BIOLOGY OF REPRODUCTION 57, 295-302 (1997)
Pharmacokinetics and Ovarian-Stimulatory Effects of Equine and Human Chorionic
Gonadotropins Administered Singly and in Combination in the Domestic Cat'
William F. Swanson, 23 Barbara A. Wolfe,3 Janine L. Brown,3 Tomas Martin-Jimenez, 4 Jim E. Riviere,4
Terri L. Roth,3 and David E. Wildt3
Conservation & Research Center,3 National Zoological Park, Smithsonian Institution,
Front Royal, Virginia 22630
College of Veterinary Medicine,4 North Carolina State University, Raleigh, North Carolina 27606
ABSTRACT
Pregnancy success and embryo survival are low with the use
of assisted reproduction in felids treated with exogenous gonad-
otropins. In this study, the pharmacokinetics and ovarian-stim-
ulatory effects of eCG and hCG were evaluated in the domestic
cat. Catheterized anestrual queens (n = 4 per treatment [ITrt]
group) were given 100 IU eCG i.v. (Trt 1), 100 IU eCG i.m. (Trt
2), 75 IU hCG i.v. (Trt 3), 75 IU hCG i.m. (Trt 4), or 100 IU
eCG i.m. followed 80 h later by 75 IU hCG i.m. (Trt 5). Blood
samples were collected at 0, 5, 30, and 60 min and 4, 8, 12,
24, 36, 48, 72, 96, 120, 144, and 168 h postinjection, and serum
samples were analyzed for estradiol-1703, progesterone, eCG,
and hCG. Pharmacokinetic traits (volume of distribution, Vd;
elimination half-life, t,,2,; clearance rate, Clr) were calculated
for eCG and hCG. When i.v. and i.m. administration were com-
pared, no differences (p > 0.05) were observed in follicle or
corpus luteum (CL) number or hormone concentrations for
queens receiving eCG or hCG alone. Number of mature ovarian
follicles ( 2 mm diameter) observed at 168 h postinjection did
not differ (p > 0.05) for eCG (mean SEM, 10.5 2.0) vs.
hCG (11.1 3.0), indicating that these were equally effective
in inducing follicular growth. In most queens (> 90%) given
single gonadotropins (i.m. or i.v.), eCG and hCG persisted in
circulation for at least 120 h and 96 h after injection, respec-
tively, reflecting similar (p > 0.05) pharmacokinetic (i.v.) values
for Vd (eCG, 91.4 ? 24.8 ml/kg; hCG, 59.1 + 7.9 ml/kg), t,,,,
(eCG, 23.0 ? 2.4 h; hCG, 22.9 ? 4.1 h), and Cir (eCG, 2.7 +
0.5 ml/h per kg; hCG, 1.8 ? 0.1 ml/h per kg). Sequential treat-
ment with eCG+hCG did not affect (p > 0.05) the t,,,, of in-
dividual gonadotropins. In summary, eCG and hCG have com-
parable pharmacokinetics and ovarian-stimulatory activity when
administered alone to the domestic cat. These findings suggest
that hCG promotes the ancillary follicle formation that is fre-
quently observed after ovulation in cats treated with eCG+hCG
regimens, possibly disrupting the maternal environment and de-
creasing fecundity following assisted reproductive procedures.
INTRODUCTION
Exogenous gonadotropin regimens, consisting of se-
quential treatment with eCG and hCG, are frequently used
in domestic and nondomestic felids as one component of
in vitro fertilization or artificial insemination procedures
[1-8]. Typically, eCG is administered to stimulate ovarian
Accepted March 21, 1997.
Received January 21, 1997.
'This research was supported, in part, by NIH grants K01 RR0009801
from the National Center for Research Resources, R01 HD 23853 from
the National Institute of Child Health and Human Development, and the
Philip Reed Foundation.
2Correspondence: William Swanson, Department of Reproductive
Physiology, Conservation & Research Center, 1500 Remount Road, Front
Royal, VA 22630. FAX: (540) 635-6571;
e-mail: nzpcrcl8@sivm.si.edu
295
follicular growth followed several days later by hCG to
induce final follicular maturation and ovulation. Applied
usage, however, has not consistently translated into high
reproductive efficiency, as measured by embryo survival
rates after embryo transfer or pregnancy success after ar-
tificial insemination [2-8]. Low fecundity after exogenous
gonadotropin treatment possibly is related to a maternal en-
vironment unsuitable for supporting embryo development.
It is well known that queens treated with eCG/hCG com-
binations typically develop ancillary ovarian follicles sev-
eral days after induced ovulation and that these follicles
subsequently form secondary corpora lutea (CL), possibly
disrupting the maternal environment [2, 4, 9, 10]. Persis-
tence of eCG after i.m. injection has been implicated in
possibly contributing to postovulatory follicle growth; how-
ever, attempted neutralization of residual eCG was ineffec-
tive in preventing ancillary follicle and secondary CL de-
velopment [10]. Therefore, perhaps hCG, rather than eCG,
is responsible for ancillary follicle development after ovu-
lation. This speculation is consistent with our earlier ob-
servations that hCG given to naturally estrual cats prolongs
behavioral estrus [11] and may promote folliculogenic ac-
tivity after natural mating [12].
