BIOLOGY OF REPRODUCTION 49, 181-185 (1993)
Repeated Administration of Prostaglandin F2, during the Early Luteal Phase Causes
Premature Luteolysis in the Pig'
CHARLES T. ESTILL,4JACK H. BRITT,5and JOHN E. GADSBY4
College of Veterinary Medicine4and Department of Animal Science,5North Carolina State University
Raleigh, North Carolina 27606
Previous investigators considered pig corpora lutea refractory to the luteolytic effects of prostaglandin (PG) F2, before Day
12 of the estrous cycle. This study was designed to determine whether multiple injections of PGF,, would result in a sustained
reduction of serum progesterone and luteolysis, leading to significant shortening of the estrous cycle and interestrous interval.
On Days 5-10 of an estrous cycle, gilts (n = 4) received injections of 12.5 mg PGF2, (dinoprost tromethamine) i.m. every 12
h, or vehicle (PBS; n = 4) according to the same schedule. Mean interestrous interval in PGF,,-treated gilts was reduced (p <
0.001) to 13.3 ± 0.5 days compared with 19.8 ± 0.6 days for control gilts. Serum progesterone declined below 1 ng/ml by Day
10.5 in PGF2,,-treated gilts compared to Day 17.5 in control animals. Serum concentrations of estradiol-17 (E,) reached maximal
levels in PGF,,-treated gilts earlier (Day 12.5) in the cycle than in control gilts (Day 19.5). Peak E, and LH concentrations
coincided with the periestrous period, suggesting that PGF,,-induced estrus is accompanied by normal follicular development
and ovulation. These results demonstrate that the pig is susceptible to the luteolytic effects of PGF2,, before Day 12 if repeated
injections are given from Day 5 through Day 10.
Corpora lutea of the cycling pig are generally consid-
ered to be resistant to prostaglandin (PG) F2a-induced lu-
teolysis prior to Day 12 of the estrous cycle. Several inves-
tigators have attempted to induce premature luteolysis in
cycling pigs by administration of single or multiple doses
of PGF2. or PGF2
analogues at various times during the
luteal phase [1-7]. Although in previous studies, adminis-
tration of PGF2a or a PGF2. analogue prior to Day 12 has
not consistently reduced interestrous interval, there is evi-
dence that these compounds can transiently affect luteal
function, as manifested by a temporary reduction in serum
progesterone concentrations, without inducing complete
luteolysis [2, 5, 7]. The purpose of this study was to test the
hypothesis that repeated multiple injections of PGF2, ad-
ministered during the early luteal phase would cause pre-
mature luteolysis in the cycling pig.
MATERIALS AND METHODS
Crossbred pubertal gilts ([Landrace x Large White] x
[Duroc x Hampshire]) were obtained from and housed at
the North Carolina Agricultural Research Service, Dept. of
Accepted March 10, 1993.
Received January 7, 1993.
'Research supported by USDA grant no. 89-37240-4680 (J.E.G. and J.H.B.), the
State of North Carolina
.E.G.) and graduate student stipend (C.T.E.) from North
Carolina State University, College of Veterinary Medicine.
2Correspondence: Dr. John E. Gadsby, North Carolina State University, College
of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606. FAX: (919) 515-
3Current address: College of Veterinary Medicine, North Carolina State Univer-
sity, Raleigh, NC 27606.
Animal Science, Unit II, swine facility. A standard corn/soy-
bean-based gestation ration was fed (1.8 kg/gilt/day)
throughout the study.
Eight gilts were randomly assigned to either PGF2a-treat-
ment (n = 4) or control (n = 4) groups. In order to de-
termine estrus, each morning, a sexually mature boar was
introduced into the gilt pens and allowed to interact with
the gilts for up to 10 min. The caretaker also performed a
back pressure response test on each gilt in the presence of
the boar. The first day during which an immobilization re-
flex was observed was designated as the onset of estrus
(Day 0). On Day 4 of an estrous cycle, an indwelling jugular
catheter was fitted to permit chronic blood sampling .
Gilts assigned to the PGF,,-treatment group received i.m.
injections of 12.5 mg PGF2a (2.5 ml dinoprost trometha-
mine; Lutalyse, Upjohn Co., Kalamazoo, MI) beginning at
0900 h on Day 5. Injections were repeated every 12 h through
Day 10 (total of 12 injections). Control gilts were handled
similarly except they were given i.m. injections of 2.5 ml
phosphate-buffered saline (PBS) twice daily during Days 5-
10. Blood (5 ml) was taken just prior to each injection and
sampling continued every 12 h until the onset of subse-
Blood Samples and Hormone Assays
Blood samples were allowed to clot at room tempera-
ture and then were stored at 4C for 12-24 h. Serum was
collected after centrifugation at 2000 x g for 20 min and
stored at -200C until assayed for concentrations of pro-
gesterone, estradiol-173, and LH.
