Available online at www.sciencedirect.

com

Theriogenology 76 (2011) 1501–1507

www.theriojournal.com

Alternatives to improve a prostaglandin-based protocol for timed

artificial insemination in sheep
J. Olivera-Muzantea,*, J. Gilb, S. Fierroa, A. Menchacac, E. Rubianesd
a
Departamento de Salud en los Sistemas Pecuarios, Área de Producción y Sanidad Ovina, Facultad de Veterinaria, EEMAC. Ruta 3 Km
363. 60000 PO Box 57072 Paysandú, Uruguay
b
Departamento de Salud en los Sistemas Pecuarios, Área de Teriogenología, Facultad de Veterinaria, EEMAC. Ruta 3 Km. 363. 60000 PO
Box 57072. Paysandú, Uruguay
c
Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Camino Cruz del Sur 2250, 12200, Montevideo, Uruguay
d
Departamento de Producción Animal y Pasturas, Facultad de Agronomía, Garzón 780, 12900 Uruguay
Received 22 April 2011; received in revised form 14 June 2011; accepted 17 June 2011

Abstract
The objective was to improve the reproductive performance of a prostaglandin (PG) F2␣-based protocol for timed artificial insemination
(TAI) in sheep (Synchrovine®: two doses of 160 ␮g of delprostenate 7 d apart, with TAI 42 h after second dose). Three experiments were
performed: Experiment 1) two doses of a PGF2␣ analogue (delprostenate 80 or 160 ␮g) given 7 d apart; Experiment 2) two PGF2␣ treatment
intervals (7 or 8 d apart) and two times of TAI (42 or 48 h); and Experiment 3) insemination 12 h after estrus detection or TAI with concurrent
GnRH. Experiments involved 1131 ewes that received cervical insemination with fresh semen during the breeding season (32/34 °S–58 °W).
Estrous behaviour, conception rate, prolificacy, and fecundity (ultrasonography 30–40 d), were assessed. In Experiment 1, ewes showing estrus
between 25 and 48 h or at 72 h after the second PGF2␣ did not differ between 80 and 160 ␮g of delprostenate (73 vs 86%, P ⫽ 0.07; and 92
vs 95%, P ⫽ NS, respectively). Conception rate and fecundity were lower (P ⬍ 0.05) using 80 vs 160 ␮g (0.24 vs 0.42, and 0.27 vs 0.47,
respectively). In Experiment 2, giving PGF2␣ 7 d apart resulted in higher (P ⬍ 0.05) rates of conception (0.45 and 0.51) and fecundity (0.49
and 0.53) than treatments 8 d apart (conception: 0.33 and 0.29; fecundity: 0.33 and 0.34) for TAI at 42 and 48 h, respectively. In Experiment
3, rates of conception, prolificacy and fecundity were similar (NS) between Synchrovine® with TAI at 42 h (0.50, 1.13, and 0.56) and AI 12 h
after estrus detection (0.47, 1.18, and 0.55), and Synchrovine® plus GnRH at TAI (0.38, 1.28, and 0.49). However, all TAI treatments had lower
(P ⬍ 0.05) prolificacy and fecundity compared to AI following detection of spontaneous estrus (1.39 and 0.83, respectively). In conclusion, the
Synchrovine® protocol was: a) more successful using 160 vs 80 ␮g delprostenate; b) more successful with a 7 d than 8 d PGF2␣ interval; c)
similarly effective for TAI versus AI 12 h after estrus detection; and d) not improved by giving GnRH at TAI.
© 2011 Elsevier Inc. All rights reserved.

Keywords: Ewes; Estrus synchronization; AI; Conception

1. Introduction superior males [1]. Currently, widespread application

Timed artificial insemination (TAI) is an important
tool when estrus detection is not feasible. It provides
synchronized inseminations and more efficient use of
* Corresponding author. Tel.: ⫹⫹598 47241282; fax: ⫹⫹598
47227950.
E-mail address: joliveramuz@gmail.com (J. Olivera-Muzante).
of these biotechnologies under commercial field condi-
tions requires easy implementation procedures, accept-
able pregnancy rates, and low environmental impact
[2]. Conventional TAI protocols involve intravaginal
devices impregnated with progestins, in conjunction
with eCG, yielding acceptable pregnancy rates both
within and outside the physiological breeding season
[3,4]. Nevertheless, repeated use of eCG has been as-