Investigations of the normality of the cat maternal en-
vironment resulting from exogenous gonadotropin treat-
ment have been hindered by two deficiencies. First, there
has been no comprehensive, normative database describing
embryogenesis and early maternal reproductive character-
istics in naturally estrual, naturally mated cats-information
that is essential for comparative purposes. Second, our un-
derstanding of the pharmacokinetic activity and ovarian-
stimulatory effects of individual exogenous gonadotropins
in cats is limited, and this limitation interferes with our
ability to design effective ovarian stimulation protocols. To
resolve the first shortcoming, we recently completed a se-
ries of studies characterizing developmental [13, 14], his-
tological [15], and endocrine changes [16] associated with
the periovulatory, conceptive, and preimplantation/early-
implantation interval in the cat. From these studies, we now
have a much broader understanding of what constitutes a
"normal" maternal environment.
The present study was designed to address the second
deficiency and to provide additional insight into the devel-
opment of gonadotropin treatments that will more consis-
tently produce a physiologically normal maternal state. Our
specific objectives were to 1) evaluate ovarian responses
and the dynamics of circulating estradiol-173 and proges-
terone secretion in response to eCG and hCG, 2) charac-
terize the pharmacokinetics of each gonadotropin after i.v.
administration, and 3) assess the circulatory persistence of
each gonadotropin alone and in combination following i.m.
delivery.
SWANSON ET AL.
MATERIALS AND METHODS
Laparoscopy and Jugular Catheter Placement
Adult female cats (Liberty Research Inc., Waverly, NY)
were housed in communal pens under controlled artificial
lighting (12L: 12D) and provided a commercial cat food diet
(Purina Cat Chow; Ralston Purina, St. Louis, MO) and wa-
ter ad libitum. Queens (n = 23) were evaluated daily for
overt estrual behavior [13, 17] to identify the anestrual
phase of the reproductive cycle before administration of
gonadotropins. To verify ovarian inactivity, queens were
anesthetized and both ovaries were examined for absence
of mature follicles ( 2 mm in diameter, vesicular, raised
above ovarian surface) and CL via laparoscopy [13, 18].
Only queens without mature follicles or CL were catheter-
ized for blood sample collection. None of the study queens
had been treated previously with gonadotropins.
For catheter placement, the ventral neck region of anes-
thetized queens was surgically prepared using 70% isopro-
pyl alcohol and povidone-iodine solution (Betadine Surgi-
cal Scrub; The Purdue Frederick Company, Norwalk, CT),
and a "through the needle" polymer resin catheter (Intra-
cath Intravenous Catheter Placement Unit; Deseret Medical
Inc., Parke, Davis and Company, Sandy, UT; 19 gauge,
20.3-cm length) was inserted percutaneously into the left
or right external jugular vein. Each catheter was advanced
(-6-8 cm) in the vein, the catheter hub and distal catheter
(-4-6 cm) were excised, and the catheter needle was re-
moved. A new catheter hub (Intramedic Luer Stub Adapter;
Becton Dickinson and Co., Clay Adams Division, Franklin
Lakes, NJ; 20 gauge), with attached PRN adapter (Becton
Dickinson Vascular Access), was placed on the distal end
of the catheter and flushed with sterile saline (1 ml) con-
taining heparin sodium (30 U/ml; Lyphomed, Division of
Fuijisawa USA, Inc., Deerfield, IL). The catheter was su-
tured in place, and the entry site was treated with antibiotic
ointment (bacitracin zinc-neomycin sulfate-polymyxin B
sulfate ointment; E. Fougera & Company, Melville, NY)
and covered with sterile gauze. The external catheter was
extended dorsolaterally to the dorsal aspect of the neck and
thoroughly bandaged using stretch roll gauze, elastic band-
age material (Vetrap Bandaging Tape; Animal Care Prod-
ucts, 3M Company, Saint Paul, MN), and elastic tape (Elas-
tikon; Johnson and Johnson Medical Inc., Arlington, TX).
All catheterized queens received s.c. penicillin (Flo-cillin,
penicillin G benzathine, and penicillin G procaine; Fort
Dodge Laboratories Inc., Fort Dodge, IA) prophylactically.
After catheter placement, animals were weighed ( 0.01 g)
and then housed singly for the next 3 days to reduce the
likelihood of accidental catheter displacement.
