
by weight (w/w) polymers, 72% w/w plasticizers (a mixture of 10% triethyl citrate
and 90% N-methylpyrrolidone) and 4% (formulation 1,2, 3) or 6% (formulation 4)
(w/w) LNG (Sigma-Aldrich, N2260). Approximately 1 ml of the LNG loaded gels
was placed into small glass sample vials capped with an aluminum vial seal. Prior to
injection, all individual dose vials of LNG-loaded gels were sterilized by Gamma
irradiation at a minimum dose of 25 kGy (Cobalt 60 Gamma irradiation, Food
Technology Service Inc., Mulberry, FL).
Gel formulation development

LNG gel formulations were assessed and modified following each new
formulation. Changes in gel formulation composition were made considering gel
injectability (gel viscosity, optimal needle gauge and length), and improvement of
fecal LNG clearance profile characteristics (minimized peak LNG concentration
immediately following injection, LNG fecal profile pattern, LNG plateau
concentrations and decay to zero concentration). Following each trial, the gel
composition was adjusted to increase the viscosity of the injectate to reduce initial
burst release and eliminate the secondary release peak of LNG to extend the release
of LNG from the biodegradable gel.
LNG dosage
In formulations 1, 2 and 3, 4% (w/w) LNG was loaded in the gels. In
formulation 4, the LNG loading was increased by 50 to 6% LNG (w/w) to increase
the duration of contraception efficacy (ovarian cycle suppression).
After approximately one year of storage of formulation 4 (6%) LNG vials
(March 2006 to April 2007), a white residue or precipitate was observed at the
bottom of the LNG vials after injection (N 5 2 females). By comparison, no
precipitate was observed in blank gel or in any of the 4% (w/w) LNG formulations
after up to 7 years of storage. Formulation 4 contained a higher concentration of
LNG than the other gel formulations, thus it is likely that the precipitate was
crystallized LNG. This potentially altered the accuracy and consistency of estimated
injected LNG dose in formulation 4. To address these issues, in subsequent
injections, the individual LNG dose vials were heated to 241C to re-dissolve the
precipitate prior to injection of the gel. Duration of contraception efficacy and post-
injection peak fecal LNG concentrations were compared within this formulation to
account for possible differences in injected LNG dose in gels with precipitation or
gels that were heated to re-dissolve the LNG. Details of changes in gel composition
for each gel formulation are summarized in Table 2.
Veterinary Considerations
Optimization of gel volume and placement
DAK Males (No, Ce, Ho) were used to test optimal gel injection volume and
placement using gel formulation 1 (vehicle only). Volumes tested ranged from 0.5 to
1.1 ml placed in a pocket bluntly dissected between the tissues in the subcutaneous
Zoo Biology
Zoo Biology
508 Wheaton et al.
TABLE 2. Composition of LNG Gel Depot Formulations Tested
LNG (%w/w) Plasticizers (%w/w) Polymer composition (%w/w)
10% triethyl citrate 90% 50/50 Polylactic-co- 85/15 Polylactic-co- Polylactic

d( )-Norgestrela N-methylpyrrolidone glycolic acid glycolic acid acid DL-Polylactide
b
Viscosity – – 0.59 0.62 0.65 0.65
Formulation
F1 4 72 24 – – –
F2 4 72 8 8 8 –
F3 4 72 4.8 – – 19.2
F4c 6 70.5 4.7 – – 18.8
a
Levonorgestrel (LNG; Sigma-Aldrich ]N2260).
b
Inherent viscosity (dl/g).
c
Formulation four (F4) was prepared using the same gel composition as formulation three (F3) but with increased LNG dose (6% LNG, 94% blank
gel (%w/w)).
space just medial and distal to the right scapula. Immediately following gel
placement, the injection site was inspected for leakage to surrounding tissues or back
through the injection site. Visual and manual (palpation) inspection was repeated
during the first two months following injection of the gel.
Subcutaneous placement of LNG gel depot

All animals were anesthetized for LNG gel injectate insertion. Two anesthetic
induction protocols were used during the study. Cotton-top tamarins (N 5 20
injections) housed at DAK were trained to enter a clear PlexiglasTM induction
chamber. Female Te (CFZ) was captured by net and transported to the veterinary
hospital where anesthesia induction was induced using 5% isoflurane in oxygen
administered through plastic tubing attached to the chamber. Once heavy sedation
to light anesthesia was achieved, the tamarin was removed from the chamber and
weighed (g). Isoflurane was administered by facemask to maintain anesthesia for the
duration of the procedure. Alternatively, for N 5 9 LNG injections, cotton-top
tamarins were captured by net and transported to the hospital where anesthesia was
induced and maintained with Ketamine and Midazolam (6–10 mg/kg).
A volume of 0.8–0.9 cc of LNG gel was drawn from each individual dose vial
using a 20-gauge 1-in. (2.54 cm) needle attached to a 3 cc luer-lokTM syringe to
ensure the delivery of 0.6–0.7 cc. For gel insertion, the cotton-top tamarin was placed
in ventral recumbency with legs and arms extended. Injection into the skin layers was
avoided. A 20-gauge (N 5 14) or 18-gauge (N 5 7) 2.5-inch (6.35 cm) spinal needle
with stylet in place was introduced through the skin into the subcutaneous space just
medial and distal to the right scapula. The spinal needle was used to dissect a pocket
between the tissue for placement of the gel injectate in order to concentrate
the location of the gel for hardening and to prevent seepage within the injectate
location and out of the injection site. The skin was tented to maintain a
subcutaneous pocket. The needle was slowly advanced caudally and parallel to the
spine until approximately 2 in. (5.08 cm) of the needle was buried. The stylet was
removed and the syringe containing the LNG gel was attached.
Calculating for residual volume remaining within either the 20 gauge or 18
gauge needles, 0.65 cc of the gel (26 mg of active LNG) was deposited into the
subcutaneous pocket on the right side of the thoraco-lumbar region. The mean
dosage of LNG administered in gel formulations 1, 2, 3 and 4 were 45.5372.52,
46.6073.30, 48.0873.39 and 71.8978.90 mg/kg, respectively. Following deposition
of the injectate, the spinal needle was slowly extracted, taking care not to deposit any
residual matrix at the site of skin penetration.
Adjunct therapy and posttreatment monitoring