0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2011.06.020
1502 J. Olivera-Muzante et al. / Theriogenology 76 (2011) 1501–1507
sociated with a humoral immune response in ewes and
goats [5,6] and development of ovarian follicular cysts
[7], followed by low pregnancy rates. In addition, use
of a progestin and eCG has been limited in some coun-
tries due to public health concerns [8] and animal wel-
fare issues [9]. However, prostaglandin-based protocols
may be a viable alternative to the use of progestagens.
Conventional PGF2␣ treatments consist of two doses
given 9 to 12 d apart [4]. Using this protocol, a high
proportion of ewes are detected in estrus after the sec-
ond dose, but over a 4 –5 d interval [10,11], making
estrus detection necessary and rendering this protocol
inappropriate for TAI. Conversely, Rubianes et al [12]
and Contreras-Solis et al [13] demonstrated that the
refractoriness of a recently formed ovine CL to PGF2␣
might be restricted to the first 2 d after ovulation, with
a consistent interval from treatment to ovulation (60.8 ⫾ 1.8
or 61.1 ⫾ 1.1 h, respectively). Based on these obser-
vations, a protocol to synchronize estrus and ovulations
(Synchrovine®, MIEM - Cámara Nacional de Regis-
tros, Montevideo Uruguay), with two large doses of
PGF2␣ given 7 d apart was proposed [1]. Synchrovine®
promotes highly synchronous estrus during the first
72 h, with 80% of the ewes in estrus within 25 to 48 h
after the second PGF2␣ dose, making it practical for use
in TAI programs. However, this protocol yielded lower
conception rates after cervical or intrauterine TAI com-
pared to the conventional P4-eCG protocol [14] or
spontaneous estrus [15]. Thus, other alternatives to im-
prove this protocol need to be studied.
A substantive reduction of the cloprostenol dose was
effective in inducing luteolysis, estrus onset, ovulation,
and subsequent development of normal CLs [16,17].
The reduced dose might decrease side effects, including
changes in cervical mucus and uterine contractions
[18,19]. A longer interval between PGF2␣ treatments
increased the sensitivity of young CLs to this hormone
[20] and increased progesterone concentrations during
development of the designated preovulatory follicle.
When feasible, AI associated with estrus detection may
be used to improve Synchrovine® results. However, a
protocol including GnRH treatment 36 h after PGF2␣
resulted in an LH surge, ovulation within 48 h, and a
fully functional CL [21]. Therefore, administration of
GnRH at AI would be a practical option if it were to
improve ovulation and fertility, as demonstrated in
dairy cows [22,23].
The aims of the present study were to determine
whether either a reduction of PGF2␣ luteolytic dose, an
increase in the interval between PGF2␣ treatments, AI
of ewes detected in estrus, or giving GnRH at TAI,
improved reproductive outcomes for a Synchrovine®
synchronization protocol.
2. Materials and methods
2.1. Animals
All procedures were approved by the University’s
Animal Experimentation Committee (CHEA-Ude-
laR). The studies were done during the physiologic
breeding season (March–April), involving 1131 clin-
ically healthy, nulliparous and multiparous ewes
(1.5–5.5 y old), with good body condition (score 3.4 ⫾ 0.4,
mean ⫾ SD [24]), grazing natural pastures at three
locations (Experiment 1: “El Coraje” Farm, Lavalleja
Uruguay, 34 °S/56’ W; Experiment 2: “Piedra Mora”
Farm, Paysandú Uruguay, 32 °S/ 57’ W; and Experiment
3: “Mario A. Cassinoni” Experimental Station, Paysandú
Uruguay, 32 °S/ 58’ W). On these farms, 13 clinically
healthy rams aged 1.5–5.5 y old, grazing natural improved
pastures (with lotus and rye grass) were used as semen
donors.
2.2. Experiments
Three experiments were done. Ewes and rams were
either randomly assigned to treatments (Experiment 1
and 2) or allocated to treatments, on the basis of breed,
parity, and body condition score (Experiment 3).
2.2.1. Experiment 1
Corriedale multiparous ewes (n ⫽ 127) were sub-
jected to cervical TAI (April 12 and 13, Year 1) with
fresh semen from two Corriedale rams. Two PGF2␣
doses for Synchrovine® were compared:
● Synchrovine®: Two doses of delprostenate (160
␮g im; Glandinex®, Universal Lab, Montevideo,
Uruguay) given 7 d apart, with TAI 42 h after the
second dose (Control).
● Synchrovine®-LD: As above, but using 80 ␮g
delprostenate.
2.2.2. Experiment 2
Australian Merino multiparous ewes (n ⫽ 583) were
cervically TAI (April 11 and 12, Year 2), with fresh
extended semen from five rams (three Australian Me-
rino and two Suffolk). In a factorial design, two PGF2␣