Progesterone in serum was quantified by direct assay
(without extraction) using Coat-a-count progesterone RIA
ESTILL ET AL.
kits (Diagnostic Products, Los Angeles, CA) as previously
described . All samples were assayed within the same
assay with an intraassay coefficient of variation of 3.3%.
Estradiol was assayed according to the procedure of Cox
and coworkers . Briefly, 200-RlI aliquots (in duplicate)
of serum were extracted with ethyl acetate, dried, and re-
constituted in 200 l PBS gel. Recovery of labeled estradiol
was 87.4 5.3% (mean + SD), and values were adjusted
for extraction efficiency. For RIA, 100 l of sample and 200
Il of rabbit anti-estradiol serum diluted 1.5 x 106 was added
to each assay tube and incubated overnight at 4C. On the
next day 100
l of estradiol-6-(o-carboxymethyl) oximino-
(2-['25I]iodohistamine (Amersham Co., Arlington Heights, IL;
sp. act. 2000 Ci/mmol), adjusted to 8000-9000 cpm/100
Rl, was added. After a 6-h incubation, bound ligand was
separated from unbound ligand using dextran-coated char-
coal followed by centrifugation. Intra- and interassay coef-
ficients of variation for four assays were 13.9% and 12.8%,
LH was assayed in serum using the double-antibody RIA
procedure described by Armstrong and coworkers . This
procedure employed anti-porcine LH serum (GDN 566,
supplied by Dr. G.D. Niswender, Colorado State Univ., Fort
Collins, CO) at 1:80 000 as the primary antibody and pu-
rified porcine LH (LER 786-3, supplied by Dr. L.E. Reichert,
Jr., Albany Medical College, Albany, NY) for standards and
iodination. All sera were analyzed in duplicate aliquots of
200 Il in a single assay. Intraassay coefficient of variation
Differences in estrous cycle length caused by treatment
with PGF2. were tested by Student's t-test . Effects of
treatment on hormone concentrations were evaluated by
ANOVA for a split-plot design with repeated measures using
the General Linear Models procedures of the Statistical
Analysis System . For progesterone concentrations, sam-
ple data obtained during Days 5-14 of the estrous cycle
were evaluated for overall treatment difference, animal-
within-treatment difference, and treatment x day of cycle
interaction. Because the treatment x day interaction was
significant, progesterone values were sorted by day and t-
tests were carried out for mean values at each sampling
time between Days 5 and 14. Differences in estradiol and
LH concentrations between treatment groups were evalu-
ated relative to the peak concentration for each treatment
group in order to examine area under the curve during the
periestrual period. Effect of treatment on concentrations at
sampling times before and after peak values were com-
pared using the Student Newman-Keuls test.
The interestrous interval (Table 1) was significantly (p
= 0.0002) reduced in the PGF2,-treated gilts compared to
TABLE 1. Interestrous intervals of gilts treated with either PGF2or
vehicle (Control) at 12-h intervals on Days 5-10 of an estrous cycle.
Cycle length (days)
*p < 0.01, cycle length different in PGF2,-treated gilts.
19.75 -+ 0.63*
13.25 ± 0.49*
In control gilts, serum progesterone rose gradually
through Day 11, remained elevated through Day 14.5, and
then decreased rapidly, reaching baseline (< 1 ng/ml) lev-
els by Day 17.5 (Fig. 1). In PGF2,,-treated gilts, serum pro-
gesterone concentrations were maximal by Day 7.5 and then
fell rapidly to a baseline concentration by Day 10.5. Com-
pared with controls, serum progesterone was significantly
lower (p < 0.05) in PGF2,,-treated gilts beginning at Day
8.5 and continuing through Day 11.5; beyond Day 11.5 the
number of animals remaining (those which had not come
into estrus) was too small to test for statistical differences
between treatment groups.
In both PGF2a-treated and control gilts, serum estradiol
concentrations were low initially and then gradually in-
creased to peak levels just prior to estrus in both treatment
groups (Fig. 2). Thus, peak concentrations were observed
on Day 12.5 in PGF2,-treated gilts compared with Day 19.5
in control gilts. The area under the curve during Days 6.5-
12.5 in PGF2,-treated gilts was not different than the area
for Days 13.5-19.5 in control gilts (p = 0.5).
6 B 10
Day of Cycle
16 18 20 22
FIG. 1. Serum progesterone concentrations in gilts treated with either
12.5 mg PGF2, (n = 4) or vehicle (controls; n = 4) at 12-h intervals begin-
ning at 0900 h on Day 5 of an estrous cycle. Each value represents mean
+ SEM for gilts that had not displayed estrus by the indicated day of the
cycle. The symbol (e) above error bars represents a gilt in estrus on the
day indicated. Day 8.5 was the first day that mean serum progesterone was
significantly less (p = 0.0116) for PGF2,-treated gilts.