Prior to recovery from anesthesia, each cotton-top tamarin received 15 ml
(range 24.1–28.6 ml/kg) of lactated Ringer’s solution subcutaneously for fluid
support, and 0.5 mg Ketoprofen (range 0.80–0.95 mg/kg) or Meloxicam (0.02 cc)
intramuscularly for analgesia. Anti-emetic therapy was administered with 0.1 mg
Metoclopramide (range 0.19–0.20 mg/kg) used intramuscularly in LNG gel
formulations 1 and 2, and Chlorpromazine (0.5–1.0 mg/kg) used intramuscularly
in LNG gel formulations 3 and 4.
Zoo Biology
510 Wheaton et al.
For the first 48 hr post-procedure, all cotton-top tamarins were monitored for
changes in appetite and behavior. Over the duration of the study all animals were
observed for evidence of swelling, abscessation and hair loss at the injection site.
Reversibility
Ovarian cycles
End of period of contraception efficacy was defined as the first day of increased
fecal PdG and E1C concentrations to values normally associated with ovarian cycles
[Heistermann et al., 1993; Ziegler et al., 1989]. Paired females were allowed one to
five ovarian cycles between LNG injection treatments. Females Ge, Pr and Ro were
monitored for 1.5–2 years following their last LNG treatment to confirm continued
ovarian cycles (long-term reversibility).
Pregnancy
Multiparous female (Te) housed with an adult, intact male, and 2.1 offspring
(CFZ) was used to test the reversibility of LNG contraception (Table 1). Removal of
the MGA implant occurred on the same day as her LNG injection. Since females
resume ovarian cycle activity within 10–14 days of MGA implant removal, the
duration of LNG suppression in female Te was not considered to be affected by her
previous MGA treatment.
Female Te received 44.83 mg/kg of LNG formulation 1. Following injection
female Te was taken back to her family group. First, morning fecal samples were
collected a minimum of three times per week and frozen onsite for later shipment to
DAK for steroid hormone analysis. Fecal PdG, E1C and LNG were monitored at
regular intervals during the course of her treatment. Female Te remained housed
with the intact male and allowed to copulate normally during the course of the study
and during the final clearance stage of the LNG and resumption of ovarian cycles.
Pregnancy was determined by fecal hormone analysis. Fecal sample collection and
steroid analysis continued throughout pregnancy until parturition.
Statistical Analysis
All EIA analyses of immunoreactive PdG, E1C and LNG in feces are reported
as mean7SD in ng or mg/g feces (wet weight). Serum LNG concentrations for the
LNG challenge and fecal LNG concentrations during the return to ovarian cycles
are reported in ng/ml. Duration of cycle suppression was calculated from day of
LNG gel injection (day 0) to the last sample before the elevated hormone
concentrations were equivalent to baseline or the follicular phase. The effects of
LNG gel formulation composition on post-injection peak LNG concentration and
duration of ovarian cycle suppression (days) were analyzed using the Mann–Whitney
U test using one-tailed significance, the Kruskal–Wallis one-way analysis of variance
or the median test (LNG post injection peaks) for comparison of multiple
formulations where appropriate. Pearson product moment correlation tests were
used to analyze the relationship between LNG treatment dose and duration of
ovarian cycle suppression. Mann–Whitney U tests were used to analyze the
differences in mean behavior scores (reproductive and affiliative behavior observa-
tions) in suppressed vs. cycling treatment conditions. All results were analyzed using
SPSS (version 15.0) with P 5 0.05 level of significance.
Zoo Biology
RESULTS
LNG Challenge
Immunoreactive LNG was readily detectable by EIA in serum and fecal
methanol extracts. Serum LNG concentrations were elevated for approximately
48 hr, with peak concentrations occurring at 24 hr following injection of 1 mg/kg of
LNG in oil. Fecal LNG concentrations also increased following LNG administra-
tion. Analysis of fecal extracts revealed a range of 0–24 hr time lag between the peak
increase in LNG observed in circulation and that observed in the feces (Fig. 1). The
reduced lag time in female Eu may be due in part to diarrhea experienced by this
female. There were no detectable concentrations of LNG measured in urine. All
subsequent analyses used fecal samples for the evaluation of efficacy of each LNG
formulation.
LNG Gel Injectate Formulation Development and Dosage

Optimization of gel volume and placement
Results of injection volume (0.5, 1.0 and 1.1 ml) test using blank gel
(formulation 1) on N 5 3 cotton-top tamarin males indicated that a maximum of
0.5–0.7 ml injection was optimal using an 18-gauge 2.5-in. needle. Following initial
hardening, the gel was barely palpable and virtually absent at one month upon
follow-up.
8 30
Ge
6
20
4
10
Serum LNG (ng/ml)
Fecal LNG (µg/g)
0 0
Eu
6
20
4
10
2
0 0
0 24 48 72 96 120 144 168
Time after LNG injection (hrs)
Fig. 1. Fecal (open circles; mg/g) and serum (closed circles; ng/ml) levonorgestrel (LNG)
concentrations following the administration of 1 mg/kg LNG i.m. in oil to females Ge (top
panel) and Eu (bottom panel). The reduced lag time in fecal LNG clearance in female Eu may
be partly due to diarrhea experienced by this female. A blood sample could not be collected
from Eu at 72 hr. Instead, a blood sample was collected at 1-week post injection (168 hr) to
confirm clearance of the LNG.
Zoo Biology
512 Wheaton et al.
Suppression of ovarian cycles

All tested LNG gel formulations successfully suppressed ovarian steroid
hormone production, reducing fecal E1C and PdG concentrations to near zero. Fecal
PdG and E1C concentrations were reduced to values below normal ovarian cycle
concentrations (PdGo1 mg/g feces, E1Co0.1 mg/g feces) within 9.675.6 days
following injection. Once ovarian cycles were fully suppressed, maximum PdG
concentrations rarely exceeded 0.2 mg/g feces, and E1C rarely exceeded 0.02 mg/g feces.
A typical example profile of fecal PdG and E1C ovarian cycles followed by LNG
injection suppression of cycles, and return of ovarian cycles is provided in Figure 2.
All females were observed to resume ovarian cycles after clearance of the LNG.
LNG Peak Concentrations, Elimination Profiles and Duration of Cycle

Suppression
4% LNG formulations
Injection of LNG gel formulation 1 produced high fecal LNG concentrations
with maximal peaks of 25.78710.64 mg/g at 2.872.0 days post injection. In this
formulation, fecal LNG elimination profiles showed a strong secondary LNG
elimination peak at 4–8 weeks after injection before an exponential decline to a
plateau (typically 200–500 ng/g feces), followed by terminal decline to values below
LNG injection Ovarian cycle suppression
8 2.0
Fecal E1C (µg/g)

Fecal PdG (µg/g)
6 1.5
4 1.0
2 0.5
300 0.0
Fecal LNG (µg/g)
20
10
0
4/04 6/04 8/04 10/04 12/04
Date
Fig. 2. Representative profile of fecal PdG (closed triangles) and E1C (open triangles)
demonstrating ovarian cycle suppression (top panel) observed during levonorgestrel (LNG)
gel depot injection treatment (bottom panel; open circles). Sample concentrations are reported
in mg/g wet feces. Shown here is LNG injection ]12 (formulation 2; female Ge; 43.48 mg/kg).
The day of LNG injection is indicated by the vertical arrow (6/11/04). In this example, fecal
LNG concentrations peaked immediately following injection (6/12/04; 23.6 mg/g) followed by
a gradual decline to nadir concentrations. Fecal PdG and E1C concentrations were reduced to
nadir 11 days following injection. Successive samples with PdG or E1C concentrations greater
than cycling baseline concentrations, followed by return of ovarian cycles, were considered the
endpoint of cycle suppression (104 days in this example).
Zoo Biology
10
Formula 1 Formula 2 Formula 3 Formula 4
8
Fecal LNG (ug/g)
0
0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 40
Number of weeks post-LNG injection