Gonadotropin Administration and Blood Sampling
Lyophilized gonadotropins (eCG and hCG; Sigma
Chemical Company, St. Louis, MO) were reconstituted in
sterile water, immediately frozen (-80?C), and stored in
individual-dose syringes until needed. Frozen, reconstituted
gonadotropins were used within 3 mo of storage, and the
same gonadotropin lots (eCG, lot no. 113H07851, 2620
IU/mg, nonbuffered; hCG, lot no. 63430321, -3000
IU/mg, buffered with 0.01 M Na2 HPO 4) were used through-
out the study. Catheterized, anestrual females were assigned
randomly to one of five treatment (Trt) groups; and on the
day after catheter placement, queens (n = 4 per Trt) were
given either 100 IU eCG i.v. (Trt 1), 100 IU eCG i.m. (Trt
2), 75 IU hCG i.v. (Trt 3), 75 IU hCG i.m. (Trt 4), or 100
IU eCG i.m. followed 80 h later with 75 IU hCG i.m. (Trt
5). The latter (Trt 5) is the standard eCG/hCG combination
regimen used with artificial insemination procedures in the
domestic cat [4]. Gonadotropin preparations and standard
dosages were chosen based on this current applied usage.
A blood sample (-2 ml) was collected from each un-
anesthetized queen via the jugular catheter immediately be-
fore gonadotropin injection (Time 0) and then at 5, 30, and
60 min and 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168
h after administration. Samples were centrifuged (1100 x
g, 10 min), and recovered serum was stored at - 80?C until
analysis. For i.v. (Trt 1, 3) and i.m. (Trt 2, 4, 5) treatments,
thawed gonadotropin doses were injected via the jugular
catheter and into the caudal thigh muscles, respectively.
After i.v. gonadotropin injection and each blood sample
collection, catheters were flushed with heparinized saline
(0.5-1.0-mi volume; 30 U heparin/ml). Jugular catheters
were removed from all females after the 48-h time point,
and subsequent blood samples were obtained from unanes-
thetized cats via jugular venipuncture. Concurrent with the
final blood sampling at 168 h, queens were anesthetized
and reevaluated laparoscopically for mature ovarian folli-
cles or CL.
Gonadotropin and Steroid Hormone RIAs
Concentrations of eCG were measured in unextracted cat
serum using a double-antibody RIA. This assay used a
mouse monoclonal antibody against bovine LH (no. 518-
B7; provided by Dr. Jan Roser, University of California,
Davis), eCG standards (PM-230GB; provided by Dr. Har-
old Papkoff, University of California, San Francisco), and
125I-labeled equine LH (E-263B; Dr. Harold Papkoff) in a
phosphate buffer-based system (PBS; 0.01 M P0 4, 0.5%
BSA, 2 mM EDTA, 0.9% NaCl, 0.01% thimerosal, pH 7.4).
Duplicate standards (100 i1, 2.6-327.5 mIU/ml) and serum
samples (100 jil; neat or diluted 1:1-1:40 in PBS) were
incubated at room temperature (22?C) with antibody (1:500
000 in 100 l) for 24 h; then tracer (20 000 c.p.m. in 100
Ipl) was added and incubation continued for an additional
24 h. Antibody-bound complexes were precipitated after a
1-h incubation with goat anti-mouse gamma globulin (1:
200 in 1 ml containing 5% polyethylene glycol) and cen-
trifugation for 30 min at 1500 g. Radioactivity in the
pellets was determined by gamma spectrometry. The anti-
body generally bound -30% of the 2 5I-equine LH with <
7% nonspecific binding. The assay was validated by 1)
demonstrating parallelism between serial dilutions of cat
serum pools and the standard curve and 2) significant re-
covery of exogenous eCG added to serum before analysis
(y = 1.01x - 0.282; r = 0.99). Assay sensitivity was 2.6
mIU/ml at 90% of maximum binding, and the intra- and
interassay coefficients of variation were < 10%, based on
average variation within samples (n = 180) and internal
controls (n = 2), respectively. The anti-bovine LH antibody
cross-reacts -39% with eCG and -10% with hCG [19].
Because of hCG cross-reactivity, serum eCG concentrations
for females receiving both gonadotropins (Trt 5) were ad-
justed by decreasing values by 10% of the measured hCG
concentration (only for samples collected after hCG injec-
tion). Accordingly, these latter values should be considered
estimates.
Concentrations of hCG were determined in unextracted
serum using a double-antibody RIA (Diagnostic Products
Corporation, Los Angeles, CA). This assay uses an anti-
body against the hCG subunit. Serum samples (100 pl1;
296
PHARMACOKINETICS OF eCG AND hCG IN CATS
neat or diluted 1:1-1:30 with pooled cat serum) or stan-
dards (3.9-250 mIU/ml diluted in cat serum) were coin-
cubated with [3-hCG antiserum (50 ?l1) for 30 min at 37?C
followed by addition of 125I-labeled hCG tracer (30 000
cpm in 50 1) and further incubation at 37?C for 30 min.