treatment intervals (7 or 8 d) and two AI-times (42 or
48 ⫾ 1 h after the second dose of PGF2␣) for Syn-
chrovine® were compared.
● Synchrovine®: as described in Experiment 1.
● Synchrovine®-48: as above, with TAI at 48 h
after second dose.
 J. Olivera-Muzante et al. / Theriogenology 76 (2011) 1501–1507
● Synchrovine®8-42: two doses of PGF2␣ 8 d apart,
with TAI 42 h after the second dose.
● Synchrovine®8-48: two doses of PGF2␣ 8 d apart,
with TAI 48 h after the second dose.
2.2.3. Experiment 3
Four hundred and twenty one ewes (345 multiparous
and 76 nulliparous) Corriedale and crosses (x Texel, x
Milchschaf, or x Ile de France; n ⫽ 209, 58, 74 and 80,
respectively) were cervically AI (March 18 to 21, Year
1) with fresh semen from six rams (two Corriedale,
three Southdown and one Poll Dorset). Four treatments
were compared:
● Synchrovine®: as described in Experiment 1.
● Synchrovine®-DE: Synchrovine®, with AI 12 h
after detected estrus.
● Synchrovine®-GnRH: Synchrovine® plus GnRH
(busereline acetate 8 ␮g im; Receptal®, Intervet
International GmbH, Unterschelei␤heim, Germany)
at TAI, 42 h after the second PGF2␣.
● Spontaneous Estrus: ewes were pre-synchronized
with one dose of PGF2␣ (delprostenate 160 ␮g
im) 17 d in advance, ignoring the induced estrus,
with AI 12 h after detection of the following
spontaneous estrus.
2.3. Semen processing
Semen was collected with an artificial vagina and
estrous teaser ewes. All rams delivered ejaculates
with acceptable volume (0.75–2 mL), concentration
(⬎ 3.0 ⫻ 109 sperm/mL), total motility (⬎ 70%), and
sperm morphology (⬍ 10% total sperm abnormali-
ties). During the experiment, two consecutive ejacu-
lates from each ram were collected within 10 min,
pooled, and treated as a single ejaculate. Ejaculates
from each ram were placed in a portable water bath
at 33 °C and subjectively assessed (motility, volume
and color). Sperm concentration was assessed with a
photometer (Spermacue®, Minitub, Landshut, Ger-
many). Fresh undiluted AI doses (200 ⫻ 106 sperm/
ewe; Experiments 1 and 3) or extended in UHT skim
milk with antibiotics (final concentration 1000 ⫻ 106
sperm/mL, insemination dose 0.2 mL/ewe; Experi-
ment 2) were maintained at 30 °C and used within 30
min after collection.
2.4. Estrus detection and AI
The onset of estrus was determined twice daily dur-
ing the first 72 h after the second PGF2␣ treatment in
ewes in Experiment 1 (Synchrovine® and Syn-
chrovine®-LD), and Experiment 3 (Synchrovine®-
1503
DE), or 17 d after PGF2␣ dose in Experiment 3 (Con-
trol, spontaneous estrus). Five vasectomized rams/100
ewes (Experiment 1) or 10 androgenized wethers/100
ewes (Experiment 3) were used as painted teaser ani-
mals. Wethers were treated with 100 mg testosterone
cyclopentylpropionate im (Testosterona Ultra Fuerte®,
Laboratorios Dispert, Montevideo, Uruguay), given on
three occasions (14, 7 and 1 d, respectively, before the
beginning of the estrus detection period). Paint was
applied twice daily.
Cervical AI was done using a speculum equipped
with a light source and an insemination gun (Walmur
Veterinary Instruments, Montevideo, Uruguay), slowly
depositing the semen as deep as possible into the cer-
vix.
2.5. Reproductive performance
Estrous behaviour (ewes in estrus/treated) and its
distribution within 72 h after the second PGF2␣ treat-
ment was assessed (Experiments 1 and 3). Rates of
conception (pregnant/inseminated ewes), prolificacy
(fetuses/pregnant ewe), and fecundity (fetuses/insemi-
nated ewes), were evaluated 30 d (Experiments 1 and 3)
or 40 d (Experiment 2) after TAI by transrectal or
transabdominal ultrasonography (Aloka® 500, Tokyo,
Japan; 5.0 MHz linear-array or 3.5 MHz convex-array,
respectively).
2.6. Statistical analyses
Differences in estrous behaviour and its distribution,
and rates of conception, prolificacy and fecundity
among synchronization protocols or spontaneous estrus
after AI were analyzed by ANOVA for categorical
variables (PROC CATMOD; SAS). Data are presented
as means, with P ⬍ 0.05 considered significantly dif-
ferent.
3. Results
3.1. Experiment 1
The results are shown in Fig. 1 and Table 1. The
PGF2␣ dose did not significantly affect the proportion
of ewes coming into estrus up to 72 h after the second
PGF2␣ (95 vs 92%; P ⫽ NS), with a high concentration
between 25 and 48 h (86 vs 73%, P ⫽ 0.07) for
Synchrovine® and Synchrovine®-LD respectively.
However, reproductive performance was better (P ⬍
0.05) in Synchrovine® treated ewes compared to Syn-
chrovine®-LD.
1504 J. Olivera-Muzante et al. / Theriogenology 76 (2011) 1501–1507