Fig. 3. Fecal levonorgestrel (LNG) elimination profiles (mean7SD mg/g feces; open circles) by
week post-injection for LNG gel formulations 1, 2, 3 (4% LNG w/w) and 4 (6% LNG w/w).
Mean (7SD) number of weeks of ovarian cycle suppression (open squares with horizontal error
bars) are provided for each formulation. N.B. The ordinate axis is truncated at 10 mg/g feces.
Maximum LNG peaks are reported elsewhere in the text.
the sensitivity of the LNG assay (panel F1, Fig. 3). In formulation 1, mean fecal
LNG concentrations were 27.02725.03 ng/g feces when ovarian cycle activity
resumed (102.0720.9 days; F1, Table 3).
In formulations 2 and 3, the type and inherent viscosity of the biodegradable
polymers used to prepare the gel matrix were adjusted to alter the viscosity of the gel
at injection and the degradation characteristics of the gel depot in the body after
injection. Changes in gel composition in formulation 2 eliminated the secondary
peak in the LNG elimination profile (panel F2, Fig. 3), but did not reduce maximum
concentrations observed in postinjection LNG peaks (23.5179.97 mg/g, Mann–
Whitney U 5 26.0, n1 5 8, n2 5 7, P 5 0.867), or result in longer suppression of
ovarian activity (120.4746.0 days, Mann–Whitney U 5 20.5, n1 5 8, n2 5 7,
P 5 0.397). Changes made in LNG gel formulation 3 reduced initial peak LNG
concentrations to 57% of formulation 1. However, comparison of median peak
LNG concentrations in formulations 1 to 3 (22.92, 23.64 and 13.48 mg/g feces,
respectively) indicated no significant differences in immediate LNG peak concentra-
tions due to changes in gel composition (Kruskal–Wallis K(2, 21) 5 3.914,
P 5 0.141). Although duration of cycle suppression in formulation 3 was 12%
longer than formulation 2 and 31% longer than formulation 1 (134.8732.8 days,
Table 3), changes in gel matrix composition or inherent viscosity for the three 4%
Zoo Biology
514 Wheaton et al.
TABLE 3. LNG Gel Depot Injection Number, Date, Dosage, Duration of Cycle Suppression,
and LNG Concentration at Resumption of Ovarian Cycles for LNG Gel Formulations F1 to F4
Cycle LNG
LNG suppressiona concentration
formulation Injection Injection Dose duration at return of
no. no. date Female (mg/kg) (days) cycles (ng/g)
F1 (4%) 1 11/26/2002 Ro 44.83 142 6.26

2 8/6/2003b Te 44.83 117 69.06c
3 8/12/2003 Pr 50.98 104 15.21
4 8/12/2003 Al 43.41 94 63.38
5 8/14/2003 Ge 44.83 84 6.97
6 8/14/2003 Ro 44.83 107 5.13
7 11/12/2003 Eu 47.27 93 22.42
8 1/8/2004 Ro 43.26 75 27.73
Avg7SD 45.5372.52 102.0720.9 27.02725.03
F2 (4%) 9 6/3/2004 Eu 50.49 134 163.46
10 6/3/2004 Pr 49.62 106 76.63
11 6/11/2004 Al 41.73 98 65.78
12 6/11/2004 Ge 43.48 104 166.39
13 4/30/2004 Da 48.42 57d 14.04
14 4/30/2004 Ro 44.83 139d 218.22
15 8/5/2004 Da 47.62 205d 10.55
Avg7SD 46.6073.30 120.4746.0 102.15781.12
F3 (4%) 16 2/22/2005 Al 47.1 99 9.16
17 2/22/2005 Pr 46.43 94 18.66
18 3/1/2005 Eu 54.05 154e n/a
19 3/4/2005 Ge 47.27 139 10.67
20 6/3/2005 Da 44.14 179 11.35
21 6/3/2005 Ro 49.52 144 58.55
Avg7SD 48.0973.39 134.8732.8 21.68720.94
F4 (6%) 22 3/14/2006 Ge 61.22 200 24.44
23 4/5/2006 Al 73.17 138 286.99
24 4/7/2006 Pr 62.72 157 73.66
25 4/12/2006 Da 82.35 199 20.05
26 4/12/2006 Ro 73.17 350 63.83
27f 4/18/2007 Ge 60.2 242 36.18
28f 4/18/2007 Pr 59.41 148 72.58
29g 4/24/2007 Al 75.27 156 83.36
Avg7SD 68.4478.61 198.8770.3 82.64785.99
a
Cycle suppression was calculated from day of injection to the date of the last sample
preceding first hormone activity (PdG or E1C) at normal cycling concentrations (see text for
details).
b
A melengestrol acetate (MGA) implant was removed on the same day as the levonorgestrel
(LNG) injection.
c
Female Te was allowed to conceive following end of LNG treatment suppression and conceived
immediately following resumption of ovarian activity.
d
Social conditions may have influenced duration of suppression in this family group
(Mother 5 Ro, daughter 5 Da).
e
Female Eu was suppressed for 154 days at the time of her death from gastrointestinal cancer.
f
Crystallized LNG residue was observed in the vial after the LNG gel was drawn.
g
LNG vial was heated in a water bath to 751F, and sonicated to re-dissolve the LNG prior to
injection.
Zoo Biology
LNG formulations did not increase duration of efficacy (Kruskal–Wallis