PGF2,, PREMATURE LUTEOLYSIS IN THE PIG
O 50 -
Day of Cycle
12 14 16
18 20 22
FIG. 2. Serum estradiol-17p concentrations in gilts treated with either
12.5 mg PGF2. (n = 4) or vehicle (controls; n = 4) at 12-h intervals begin-
ning at 0900 h on Day 5 of an estrous cycle. Each value represents mean
+ SEM of gilts which had not displayed estrus by the indicated day of the
cycle. The symbol (e) above error bars represents a gilt in estrus on the
Serum LH concentrations rose just prior to or coincident
with the onset of estrus in a similar fashion in control and
PGF2a-treated gilts (data not shown). The area under the
curve during Days 6.5-12.5 in PGF2,-treated gilts was not
different than the area for Days 13.5-19.5 in controls (p =
0.5). A distinct LH surge was not demonstrated in each gilt
because blood sampling was terminated at the onset of es-
This is the first report, in the pig, of consistent PGF2,-
induced premature luteolysis and reduction of estrous cycle
length by more than 1-2 days using this agent or its ana-
logues. We conclude that shortening of the estrous cycle
length was due to PGF2,-induced luteolysis based on the
sustained reduction in serum progesterone. It is likely that
multiple injections of PGF2a are necessary to achieve pre-
mature luteolysis but neither the minimum number of in-
jections nor the minimum dose is known; these questions
require further investigation.
Attempts by several investigators to cause premature lu-
teolysis and reduce estrous cycle length in the cycling pig
have been unsuccessful. For example, Diehl and Day 
reported that a single intrauterine dose of PGF2, (2 or 5
mg) on Day 10 or i.m. dose on Day 12 of an estrous cycle
had no effect on plasma progesterone or interestrous in-
terval. Similarly, Hallford  reported that gilts treated four
times (20 mg PGF2a i.m.) at 12-h intervals beginning on Day
4 of an estrous cycle showed no significant alteration in
plasma progesterone. On the other hand, Connor and col-
leagues  demonstrated that when gilts were injected with
PGF2,, (20 mg i.m.) on Day 9 or Days 9 and 10, serum pro-
gesterone was significantly decreased from pre-injection
concentrations by 8 h post-injection; however, by 96 h post-
injection the serum progesterone concentrations had re-
turned to normal levels and followed a pattern similar to
controls during the remainder of the cycle. Guthrie and
Polge  suggested that multiple injections of a PGF2a an-
alogue were more likely to be luteolytic than a single dose.
In their study, four injections of synthetic PGF2a analogue
(ICI 79 939) administered every 12 h beginning on Day 10
was more likely to induce luteolysis than a single injection
on Day 10. The suggestion that pig corpora lutea are ca-
pable of responding to PGF2. prior to Day 12 is further
supported by Kryzmowski and coworkers  who dem-
onstrated a reduction in serum progesterone when PGF2a
was infused (10-h infusion containing 1.5 mg PGF2.) into
the anterior uterine vein on Days 6, 8, 10, 12, 14, and 15
of the estrous cycle. At each stage of the cycle, PGF2a sup-
pressed luteal function, although the fall in progesterone
secretion was much greater and statistically significant when
the infusion was performed on Days 12, 14, and 15 of an
estrous cycle compared with Days 6, 8, and 10. Finally,
Berghorn and coworkers  found that multiple injections
of PGF2. (25 mg at 1 h and at 4 h) started on Day 6 sig-
nificantly reduced cycle length in sows; however, cycle length
was not reduced in sows treated with multiple PGF2a in-
jections on either Days 10 or 14 . These investigators
suggested that transport of animals during the study may
have interacted with treatment to reduce cycle length .
In the present study, a significant difference in serum
progesterone between PGF2,-treated gilts and controls was
first apparent by Day 8.5. It is unknown if the PGF2,, injec-
tions administered beyond this time contribute substan-
tially to luteolysis. If luteolysis continues in the absence of
PGF2, injections after Day 8.5, it is possible that the number
of PGF2, injections could be reduced and still constitute an
effective treatment for shortening the estrous cycle. Fur-
thermore, use of longer-acting PGF2a analogues may allow
this treatment regimen to be refined further.
The rise in concentrations of serum estradiol and LH prior
to the onset of PGF2,-induced estrus indicated that follic-
ular maturation and ovulation occurred normally. Corpus
luteum life span following the PGF2,-induced estrus was
also of normal length (22 days) in the two PGF2,-treated
gilts which were not removed from the swine facility prior
to next estrus. Since it appears that the estrous cycle sub-
sequent to PGF2,,-induced premature luteolysis is normal
with respect to behavior, ovarian steroid and LH profiles,
and luteal life span, we speculate that conception rate would
also be expected to be normal. This notion is supported
by the study conducted by Hallford and colleagues in which
gilts receiving PGF2,, injections at 12-h intervals on Days 12
and 13 of the estrous cycle had normal conception rates at
the estrus subsequent to treatment .