Antibody-bound complexes were precipitated by addition
of 0.5 ml goat anti-rabbit gamma globulin and centrifuga-
tion for 30 min at 1500 x g. Cross-reactivity of hCG an-
tisera with eCG and human LH is less than 0.1% and 0.2%,
respectively. Serial dilutions of cat serum pools were par-
allel to the standard curve. Addition of exogenous hCG to
cat serum resulted in significant net recovery (y = 1.18x
- 8.7; r = 0.99). Assay sensitivity was 5 mIU/ml, and the
intra- and interassay coefficients of variation were < 10%
on the basis of average sample (n = 144) variation and
internal controls (n = 2), respectively.
Estradiol-17 and progesterone concentrations were
measured in unextracted serum using validated, solid-phase
125I RIAs (Coat-a-Count; Diagnostic Products Corporation)
as described previously [16]. Assay sensitivities for estra-
diol-17 and progesterone were 5.0 pg/ml and 0.05 ng/ml,
respectively. Intra- and interassay coefficients of variation
were < 10% on the basis of average sample (n = 300)
variation and internal controls (n = 2), respectively.
Pharmacokinetic and Statistical Analysis
For i.v. gonadotropin treatment groups (Trt 1, 3), log
serum eCG and hCG concentrations (mIU/ml) of individual
animals were plotted against time and fitted to various com-
partmental models using a computer modeling program
(SAAMII; SAAM Institute, University of Washington, Se-
attle, WA). After curve fitting to the most appropriate mod-
el, disappearance curves for each gonadotropin were sub-
jected to linear regression analysis in a semilogarithmic
scale to determine y intercepts (A and B) and slopes ( and
3) of distribution and elimination components, respectively.
For each gonadotropin, theoretical initial serum concentra-
tion (Co) was calculated by summing the y intercepts, and
distribution (t1 /2 ) and elimination (t1 /2.) half-lives were cal-
culated as t1/2, = 0.693/ot and t1/2, = 0.693/3, respectively.
Area under the curve (AUC) for each disposition curve was
determined using AUC = A/ + B/3. Mean residence
times (MRT) were calculated as MRT = AUMC/AUC,
with the numerator AUMC = A/at2 + B/[32. Volume of
distribution (Vd) was determined using Vd = dose/
(AUC)3, and clearance rate (Clr) was ascertained using Clr
= x Vd [20].
For i.m. treatment groups (Trt 2, 4, 5), log serum eCG
and hCG concentrations (mIU/ml) of individual animals
were plotted against time and slopes () of the postabsorp-
tive elimination curves determined using linear regression
analysis in a semilogarithmic scale. Elimination half-lives
(t11/2) were calculated from t/2 = 0.693/3, and AUCs were
calculated by the log trapezoidal method, extrapolated to
infinity. Absolute bioavailability (F) for each gonadotropin
was determined as F = AUCi.m./AUCi v., using mean values
for AUCi.m and AUCi.v.
For each treatment group, serum estradiol-17 concen-
trations over time were assessed by calculating AUC using
the trapezoidal method, extrapolated to infinity [20]. Mean
(? SEM) values were determined for pharmacokinetic pa-
rameters (Co, A, B, t/2, tl/2, MRT, AUC, Vd, Clr), estra-
diol concentrations over time (AUC), progesterone concen-
trations (for anovulatory and ovulatory queens), and num-
ber of ovarian follicles and total ovarian structures (mature
FIG. 1. Mean ( SEM) number of follicles (- 2 mm) in anovulatory
queens and total ovarian structures (follicles + CL) in all queens observed
via laparoscopy at 168 h after gonadotropin injection. Anestrual queens
received either 100 IU eCG (i.v. or i.m.), 75 IU hCG (i.v. or i.m.), or 100
IU eCG i.m. followed 80 h later with 75 IU hCG i.m. Numbers in paren-
theses indicate the number of queens included in each mean value cal-
culation.
follicles + CL) at the 168-h time point. Values for dosage-
independent (t1 /2,, t1/20, MRT, Vd, Clr) and dosage-depen-
dent (AUC) pharmacokinetic parameters were compared
between gonadotropins and/or routes of administration us-
ing a Student's t-test [21], when appropriate. Across treat-
ment groups, mean values for estradiol concentrations,
number of ovarian follicles, and total ovarian structures
were compared using analysis of variance [22]. Correlation
coefficients were calculated between estradiol (AUC) and
total number of ovarian structures [21].