K(df 5 2) 5 3.324, P 5 0.190). In support of these findings, injected LNG dosage
in the three 4% LNG formulations ranged from 41.73 to 54.05 mg/kg. Mean LNG
dosage did not differ between formulations (Kruskal–Wallis K(df 5 2) 5 1.868,
P 5 0.393), and was not correlated with increased cycle suppression duration
(r2 5 0.051). Because gel formulation 3 exhibited maximum mean suppression
duration, while reducing initial postinjection LNG peaks and eliminating secondary
peaks, formulation 3 was selected for use as the base injectate gel composition for
formulation 4. The LNG concentration was increased to 6% (w/w) in this
formulation to test for increased cycle suppression duration.
Comparison of 4% vs. 6% LNG dosage
Mean postinjection peak LNG values in the 6% gel (formulation 4) were 4.3 times
higher than the comparable 4% formulation (formulation 3), but the difference was not
significant, which was likely due to the extreme variability in LNG peak values in
this formulation (range 5.20–268.56 mg/g, Mann–Whitney U 5 14.0, n1 5 6, n2 5 7,
P 5 0.228). Peak fecal LNG concentrations were not always observed on the same day
and ranged from 1 to 11 days postinjection. Plateau LNG concentrations were also
variable with females averaging 0.7870.63 mg/g and ranged from 0.30 to 3.51 mg/g feces.
Comparing all four gel formulations, the increased LNG dose in formulation 4
produced a weak positive correlation with increased ovarian cycle suppression
(r2 5 0.372) and a significant main effect of treatment formulation on suppression
duration (Kruskal–Wallis K(df 5 3) 5 13.57, P 5 0.004). Mean duration of suppres-
sion in formulation 4 (6% LNG) was 47% longer than in formulation 3 (4%) (range
138–350 days, Mann–Whitney U 5 8.0, n1 5 6, n2 5 8, P 5 0.043), 65% longer than
formulation 2 (Mann–Whitney U 5 7.0, n1 5 7, n2 5 8, P 5 0.014) and almost twice
the duration of formulation 1 (Mann–Whitney U 5 1.0, n1 5 8, n2 5 8, P 5 0.000)
(see Fig. 3). Mean latency from LNG gel depot injection to cycle suppression for all
four formulations was 9.074.4 days (range 3–17 days).
Individual variability in the duration of cycle suppression was evident in all
four formulations. This could be due to variance of the apparent surface area
exposed to the body after the gel hardened, which is determined by the geometric
shape of the gel at the injection site. Differences in LNG metabolism and response to
LNG among tested animals could also contribute to the variability. For contra-
ceptive considerations we must consider the minimum duration of efficacy for
maximum safety and minimal risk of return of ovarian cycles and pregnancy in this
species. The shortest duration of efficacy was observed in formulation 2 with 57 days
(8.1 weeks), followed by formulation 1 (10.7 weeks), formulation 3 (13.4 weeks) and
formulation 4 (6%; 19.7 weeks; see Table 3 and Fig. 3).
Females Ge and Pr (injection ]27, 28) received injection of the 6% gel that was
observed to contain a residue or precipitate at the bottom of the vial. Duration of
suppression in these two females (34.6 and 21.1 weeks) did not differ from their
treatment group in duration (range 19.70–50.0 weeks), or in LNG peak
concentrations (34.2 and 34.4 mg/g LNG; group range 5.2–268.6 mg/g LNG). Heating
the gel to re-dissolve the precipitate (Table 3, injection ]29, female Al) produced the
highest postinjection peak LNG concentrations of this treatment group (268.6 mg/g
LNG) but did not alter duration of efficacy compared to the earlier injection trials in
this group (22.3 weeks, see Table 3). Using the most conservative result in this
Zoo Biology
516 Wheaton et al.
formulation, maximum recommended safety margin of efficacy for the best formu-
lation (formulation 4) is 18–19 weeks where the priority is to maintain effective
contraception.
Affiliative Behavior
Suppression of ovarian endocrine steroidal function by LNG treatment did not
alter affiliative behavior. Comparison of frequencies of observed behaviors during
normal ovarian cycles (90 day pre-LNG injection or return of ovarian cycles
following LNG elimination) with LNG treatment (suppression of ovarian PdG and
E1C activity) across injections revealed no significant differences in grooming
frequency or duration, scent marking, copulatory behaviors (mounts and attempted
mounts) or proximity to the male (all P values greater than 0.05).
Reversibility
Ovarian cycles
All LNG gel depot treated females were observed to return to normal ovarian
cycles following clearance of LNG after each injection in this study. Following the
final LNG injection (formulation 4) for females Ge, Pr and Ro, fecal sample
collection and steroid analysis continued for 0.6, 0.8 and 1.3 years, respectively to
assess long-term effects on ovarian cycles. Fecal PdG and E1C analysis demonstrated
continued, normal ovarian cycles in all three females.
Pregnancy
One female (Te) was permitted to breed upon resumption of ovarian cycles at
the CFZ. She resumed cycling at 117 days postinjection and conceived immediately.
Following a pregnancy loss at approximately week 19 to 20, Te immediately
conceived again and successfully produced a single offspring after a normal
pregnancy.
Veterinary Considerations
LNG gel insertion
The most significant adverse reaction noted during this study was development
of significant swelling, inflammation and abscess formation at the injection (N 5 1;
female Me) and deposition sites (N 5 1; male control). Histologic evaluation of the
swollen tissue identified granulomatous inflammation (histiocytes, multinucleated
cells, mononuclear cells and neutrophils) within the subcutaneous adipose tissue
consistent with a foreign body reaction. Both of these cases support the need for
careful passage of the needle within the subcutaneous space and deposition of the
gel, a process that would likely be difficult without completely anesthetizing the
tamarin. A spinal needle with stylet in place is recommended to dissect a pocket
between the tissue for placement of the gel in order to concentrate the location of the
gel and limit damage and irritation of surrounding tissue.
Anesthesia, adjunct therapy and post-treatment monitoring
Gastrointestinal (GI) signs including excessive salivation, retching, and
vomiting were also observed during this study. However, these symptoms were
observed in all trials (blank gel, LNG loaded gel) and with all anesthetic induction
Zoo Biology
and maintenance protocols (isoflurane induction box, ketamine injection followed by