ESTILL ET AL.
The explanation for the relative insensitivity of porcine
corpora lutea to the luteolytic effect of PGF2a prior to Day
12 of the estrous cycle is unknown. However, previous data
from this laboratory have demonstrated that relatively low
numbers of specific high-affinity PGF2,-binding sites (re-
ceptors) were detected on large luteal cells prior to Day
12 . In addition, it was reported that the number of high-
affinity binding sites increased dramatically on Day 13 ,
coinciding with the apparent onset of enhanced sensitivity
to the luteolytic effects of PGF2, between Days 12 and 13
[1-3, 5-7,15]. The possibility exists that premature luteol-
ysis, in response to repeated exogenous PGF2, injections
administered during the early luteal phase, is mediated
through specific PGF2,, receptors [14,16] and subsequent
activation of post-receptor mediators (i.e., phosphoinositol
metabolism, Ca2 +
mobilization and activation of protein ki-
nase C) [17-20]. How the mechanisms were activated pre-
maturely in the current study is unknown. One explanation
is that PGF2 causes up-regulation of its own receptor such
receptor concentration increases earlier in the
cycle, thus rendering the corpus luteum sensitive to PGF2,.
Preliminary observations supporting this hypothesis have
been reported: in pregnant and pseudopregnant gilts, the
number of PGF2, high-affinity receptors per large luteal cell
is reduced compared to that in nonpregnant gilts , which
correlates with decreased uterine release of PGF2 in preg-
nant and pseudopregnant gilts compared with nonpregnant
gilts [22, 23]. If PGF2, receptor concentrations are not influ-
enced by exposure to PGF2,, it is conceivable that the pre-
mature luteolysis we have observed in this study is related
to a PGF2,-stimulated accumulation of cellular mediators such
as protein kinase C [18, 20] and/or calcium [17, 19] within
the luteal cells, which in turn sensitizes the corpus luteum
to luteolytic effects of PGF2 .
demonstrated, in ovine large luteal cells, a PGF2,-induced
increase in intracellular calcium, which was probably due
to the influx of extracellular calcium, and concluded that
alteration in intracellular calcium concentrations could be
one of the intracellular messengers mediating the luteolytic
action of PGF2,. In other studies it has been suggested that
PGF2-induced increases in phosphatidylinositol 4,5-bis-
phosphate hydrolysis may mediate the increased intracel-
lular calcium  and lead to activation of protein kinase
C [18, 20]. Wiltbank and colleagues have also demonstrated
that activation of protein kinase C may itself mediate the
direct antisteroidogenic effect of PGF
Another possible explanation of PGF2,-induced prema-
ture luteolysis is that repeated administration of PGF2, re-
sults in an increase of the ratio of luteolytic (PGF
teotropic prostaglandins (PGE2); when the luteolytic:
luteotropic prostaglandin ratio is high, luteal steroidogen-
esis may be decreased, leading to luteolysis and shortening
of the cycle [24, 25]. Administration of PGF2, could further
alter this ratio by increasing endogenous PGF2, release .
Wiltbank and colleagues 
,, on the large luteal
,,) to lu-
Finally, repeated injections of PGF2, also may result in
premature luteolysis, at least in part, through inducing a
change in the ratio of luteal cell types . Braden and co-
workers  demonstrated a PGF2,-induced preferential loss
of small cells in the luteal phase of cycling ewes. This co-
incided with the time of increased endogenous PGF2. re-
lease. Thus, it is possible that repeated injections of PGF2
early in the luteal phase altered the ratio of luteal cell types,
resulting in a relative increase in PGF2-sensitive large lu-
In summary, this study demonstrates that premature lu-
teolysis and shortening of the estrous cycle can be achieved
by administration of PGF2 in cycling gilts. Because of the
relative refractoriness of the porcine corpus luteum to the
luteolytic effects of PGF2,, during the first 12 days of the
cycle, multiple injections are required for consistent lute-
olysis. It appears, based on serum hormone concentrations,
that the estrus subsequent to PGF2,-induced premature lu-
teolysis is normal and likely to be fertile.
We thank Jamie Lovdal for assistance in cannulation of experimental animals,
Patricia M. Dennis for surgical assistance, Vickie Hedgepeth for technical assistance
in hormone assays, Dr. Billy Flowers for assistance with statistical analysis, Dr. Gor-
don Niswender for providing LH antisera, and Dr. Leo Reichert, Jr., for providing
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