RESULTS
Ovarian and Endocrine Characteristics
On the basis of behavioral and laparoscopic assessments,
study females were classified as anestrual on 24 occasions
and subjected to jugular catheterization. On four occasions,
catheters could not be placed appropriately or were inserted
properly but later found to have malfunctioned. Following
i.v. or i.m. administration of eCG or hCG, most females
(95%) exhibited behavioral estrus within 2-3 days, with
queens receiving i.v. injections entering estrus -1 day ear-
lier (p < 0.05) than queens injected i.m. Laparoscopy at
168 h revealed that 4 of 4 females receiving the combina-
tion eCG/hCG treatment ovulated (based on presence of
ovarian CL). However, distinct ovulatory responses also
were observed in some females given single injections of
eCG (i.v.: 2 of 4, 50%; i.m.: 1 of 4, 25%) or hCG (i.v.: 1
of 4, 25%; i.m.: 1 of 4, 25%), regardless of route of ad-
ministration. Based on laparoscopic evaluations at 168 h,
the mean number of mature ovarian follicles (range, 10.0
? 2.9-17.7 5.6) in anovulatory queens and the number
of total ovarian structures (follicles + CL; range, 9.8 
1.5-18.0 3.4) in all queens did not differ (p > 0.05)
among treatment groups (Fig. 1).
Females also exhibited similar temporal patterns in se-
rum estradiol concentrations, with values typically increas-
ing above basal levels ( 20 pg/ml) 24-72 h after gonad-
otropin injection, reaching peak values by 120-144 h, and
then declining to baseline by 168 h (Fig. 2). Across treat-
ments, there were no differences (p > 0.05) in mean estra-
diol values over time based on AUC calculations (mean
range, 1936.6 203.5-4909.6 573.0 pg/h per ml). Es-
tradiol concentrations over time were positively correlated
297
SWANSON ET AL.
7
6
5
' 4
._;4
3
WI
~jnI
0 24 48 72 96 120 144 168
Time (h)
FIG. 2. Mean (t SEM) serum estradiol-1 7[ concentrations in anestrual
queens administered either 100 IU eCG (i.v. or i.m.), 75 IU hCG (i.v. or
i.m.), or 100 IU eCG i.m. followed 80 h later with 75 IU hCG i.m.
(r = 0.80; p < 0.01) with number of total ovarian structures
at 168 h.
Serum progesterone concentrations typically remained
basal ( 2 ng/ml) in anovulatory queens but rose above
baseline in ovulatory, eCG or hCG only-treated females
beginning -96 h after injection and in ovulatory
eCG+hCG-treated queens beginning -120-144 h after
eCG injection. In ovulatory females, mean progesterone in-
creased (p < 0.01) from a nadir of 1.2 ? 0.4 ng/ml (Time
0) to 9.6 1.7 ng/ml at 168 h postinjection. Despite
eCG+hCG-treated queens having more (p < 0.05) CL
(mean, 15.0 ? 3.3) than ovulatory females given eCG or
hCG only (5.4 ? 1.4), progesterone concentrations at 168
h did not differ (p > 0.05) between the two groups (12.8
+ 2.2 and 7.1 ? 1.9 ng/ml, respectively).
Pharmacokinetics
Pharmacokinetic analysis revealed that eCG and hCG
disappearance curves after i.v. delivery best fit a two-com-
partment, pharmacokinetic model, typified by a rapid dis-
tribution phase and a slow elimination phase (Fig. 3). Phar-
macokinetic values were similar for the two gonadotropins
(Table 1), with no differences (p > 0.05) between any dos-
age-independent parameter (tl/2,, t1/20, MRT, Vd, Clr).
TABLE 1. Pharmacokinetic parameters (mean SEM) of eCG and hCG
following bolus injection in domestic cats.]
Parameterb eCG hCG
Co (mlU/ml) 1948.9 + 1028.7 1697.2 + 169.9
A (mlU/ml) 1558.4 + 943.4 1435.5 + 154.5
B (mlU/ml) 390.6 + 101.9 261.7 + 50.4
t,,, (h) 0.9 + 0.7 3.0 + 0.5
t?, (h) 23.0 + 2.4 22.9 + 4.1
MRT (h) 32.1 + 3.7 20.5 + 3.3
AUC (mlU-h/ml) 12571.4 + 2508.8 14476.7 + 2231.2
Vd (ml/kg) 91.4 ? 24.8 59.1 + 7.9
Clr (ml/h/kg) 2.7 + 0.5 1.8 + 0.1
BW (kg) 3.4 + 0.1 3.0 + 0.3
' Anestrual queens (n = 4 per Trt) were given either eCG (100 IU/dose)
or hCG (75 IU/dose) via i.v. catheters, and serial blood samples were
collected over the next 168 h for determination of serum gonadotropin
concentrations.
b, Co, theoretical initial serum concentration; A, zero intercept rapid dis-
tribution curve; B, zero intercept elimination curve; t,,,, half-life of rapid
distribution; t,,, half-life of elimination; MRT, mean residence time; AUC,
area under the curve; Vd, volume of distribution; CIr, total clearance rate;
BW, body weight.
Concentration-time curves for eCG and hCG after i.m.
injection revealed a variable absorptive phase and a pro-
longed postabsorptive elimination phase (Fig. 4). When i.m.
and i.v. delivery were compared, elimination half-lives
were similar (p > 0.05) for eCG (27.1 + 1.7 vs. 23.0 ?