facemask induction with isoflurane). These GI signs are not commonly observed
following anesthesia in cotton-top tamarins. Prior to this study, tamarins had
routinely been hand-caught or netted followed by anesthesia using isoflurane
induction delivered by facemask. In N 5 25 anesthetic events performed on nine
cotton-top tamarins using this anesthetic induction method, GI signs were only
noted two times. This suggests that the effect may be due to a combination of
reaction to the LNG gel matrix or the elevated progestins immediately following
injection. Intermittent vomiting along with partial anorexia may persist for 24 hr
after LNG treatment. Replacement fluids (subcutaneously) and anti-emetic therapy
is recommended to reduce the likelihood of symptoms.
Body weight (g) recorded during each LNG injection procedure was compared
for each injection and formulation for each tamarin. Females ranged from 488 to
623 g during the course of the study. No mean change in body weight (measured as
gain or loss between subsequent injections) was observed after multiple LNG gel
injections ( 3.3744.5 g; r2 5 0.001).
DISCUSSION
Subdermal MGA implants are the most common contraceptives used in zoo
primates [DeMatteo, 1997; Munson et al., 1995; Porton and DeMatteo, 2005]. The
implants contain synthetic progesterone mixed in a silastic polymer and a catalyst, and
they prevent reproduction by suppressing ovulation and normal ovarian cyclicity
[Kazensky et al., 1998]. MGA has showed to be effective in preventing pregnancy in
golden lion tamarins (Leontopithecus rosalia). After removal of the MGA implant,
75% of the contracepted females resumed reproduction within two years [Wood et al.,
2001]. MGA has been effective in preventing pregnancy in the common marmoset
(Callithrix jacchus) for up to 21 months. In golden headed lion tamarins
(Leontopithecus chrysomelas), MGA has been effective in preventing pregnancy, but
there is some concern about the reversibility of reproduction once the MGA has been
removed. Analyses by De Vleeschouwer et al. [2004] showed that only 34% of the
golden headed lion tamarins resumed breeding after their MGA implants were
removed or had expired. Smaller litters and high infant mortality was also observed in
females previously implanted with MGA [De Vleeschouwer et al., 2000]. While cotton-
top tamarins have been given MGA for many years in zoos [DeMatteo et al., 2002], a
formal evaluation has not been conducted on the efficacy or long-term effects of MGA
in this species. Data presented in DeMatteo et al. [2002] show that 273 cotton-top
tamarins have been given MGA implants with reports of only 14 animals having
MGA removed for reproduction. Of those, only eight females gave birth within one
year of implant removal. Information on why the remaining six females did not give
birth is not available. Because of these concerns, efforts have been underway for
several years to identify an alternative contraceptive strategy, and this report suggests
that LNG delivered as a slow release implant should be considered.
In women, LNG modes of contraceptive action include anovulation, deficient
luteal phases, suppression of follicular growth and altered cervical mucus [Alvarez
et al., 1986, 1996; Brache et al., 1985, 1990, 1999, 2001; Croxatto et al., 1981, 1982,
1988; Darney, 1994; Dunson et al., 1998; Faundes et al., 1991; Lemus et al., 1992;
Ratsula et al., 1989]. Likewise, LNG has been found to be effective in a variety of
Zoo Biology
518 Wheaton et al.
laboratory and wildlife species including mice [Shirley and Bundren, 1995], Wistar
rats [Kuhnz et al., 1995; Telleria et al., 1994], New Zealand white rabbits [Nisker
et al., 1988], striped skunk (Mephitis mephitis) [Bickle et al., 1991], domestic cat
[Baldwin et al., 1994; Looper et al., 2001; Pelican et al., 2004] Japanese quail
(Coturnix coturnix japonica) [Tell et al., 1999], eastern grey kangaroo (Macropus
eugenii) [Coulson et al., 2008; Nave et al., 2002a,b]; tammar wallaby (Macropus
eugenii) [Hynes et al 2007; Nave et al., 2000, 2002b], koala (Phascolarctos cinereus)
[Middleton et al., 2003], white-faced saki (Pithecia pithecia) [Savage et al., 1998,
2002], chimpanzees (Pan troglodytes) [Bettinger et al., 1997], Barbary macaques
(Macaca sylvanus) [Wallner et al., 2006, 2007], and brown capuchin (Cebus apellla)
[Nagle et al., 2009; Ortiz et al., 2004]. LNG was not found to be effective in white-
tailed deer (Odocoileus virginianus) [White et al., 1994; Plotka and Seal, 1989].
While LNG has not been applied extensively to New World primates, studies
suggest that it may be promising [Porton and DeMatteo, 2005]. LNG exhibits strong
progestational effects and lacks estrogen-like activity [Lemus et al., 1992], which is
likely to promote species-typical behavior [e.g. genital swelling in chimpanzees; see
Bettinger et al., 1997 for a review]. Ortiz et al. [2004] found that an oral dose of LNG
can inhibit or delay ovulation in Cebus apella. Savage et al. [1998] found that LNG
administered in a 70 mg silastic tubule implanted subdermally between the scapulae
of white-faced sakis (Pithecia pithecia) was effective in preventing pregnancy for up
to 734 days and was reversible with females conceiving between 1 and 3 cycles
following LNG removal. The challenge with the silastic tubule implant is that it can
be removed by cagemates [Porton and DeMatteo, 2005] as has been observed with
white-faced sakis [Savage et al., 1998] and in chimpanzees [Bettinger et al., 1997].
This study has demonstrated that subcutaneous LNG depot injection is an
effective and reversible contraceptive method for cotton-top tamarins and is likely to
be effective in other primate species. Depending on the needs of individual
institutions, tested formulations ranged between 14.6 and 28.4 weeks of mean
effectiveness. Since cotton-top tamarin breeding recommendations are made
annually, a single injection of 60 mg/kg LNG (formulation 4, 6% LNG) delivered
as an injectable, slow-release, cesium-irradiated biodegradable gel may be the most
useful to insure effective contraception and reversibility if breeding is desired within
one year. The 6% LNG formulation eliminated fecal PdG and E1C profiles, reducing
fecal concentrations to nadir values for up to 19 weeks (range 19.7–50.0 weeks). All
females were observed to resume normal ovarian cycles after repeated injection trials
over the course of seven years. In general, ovarian steroid activity resumed when
fecal LNG concentrations were reduced to less than 100 ng/g feces. In a further test
of contraception reversibility, one female (Te) paired with an intact male was
permitted by the SSPr to breed and successfully conceived immediately following
return of cycles. Following testing, females were monitored up to 1.3 years to
confirm continuation of ovarian cyclicity. Contraceptive treatment did not alter
reproductive or affiliative behaviors important in social or family cohesion in this
species [Savage et al., 1998].
As in human trials, LNG treatment in cotton-top tamarins produced high
individual variability in LNG elimination profiles, secondary peaks and peak LNG
concentrations following injection, and duration of ovarian cycle suppression [Kook
et al., 2002]. Initial and secondary LNG elimination peaks likely represent rapid
metabolism of the injected LNG depot reducing duration of contraception effect.
Zoo Biology
Changes in gel matrix formulation viscosity were made to alter those initial
degradation characteristics of the injected gel depot. These adjustments successfully
eliminated secondary LNG peaks [Wang et al., 2005] and reduced peak LNG
concentrations but did not increase ovarian cycle suppression duration, which was only
altered by increased LNG dosage to 6%. The gel formulations reported here are at the
limit of injectability in terms of viscosity, volume and dosage. Formulation 4 (6%
LNG) likely represented super-saturation and further matrix changes to increase the
duration of contraceptive efficacy of the LNG gel formulations could be challenging.
LNG contraception provides a valuable contraceptive option for management
of this critically endangered species in captivity. Compared to current injectable
contraception options in this species (Depo Proveras), this injectable, LNG gel
formulation is long acting. Using this biodegradable gel, LNG dosage can be
modified to suit species-specific needs to produce physiological concentrations in
circulation required for effective contraception. A 60 mg/kg dose provided 19–50
weeks of contraception compared to 4–5 weeks for Depo Proveras injection. Unlike
MGA implants, the LNG gel is unlikely to be removed by cagemates, limiting the
chance of nonrecommended births in this species.
CONCLUSIONS
This study tested four LNG-loaded biodegradable gel formulations in cotton-
top tamarins. All tested formulations successfully eliminated fecal PdG and E1C
profiles to nadir concentrations indicating the contraception efficacy of subdermal
injection of LNG gels in suppression of ovarian cycles.
Treatment was effective for 102.0720.9 days to 198.8770.3 days depending on
gel formulation and LNG dose used. Maximal duration of efficacy was observed in
formulation 4 (6% LNG) where treatment with 68.4478.61 mg/kg LNG effectively
suppressed ovarian cycles for 19.7–50.0 weeks in N 5 8 injections.
All treated females successfully resumed ovarian cycle activity (fecal PdG and
E1C profiles) following repeated trials, and for up to 1.3 years of cycle monitoring
following cessation of treatment. One female (Te) housed with an intact adult male
resumed cycling at 117 days post-injection and conceived immediately. Following a
pregnancy loss at approximately week 19–20, Te immediately conceived again and
successfully produced a single baby after a normal pregnancy.
The LNG EIA was validated for use in cotton-top tamarins as a tool to
monitor and maximize LNG gel formulation efficacy. In general, ovarian cycles
resumed when fecal LNG concentrations were reduced to less than 100 ng/g feces in
24 of 29 tested injections.
ACKNOWLEDGMENTS
We thank C. Munro for preparation and provision of the PdG, E1C and LNG
antisera and label. We thank R. Phillips and two anonymous reviewers for helpful
comments on the manuscript. We also thank the DAK CS Mammals Team for their
hard work and dedication in fecal sample collection. We also thank the Central
Florida Zoo for their partnership on this project. Special thanks to H. Forde,
H. Miller and K. Fowler for their assistance in the EIAs. This project was reviewed
and approved by Disney’s Animal Care and Welfare Committee.
Zoo Biology
520 Wheaton et al.
REFERENCES
Alvarez F, Brache V, Tejada AS, Faúndes A. 1986. Coe CL, Savage A, Bromley LJ. 1992. Phyloge-
Abnormal endocrine profile among women with netic influences on hormone levels across the
confirmed or presumed ovulation during long-term primate order. Am J Primatol 28:81–100.
NORPLANTs use. Contraception 33:111–119. Coulson G, Nave CD, Shaw G, Renfree MB. 2008.
Alvarez F, Brache V, Faundes A, Tejada AS, Long-term efficacy of levonorgestrel implants for
Thevenin F. 1996. Ultrasonographic and endo- fertility control of eastern grey kangaroos
crine evaluation of ovarian function among (Macropus giganteus). Wildl Res 35:520–524.
Norplants implants users with regular menses. Croxatto HB, Dı́az S, Miranda P, Elamsson K,
Contraception 54:275–279. Johansson EDB. 1981. Plasma levels of levonor-
Asa CS. 1993. The development of contraceptive gestrel in women during longterm use of
methods for captive wildlife. USDA National Norplants. Contraception 23:197–209.
Wildlife Research Center Symposia, Contracep- Croxatto HB, Diaz S, Pavez M, Miranda P,
tion in Wildlife Management, University of Brandeis A. 1982. Plasma progesterone levels
Nebraska–Lincoln. p 235–240. during long-term treatment with levonor-
Asa CS. 2005. Types of contraception: the choices. gestrel silastic implants. Acta Endocrinol 101:
In: Asa CS, Porton I, editors. Wildlife contra- 307–311.
ception: issues, methods, and applications. Croxatto HB, Dı́az S, Pavez M, Brandeis A. 1988.
Baltimore, MD: John Hopkins University Press. Estradiol plasma levels during long-term treat-
p 29–52. ment with Norplants subdermal implants. Con-
Asa CS, Porton I, Baker AM, Plotka ED. 1996. traception 38:465–475.
Contraception as a management tool for controlling Darney PD. 1994. Hormonal implants: contra-
surplus animals. In: Kleiman DG, Allen ME, ception for a new century. Am J Obstet Gynecol
Thompson KV, Lumpkin S, editors. Wild 170:1536–1543.
mammals in captivity: principles and techniques. DeMatteo K. 1997. AZA Contraception Advisory
Chicago: Chicago University Press. p 451–467. Group contraception report. Part I: Primates.
Assocation of Zoos and Aquarium Contraceptive Saint Louis, MO: Saint Louis Zoological Park.
DeMatteo KE, Porton IJ, Asa CS. 2002. Com-
Advisory Group. 2010. Contraception recom-
ments from the AZA Contraception Advisory
mendations: new world monkeys (Accessed from
Group on evaluating the suitability of contra-
website 1/15/2010).
ceptive methods in golden-headed lion tamarins
Baldwin CJ, Peter AT, Bosu WT, Dubielzig RR.
(Leotopithecus chrysomelas). Anim Welf 11:
1994. The contraceptive effects of levonorgestrel
343–348.
in the domestic cat. Lab Anim Sci 44:261–269.
De Vleeschouwer K, Leus K, Van Elsacker L.
Bettinger T, Cougar D, Lee D, Lasley BL, Wallis J.
2000. An evaluation of the suitability of contra-
1997. Ovarian hormone concentrations and ceptive methods in golden-headed lion tamarins
genital swelling in female chimpanzees with (Leontopithecus chrysomelas), with emphasis on
Norplant implants. Zoo Biol 16:209–223. melengestrol acetate (MGA) implants: 1. Effec-
Bickle CA, Kirkpatrick JF, Turner Jr JW. 1991. tiveness, reversitlity and medical side-effects.
Contraception in striped skunks with Norplants Anim Welf 9:251–271.
implants. Wildl Soc Bull 19:334–338. De Vleeschouwer K, Leus K, Van Elsacker L.
Brache V, Faundes A, Johansson E, Alvarez F. 2004. Re-assessing the reversibility of melengestrol
1985. Anovulation, inadequate luteal phase and acetate (MGA) implants in golden-headed lion
poor sperm penetration in cervical mucus during tamarins (Leontopithecus chrysomelas): a com-
prolonged use of Norplants implants. Contra- parison with golden lion tamarins (Leontopithecus
ception 31:261–273. rosalia). Anim Welf 13:183–191.
Brache V, Alvarez-Sanchez F, Faundes A, Tejada AS, Dunson TR, Blumenthal PD, Alvarez F, Brache V,
Cochon L. 1990. Ovarian endocrine function Cochon L, Dalberth B, Glover L, Remsburg R,
through five years of continuous treatment with Vu K, Katz D. 1998. Timing of onset of
Norplants subdermal contraceptive implants. contraceptive effectiveness in norplant implant
Contraception 41:169–177. users. Part I. Changes in cervical mucus. Fertil
Brache V, Blumenthal PD, Alvarez F, Dunson TR, Steril 69:258–266.
Cochon L, Faundes A. 1999. Timing of onset of Evans S. 1983. Breeding of the cotton-top tamarin
contraceptive effectiveness in Norplants implant Saguius oedipus oedipus: a comparison with the
users. II. Effect on the ovarian function in the common marmoset. Zoo Biol 2:47–54.
first cycle of use. Contraception 59:245–251. Faundes A, Brache V, Tejada AS, Cochon L,
Brache V, Alvarez F, Faundes A. 2001. Mechan- Alvarez-Sanchez F. 1991. Ovulatory dysfunction
ism of action of levonorgestrel contraceptive during continuous administration of low-dose
implants. Gynecol Endocrinol 15:14–20. levonorgestrel by subdermal implants. Fertil
Cleveland J, Snowdon CT. 1984. Social develop- Steril 56:27–31.
ment during the first twenty weeks in the cotton- Filippi A, Gamble KC, Romagnoli S. 2004.
top tamarin (Saguinus o. oedipus). Anim Behav Melengestrol acetate (MGA) and its use in
32:432–444. non-human primates reproduction. European
Zoo Biology
Association of Zoo and Wildlife Veterinarians (Callithrix jacchus) using melengestrol acetate
(EAZWV) 5th Scientific Meeting, May 19–23, implants. J Med Primatol 28:36–47.
2004, Ebeltoft, Denmark, Ithaca, NY, IVIS, Munro C, Laughlin LS, Baldwin DM, Lasley BL.
p 1–5. 1996. An enzyme immunoassay for serum and
French JA. 1983 Lactation and fertility: an urinary levonorgestrel in human and non-human
examination of nursing and interbirth intervals primates. Contraception 54:43–53.
in cotton-top tamarins (Saguinus o. oedipus). Munson L, Harrenstien LM, Haslem CA, Stokes JE.
Folia Primatol 40:276–282. 1995. Update on diseases associated with contra-
Graham LH, Schwarzenberger F, Möstl E, ceptive use in zoo animals. 1995. Proc Joint Conf
Galama W, Savage A. 2001. A versatile enzyme Am Assoc Zoo Vet, Wildl Dis Assoc, and Am
immunoassay for the determination of proges- Assoc Wildl Vet 1995. p 398–400.
togens in feces and serum. Zoo Biol 20:227–236. Munson L, Moresco A, Calle PP. 2005 Adverse effects
Gray ME, Cameron EZ. 2010. Does contraceptive of contraceptives. In: Asa CS, Porton IJ, editors.
treatment in wildlife result in side effects? Wildlife contraception: issues, methods and applica-
A review of quantitative and anecdotal evidence. tions. Baltimore: Johns Hopkins Press. p 66–82.
Reproduction 139:45–55. Murnane RD, Zdziarski JM, Walsh TF,
Heistermann M, Tari S, Hodges JK. 1993. Kinsel MJ, Meehan TP, Kovarik P, Briggs M,
Measurement of faecal steroids for monitoring Raverty SA, Lyndsay G, Phillips J. 1996.
ovarian function in New World primates, Melengestrol acetate-induced exuberant endome-
Callitrichidae. J Reprod Fertil 99:243–251. trial decidualization in Goeldi’s marmosets
Hynes EF, Nave CD, Shaw G, Renfree MB. 2007. (Callimico goeldii) and squirrel monkeys (Saimiri