2.4 h) and hCG (26.1 3.1 vs. 22.9 + 4.1 h), and the
AUC (eCG, 6601.9 ? 1076.9 vs. 12571.4 + 2508.8 mIU/h
per ml; hCG, 8873.2 1221.2 vs. 14476.7 2231.2
mIU/h per ml) was not different (p > 0.05). Both gonad-
otropins demonstrated comparable absolute bioavailability
(eCG, 52.5%; hCG, 61.3%) after i.m. injection. After i.v.
or i.m. delivery of individual gonadotropins, eCG persisted
in circulation in all queens for at least 120 h, whereas hCG
was detectable in 7 of 8 females for at least 96 h. In one
female given hCG i.v., hCG was undetectable after 72 h.
For the eCG+hCG combination, initial absorption and
elimination curves for eCG after i.m. injection (Fig. 5) were
qualitatively similar to that of eCG administered alone, and
the elimination half-lives of eCG (23.7 + 1.2 h) and hCG
(23.2 ? 2.5 h) administered in sequential combination did
not differ (p > 0.05) from that of individual gonadotropins
40
,E 30'
20
o
.=
0
E 10
U:
3000lt
120
1 6001 
68
Time (h)
FIG. 3. Disappearance curve of eCG and hCG following bolus i.v. in-
jection in the domestic cat. Anestrual queens (n = 4 per Trt) received
either 100 IU eCG i.v. or 75 IU hCG i.v., and serial blood samples were
collected via jugular catheters and venipuncture for the next 168 h. Val-
ues are expressed as means (+ SEM). The inset graph depicts the distri-
bution phase.
400
E
300
2 300
,o
100
C
--- eCG i.m.
- hCG i.m.
96
Time (h)
FIG. 4. Disappearance curve of eCG and hCG following i.m. injection
in the domestic cat. Anestrual queens (n = 4 per Trt) received either 100
IU eCG i.m. or 75 IU hCG i.m., and serial blood samples were collected
via jugular catheters and venipuncture for the next 168 h. Values are
expressed as means (+ SEM). The inset graph depicts the absorptive and
distribution phases.
298
.
PHARMACOKINETICS OF eCG AND hCG IN CATS
400
E-
I-
2 200
E loo
1LW
300
.o 1
Time (h)
FIG. 5. Disappearance curve of eCG and hCG following sequential i.m.
injection in the domestic cat. Anestrual queens (n = 4) received 100 U
eCG i.m. followed 80 h later by 75 IU hCG i.m., and serial blood samples
were collected via jugular catheters and venipuncture during a 168-h
period. The arrow indicates time of hCG injection. Values are expressed
as means ( SEM). The inset graph depicts the absorptive and distribution
phases of eCG.
after i.m. delivery. In all females given the combination
regimen, eCG was detectable in serum samples for at least
168 h after eCG injection.
DISCUSSION
This study represents the first objective assessment of
gonadotropin pharmacokinetics and their relation to the
stimulatory effects of gonadotropins alone or in combina-
tion in the domestic cat. Although of basic comparative
value for mammalian species in general, these data are par-
ticularly important for the development of effective ovarian
stimulation protocols for endangered felid species. Es-
pecially significant were the observations of hCG follicu-
logenic effects and persistence in circulation, findings that
suggest the need to modify existing gonadotropin therapies.
When given alone, eCG and hCG had comparable ef-
fectiveness in inducing ovarian follicular development,
each stimulating the production of more than 10 mature
follicles per queen. In contrast, naturally cyclic females de-
velop about 5 mature follicles during a typical estrus [23].
Folliculogenic and luteotrophic activities of eCG have been
well documented in vivo and in vitro [24, 25]. In cats, eCG
traditionally has been used to induce follicular growth [26,
27], although high dosages or multiple injections also are
known to induce ovulation [27]. In contrast, hCG is known
for its luteotrophic bioactivity [28], but intrinsic folliculo-
genic activity has been reported in the hamster [29] and rat
[30, 31]. In the cat, hCG has a strong affinity for LH re-
ceptors [16] and has been used for almost two decades to
induce ovulation in naturally estrual and eCG-treated fe-
males [4, 32].
Earlier observations [11, 12] combined with the present
results support the assertion that hCG has luteotrophic and
folliculogenic effects in the cat, including the capacity to
stimulate growth and maturation of smaller antral follicles
(< 2 mm in diameter). Domestic cat ovaries, regardless of
female reproductive status, contain diverse, asynchronous
cohorts of developing preantral and antral follicles [33-35]
that likely differ markedly in responsiveness to individual
gonadotropins. Among polytocous species, naturally cyclic
and eCG-stimulated pigs also exhibit ovarian follicular het-
erogeneity as demonstrated by variable follicular size, go-
nadotropin-binding capacity, and steroid content [36, 37].