Effects of levonorgestrel on ovulation and sciureus). J Zoo Wildl Med 27:315–324.
oestrous behaviour in the female tammar Nagle CA, Lahoz MM, Porta MM, Torres M,
wallaby. Reprod Fertil Dev 19:335–340. Manzur T, Farinati Z. 2009. Suppression of
Kazensky CA, Munson L, Seal US. 1998. The ovulation by a synthetic progestin in the capu-
effects of melengestrol acetate on the ovaries of chin monkey. J Med Primatol 38:340–346.
captive wild felids. J Zoo Wildl Med 29:1–5. Nave CD, Shaw G, Short RV, Renfree MB. 2000.
Kook K, Gabelnick H, Duncan G. 2002. Pharma- Contraceptive effects of levonorgestrel implants
cokinetics of levonorgestrel 0.75 mg tablets. in a marsupial. Reprod Fertil Dev 12:81–86.
Contraception 66:73–76. Nave CD, Coulson G, Poiani A, Shaw G,
Kuhnz W, Fritzemeier KH, Hegele-Hartung C, Renfree MB. 2002a. Fertility control in the
Krattenmacher R. 1995. Comparative progesta- eastern grey kangaroo using levonorgestrel
tional activity of norgestimate, levonorgestrel- implants. J Wildl Manage 66:470–477.
oxime and levonorgestrel in the rat and binding Nave CD, Coulson G, Short RV, Poiani A,
of these compounds to the progesterone receptor. Shaw G, Renfree MB. 2002b. Long-term fertility
Contraception 51:131–139. control in the kangaroo and the wallaby using
Lee-Parritz DE, Roberts E. 1994. Efficacy of levonorgestrel implants. Reprod Suppl 60:71–80.
hormonal contraception in cotton-top tamarins Nettles VF. 1997. Potential consequences and
(Saguinus oedipus). Wheeling WV: AZA Regional problems with wildlife contraceptives. Reprod
Conference Proceedings. p 387–390. Fertil Devel 9:137–143.
Lemus AE, Vilchis F, Damsky R, Chávez BA, Nisker JA, Kirk ME, Nunez-Troconis JT. 1988.
Garcı́a GA, Grillasca I, Pérez-Palacios G. 1992. Reduced incidence of rabbit endometrial neo-
Mechanism of action of levonorgestrel: in vitro plasia with levonorgestrel implant. Am J Obstet
metabolism and specific interactions with steroid Gynecol 158:300–303.
receptors in target organs. J Steroid Biochem Ortiz ME, Ortiz RE, Fuentes MA, Parraguez VH,
Mol Biol 41:881–890. Croxatto HB. 2004. Post-coital administration of
Leong KM, Terrell SP, Savage A. 2004. Causes levonorgestrel does not interfere with post-
of mortality in captive cotton-top tamarins fertilization events in the new-world monkey
(Saguinus oedipus). Zoo Biol 23:127–137. Cebus apella. Hum Reprod 19:1352–1356.
Looper S, Anderson GM, Sun Y, Shukla A, Pelican KM, Brown JL, Wildt DE, Ottinger MA,
Lasley BL. 2001. Efficacy of levonorgestrel Howard JG. 2004. Ovarian suppression with the
when administered as an irradiated, slow-release progestin levonorgestrel improves ovulation in-
injectable matrix for feline contraception. Zoo duction for artificial insemination in the domestic
Biol 20:407–421. cat. Reprod Fertil Dev 16:131–131 [Abstract].
Middleton DR, Walters B, Menkhorst P, Wright P. Pérez-Palacios G, Chávez B, Escobar N, Vilchis F,
2003. Fertility control in the koala, (Phascolarctos Larrea F, Lince M, Pérez AE. 1981. Mechanism
cinereus): the impact of slow-release implants of action of contraceptive synthetic progestins.
containing levonorgestrel or oestradiol on the J Steroid Biochem 15:125–130.
production of pouch young. Wildl Res 30: Phillips R, Wheaton CJ. 2008. Urinary steroid
207–212. hormone analysis of ovarian cycles and preg-
Möhle U, Heistermann M, Einspanier A, Hodges JK. nancy in mandrills (Mandrillus sphinx) indicate
1999. Efficacy and effects of short- and medium- that menses, copulatory behavior, sexual swel-
term contraception in the common marmoset lings and reproductive condition are associated
Zoo Biology
522 Wheaton et al.
with changing estrone conjugates (E1C) and Tardif SD, Carson RL, Gangaware BL. 1990.
pregnanediol-3-glucuronide (PdG). Zoo Biol 27: Infant-care behavior of mothers and fathers in a
320–330. communal-care primate, the cotton-top tamarin
Plotka ED, Seal US. 1989. Fertility control in (Sanguinus oedipus). Am J Primatol 22:73–85.
female white-tailed deer. J Wildl Dis 25:643–646. Tardif SD, Carlson RL, Gangaware BL. 1992.
Porton IJ, DeMatteo KE. 2005. Contraception in Infant-care behavior of non-reproductive helpers
nonhuman primates. In: Asa CS, Porton IJ, in a communal-care primate, the cotton-top
editors. Wildlife contraception: issues, methods tamarin (Saguinus oedipus). Ethology 92:155–167.
and applications. Baltimore: Johns Hopkins Tell L, Shukla A, Munson L, Thosar S, Kass P,
Press. p 119–148. Stanton R, Needham M, Lasley BL. 1999.
Price EC, McGrew WC. 1990. Cotton-top tamarins A comparison of the effects of slow release,
(Saguinus (o.) oedipus) in a semi-naturalistic injectable levonorgestrel and depot medroxypro-
captive colony. Am J Primatol 20:1– 12. gesterone acetate on egg production in Japanese
Ratsula K, Toivonen J, Lähteenmäki P, Luukkainen T. quail (Coturnix coturnix japonica). J Avian Med
1989. Plasma levonorgestrel levels and ovarian Surgery 13:23–31.
function during the use of a levonorgestrel- Tellerı́a CM, Carrizo DG, Deis RP. 1994. Levo-
releasing intracervical contraceptive device. norgestrel inhibits luteinizing hormone-stimu-
Contraception 39:195–204. lated progesterone production in rat luteal cells.
Savage A, Ziegler TE, Snowdon CT. 1988. Socio- J Steroid Biochem Mol Biol 50:161–166.
sexual development, pair bond formation, and Wang SH, Zhang LC, Lin F, Sa XY, Zuo JB,
mechanisms of fertility suppression in female Shao QX, Chen GS, Zeng S. 2005. Controlled
cotton-top tamarins (Saguinus oedipus oedipus). release of levonorgestrel from biodegradable
Am J Primatol 14:345–359. poly(D,L-lactide-co-glycolide) microspheres: in vitro
Savage A, Shideler SE, Soto LH, Causado J, and in vivo studies. Int J Pharm 301:217–225.
Giraldo LH, Lasley BL, Snowdon CT. 1997. Wallner B, Dittami J, Wallis J. 2006. Influence of
Reproductive events of wild cotton-top tamarins perineal swellings on behavior and stress reaction
in levonorgestrel implanted Macaca sylvanus
(Saguinus oedipus) in Colombia. Am J Primatol
females. Neuroendocrinol Lett 27:253–256.
43:329–337.
Wallner B, Möstl E, Dittami J. 2007. Chronic
Savage A, Zirofsky DS, Shideler SE, Lasley BL.
levonorgestrel treatment in Macaca sylvanus:
1998. The use of Norplant as an effective means
effects on perineal swelling size and fecal sex
of contraception in the white-faced saki (Pithecia
steroid excretion. Neuroendocrinol Lett 28:
pithecia). Am J Primatol 45:205 [Abstract].
326–332.
Savage A, Zirofsky DS, Shideler SE, Smith TE,
Washabaugh KF, Snowdon CT, Ziegler TE. 2002.
Lasley BL. 2002. Use of levonorgestrel as an
Variations in care for cotton-top tamarin, Saguinus
effective means of contraception in the white- oedipus, infants as a function of parental experience
faced saki (Pithecia pithecia). Zoo Biol 21:49–57. and group size. Anim Behav 63:1163–1174.
Savage A, Soto L, Medina F, Emeris G, Soltis J. White L, Warren R, Fayrer-Hosken R. 1994.
2009. Litter size and infant survivorship in wild Levonorgestrel implants as a contraceptive in
groups of cotton-top tamarins (Saguinus oedipus) captive white-tailed deer. J Wildl Dis 30:241–246.
in Colombia. Am J Primatol 71:707–711. Wood C, Ballou JD, Houle CS. 2001. Restoration
Shirley B, Bundren JC. 1995. Effects of levonorges- of reproductive potential following expiration or
trel on capacity of mouse oocytes for fertilization removal of melengestrol acetate contraceptive
and development. Contraception 51:209–214. implants in golden lion tamarins (Leontopithecus
Snowdon CT, Savage A, McConnell PB. 1985. rosalia). J Zoo Wildl Med 32:417–425.
A breeding colony of cotton-top tamarins Ziegler TE, Bridson WE, Snowdon CT, Eman S.
(Saguinus oedipus). Lab Anim Sci 35:477–480. 1987a. Urinary gonadotropin and estrogen
Tardif SD, Harrison ML. 1986. Energetic demands excretion during the postpartum estrus, concep-
of infant care in the calitrichids: Species compar- tion, and pregnancy in the cotton-top tamarin
isons. Primate Report 14:78–79. (Saguinus oedipus oedipus). Am J Primatol 12:
Tardif SD, Clapp NK. 1993. Breeding the cotton- 127–140.
top tamarin in captivity. In: Clapp NK, editor. Ziegler TE, Savage A, Scheffler G, Snowdon CT.
A primate model for the study of colitis and 1987b. The endocrinology of puberty and repro-
colonic carcinoma: the cotton-top tamarin: ductive functioning in female cotton-top tamar-
Saguinus oedipus. Boca Raton, FL: CRC Press. ins (Saguinus oedipus) under varying social
p 45–53. conditions. Biol Reprod 37:618–627.
Tardif SD, Carson RL, Gangaware BL. 1986. Ziegler TE, Sholl SA, Scheffler G, Haggerty MA,
Comparison of infant care in family groups of Lasley BL. 1989. Excretion of estrone, estradiol,
the common marmoset (Callithrix jacchus) and and progesterone in the urine and feces of the
the cotton-top tamarin (Saguinus oedipus). Am J female cotton-top tamarin. Am J Primatol 17:
Primatol 11:103–110. 185–195.
Zoo Biology

Zoo Biology - 2010 - Wheaton - The Use of Long Acting Subcutaneous Levonorgestrel LNG Gel Depot As An Effective

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Zoo Biology - 2010 - Wheaton - The Use of Long Acting Subcutaneous Levonorgestrel LNG Gel Depot As An Effective

Uploaded by

Copyright:

Available Formats

10982361, 2011, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/zoo.20354 by C/O Glucksman Library, Wiley Online Library on [02/11/2022].

The Use of Long Acting Subcutaneous

Cotton-top tamarins (Saguinus oedipus) are a critically endangered species that

r 2010 Wiley Periodicals, Inc.

that subcutaneous LNG depot injection was an effective, reversible contraceptive

Keywords: contraception; biodegradable injectate; estrone conjugates; pregnanediol-

failure [R. Phillips, Cotton-top Tamarin SSPr Coordinator, pers. comm.].

Reversibility of LNG contraceptive treatment was assessed through monitoring

Validation 0% 4% 6% Observations reversibilitya

Contraception LNG Contraception Ovarian Pregnancy

DAK Ge F MGA x MGA removed x x x x x x

Eu F MGA x MGA removed x x x x

Al F MGA MGA removed x x x x x x

Ro F MGA MGA removed x x x x x x

CFZ Te F MGA MGA removed x x x

Fecal Sample Collection and Extraction

Ovarian Cycle Monitoring

male–female pairs). In paired animals, copulatory activity (including mounts and

LNG Gel Injectate Formulation Development and Dosage

Gel formulation development

TABLE 2. Composition of LNG Gel Depot Formulations Tested

LNG (%w/w) Plasticizers (%w/w) Polymer composition (%w/w)

10% triethyl citrate 90% 50/50 Polylactic-co- 85/15 Polylactic-co- Polylactic

Subcutaneous placement of LNG gel depot

Adjunct therapy and posttreatment monitoring

LNG Gel Injectate Formulation Development and Dosage

Suppression of ovarian cycles

LNG Peak Concentrations, Elimination Profiles and Duration of Cycle

LNG injection Ovarian cycle suppression

Fecal E1C (µg/g)

Number of weeks post-LNG injection

F1 (4%) 1 11/26/2002 Ro 44.83 142 6.26

LNG formulations did not increase duration of efﬁcacy (Kruskal–Wallis

and maintenance protocols (isoﬂurane induction box, ketamine injection followed by

You might also like