Similarly, hamster ovaries contain a heterologous follicle
population with variable FSH and LH receptor expression
and steroidogenic capacity [38, 39]. Although eCG is pre-
dominantly folliculogenic and hCG luteotrophic in the cat,
each exhibits duality and thus can mimic the other's pri-
mary action. Some evidence has suggested, in fact, that
dual activity of administered gonadotropins is required in
the cat, since highly purified human FSH administered
alone ineffectively promotes folliculogenesis [40]. Further-
more, the superovulatory potential of eCG and hCG is re-
lated to a capacity to "rescue" preantral and early antral
follicles from atresia [29, 41], and the domestic cat ovary
is composed of a large follicle population in various stages
of atresia [35].
In about 30% of queens, immature follicles underwent
final maturation and ovulation within 2-3 days of a single
eCG or hCG injection, as evidenced by distinct CL for-
mation and elevated circulating progesterone. Ovulation
may reflect the innate ovulatory (LH) properties of each
gonadotropin, or, alternatively, each promoted follicular
growth and ovulation occurred "spontaneously." Although
domestic cats usually are considered induced ovulators,
spontaneous ovulation has been observed in naturally es-
trual, unmated females [42]. Sporadic spontaneous ovula-
tion probably also represents another impediment to induc-
ing consistent ovarian responses in the cyclic felid, es-
pecially in nondomestic cats that are intractable and diffi-
cult to assess for estrual status [43, 44]. The present
findings reinforce our previous assertions [7, 43] that phar-
maceutical down-regulation of pituitary activity may be a
prerequisite to improving ovarian responses to exogenous
gonadotropin treatments in felids.
In a previous study [10], the elimination half-life of eCG
in the cat was estimated at -45 h, based on i.m. delivery
and a brief sampling period (i.e., 12-84 h). In the present
expanded study involving i.v. administration, extensive
blood sampling, and more sophisticated analysis, we deter-
mined that the eCG disposition curve best fits a two-com-
partment model, characterized by distinct exponential dis-
tribution and elimination phases. The half-life of eCG elim-
ination was -23 h, substantially shorter than our earlier
estimation. Compared to pharmacokinetic eCG values re-
ported for the sheep [45, 46], cow [47], and rat [48], values
for elimination half-life, volume of distribution, and clear-
ance rate in the cat generally fell within an intermediate
range. Elimination kinetics in these other species also best
fit a two-compartment model, with elimination half-lives
ranging from 6 (rat) to 120 (cattle) h. Clearance rates and
volumes of distribution are more similar among species,
ranging from 1.3 (sheep) to 2.1 (rat) ml/h per kg and from
-50 (rat) to 94.5 (sheep) ml/kg, respectively. In cattle and
sheep, eCG reportedly persists in circulation for 120-240
h after injection [46, 47]. In cats, eCG was detectable in
blood for at least 120 h, an observation consistent with its
proven capacity to promote folliculogenesis after a single
injection in multiple felid species [2, 3, 5, 7, 8].
Although hCG has a lower molecular mass than eCG
(-39 kDa vs. -64 kDa) and a lower percentage of car-
bohydrate (33% vs. 45%) [24, 49, 50], it demonstrated very
similar pharmacokinetic properties after i.v. injection. Sim-
ilar to findings in the human and monkey [51, 52], hCG
elimination in the cat best fits a biexponential model. In-
jected hCG in humans has a slower clearance rate and a
longer elimination half-life (-36 h) [51] than observed in
299
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SWANSON ET AL.
the cat. By contrast, the clearance rate and elimination half-
life of hCG for the cat were relatively prolonged compared
to values for the monkey [52] and rat [53]. Both eCG and
hCG had limited volumes of distribution relative to the
measured plasma volume of the cat (46.8 ml/kg) [54], pos-
sibly reflecting the large molecular size and low lipid per-
meability of both glycoproteins [46]. Also related to these
physicochemical properties, both gonadotropins exhibited
reduced bioavailability after i.m. injection, with only 50-
60% of injected gonadotropin entering circulation. Despite
similar pharmacokinetic traits, hCG persistence in circula-
tion (-96 h) was generally less than that of eCG, possibly
due to differences in total injected dosage (75 IU vs. 100
IU) and sensitivities of the respective RIAs. However, given
that ovulation occurs approximately 30 h after hCG admin-
istration [4, 55], this persistence indicates that hCG is pres-
ent in circulation for -66 h postovulation, likely promoting
postovulatory follicular growth and affecting luteinization
of ovulated and/or unovulated follicles.
Importantly, pharmacokinetic patterns in cats given
eCG+hCG were not appreciably different from those ob-
tained with eCG or hCG administered alone. Because go-
nadotropins influence FSH and LH receptor expression
[56, 57], the pharmacokinetics of gonadotropins adminis-
tered in sequential combination could be affected by in-
creased or decreased receptor induction and receptor-ligand
interaction. In other species, the circulatory clearance of
eCG and hCG is primarily mediated through renal, hepatic,
and plasma-associated metabolism, with the ovary playing
a limited role [58, 59]. Hepatic and renal function in go-
nadotropin clearance has not been investigated in the cat,
but humoral immune responses to these foreign proteins
can affect in vivo bioactivity, presumably by decreasing
circulatory persistence [60, 61]. Because of the naive im-
munological status of study females and the expected nat-
ural lag time associated with primary immune responses
[61], it is unlikely that immunoglobulin production signif-
icantly influenced clearance rates reported in the present
study.
Persistence of hCG postinjection and its demonstrated
folliculogenic effects when given alone suggest that it is at
least partially responsible for ancillary follicle development
after ovulation in the cat. However, eCG, when given prior
to hCG, likely stimulates some additional follicle growth
and potentiates the responsiveness of early antral follicles
to the intrinsic folliculogenic and luteotrophic activity of
hCG. A similar relationship probably occurs with
FSH+hCG regimens, because anestrual cats treated with
multiple injections of FSH followed by hCG also develop
ancillary follicles several days after ovulation [27]. A pre-
eminent role for hCG in promoting postovulatory follicle
growth is supported by our recent observation that neutral-
izing residual eCG at the time of follicular aspiration is
ineffective in preventing formation of ancillary follicles
[10]. Furthermore, the persistence of hCG in circulation and
its prolonged luteotrophic effects may explain the luteini-
zation of ancillary follicles to form secondary CL [10].
The disruptive significance of postovulatory ancillary
follicles in the cat is unknown. Ancillary follicles forming
after eCG injection and ovulation appear to be functional
in the cow, as evidenced by elevated plasma estradiol con-
centrations detected using frequent blood sampling [62, 63].
However, in contrast to the cat, the cow usually is not treat-
ed with hCG but is allowed to generate an endogenous LH
surge. It is possible that ancillary follicles in cats are not
producing significant estradiol because prolonged exposure
to the LH-like signal of hCG combined with rising proges-
terone may disengage follicular aromatase activity [64, 65].
Additionally, estradiol release in the cat is highly pulsatile,
complicating accurate longitudinal assessments [23]. Final-
ly, sustained elevations in serum estradiol that persist for
several days after ovulation in eCG+hCG-treated cats also
may represent a relatively slow transition from estradiol to
progesterone secretion in ovulated follicles [2, 9].
Recently, fecal hormone metabolite monitoring has been
used to compare estradiol production between naturally es-
trual, mated tigers (Panthera tigris) and tigers treated with
eCG+hCG [66]. In that study, mean fecal estradiol values
in the early postovulatory period were significantly greater
in gonadotropin-treated compared to "natural" females, re-
maining elevated for 15 days after administration of hCG.
Several of these females also produced a pronounced fecal
estradiol spike 1-2 wk after hCG, suggesting the formation
of ancillary follicles, previously documented laparoscopi-
cally [3], that are contributing to abnormal estradiol pat-
terns. Similar fecal metabolite patterns are observed in the
eCG+hCG-treated snow leopard (Uncia uncia) but are ab-
sent in the clouded leopard (Neofelis nebulosa) and cheetah
(Acinonyx jubatus) [43, 44, 67]. Because cats exhibit pro-
found species-specific differences in responsiveness to ex-
ogenous gonadotropins [7, 8], these variations in postovu-
latory estradiol production are likely related to natural dif-
ferences in gonadotropin sensitivities among species.
The persistence of hCG and associated ancillary follicle
formation could be managed by several approaches, in-
cluding the use of hCG-specific antisera to neutralize resid-
ual hCG after ovulation induction [28]. An alternative ap-
proach might be to alter the ovulatory signal by perhaps
substituting exogenous GnRH for hCG. Naturally estrual
cats readily ovulate in response to GnRH injection [12, 68],
but studies in progress in our laboratory are revealing that
GnRH used in combination with eCG rarely elicits ovula-
tion in the cat, possibly due to poor timing of LH release
relative to follicular maturity and the comparatively short
duration of the LH surge. The challenge is to identify an
ovulation induction protocol that provides a sufficiently
prolonged LH-like signal that is terminated with the onset
of ovulation, ideally similar to that measured in naturally
mated queens [23]. This strategy presumably would pro-
duce a more physiologically normal maternal environment,
which we suspect would translate into higher fertility and
pregnancy success with the use of assisted reproductive
procedures in felids.
ACKNOWLEDGMENTS
The authors thank Dr. Michael Stoskoff and the North Carolina State
University Environmental Medicine Consortium for assistance in arrang-
ing pharmacokinetic analysis; Laura Graham for technical support with
endocrine assays; Dr. Jan Roser at the University of California, Davis, for
donation of the anti-bovine LH monoclonal antibody; and Dr. Harold Pap-
koff at the University of California, San Francisco, for donation of iodin-
ation-grade equine LH and eCG standard preparations. We also are in-
debted to the Purina C.A.R.E.S. (The Conservation of Animals Reaching
Extinct Status) Fund for donation of cat food.
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