Fig. 1. Distribution of estrous ewes treated with two doses ( □ Synchrovine®-LD, 80 ␮g;  Synchrovine®, 160 ␮g) of PGF2␣ (delprostenate)
given 7 d apart (n⫽127; Experiment 1). There were no significant differences between experimental groups.

3.2. Experiment 2 (Synchrovine®-DE) nor GnRH at TAI (Synchrovine®-

Reproductive performance (conception, prolifi-
cacy, and fecundity) for each group is shown (Table
2). There was no AI-time effect (42 vs 48 h, P ⫽ NS)
within the PGF2␣ treatment interval (7 or 8 d apart).
Nevertheless, there was a PGF2␣ treatment interval
effect, with better reproductive performance (P ⬍
0.05) in ewes given PGF2␣ 7 d apart compared to
those treated 8 d apart. The interaction PGF2␣ treat-
ment interval*AI time was significant (P ⬍ 0.05) just
for prolificacy, but not for conception or final fecun-
dity (P ⫽ NS).
3.3. Experiment 3
The proportion of ewes showing estrus between 25
and 48 h and the total number showing estrus by 72 h
after the second PGF2␣ in Synchrovine®:DE group
were 81 and 92%, respectively. Neither estrus detection
Table 1
Reproductive responses obtained with two doses of PGF2␣
(delprostenate) and cervical insemination with fresh semen in
sheep (Experiment 1).
Conception Prolificacy Fecundity
a
Synchrovine® 0.42 (27/64) 1.11 (30/27) 0.47a (30/64)
(n ⫽ 64)
Synchrovine®-LD 0.24b (15/63) 1.13 (17/15) 0.27b (17/63)
(n ⫽ 63)
Synchrovine®, two PGF2␣ treatments 7 d apart (160 ␮g delproste-
nate) and TAI 42 h after second PGF2␣; Synchrovine®-LD, two
PGF2␣ treatments 7 d apart (80 ␮g delprostenate) and TAI 42 h after
second PGF2␣; Conception, pregnant at 30 d after AI/inseminated
ewes; Prolificacy, fetuses/pregnant ewe; Fecundity, fetuses/insemi-
nated ewe.
a,b
Within a column, rates without a common superscript differed
(P ⬍ 0.05).
GnRH) significantly improved (P⫽NS) the reproduc-
tive performance of synchronized ewes. All groups
subjected to these synchronization protocols had lower
(P ⬍ 0.05) fecundity than the Control group (sponta-
neous estrus, Table 3).
4. Discussion
In the present study, both the amount of PGF2␣ and
the interval between PGF2␣ treatments significantly af-
fected reproductive performance of synchronized ewes.
However, neither estrus detection (to determine time of
AI) nor administration of GnRH at TAI significantly
affected the reproductive outcome of the Synchrovine®
protocol.
The cumulative proportion of ewes showing estrus
by 72 h after the second PGF2␣ treatment was similar
between the full and half dose of PGF2␣ analog (Ex-
periment 1), but the ewes in estrus between 25 and 48 h
in the group treated with the full dose (Synchrovine®)
tended to be higher, with a sharp onset of estrus. This
could account for the better reproductive performance
in this group. These results contradicted other basic
experiments using a reduced dose of cloprostenol
[16,17], but agreed with other studies [20] regarding
estrus response following dinoprost tromethamine
treatment (10 vs 5 mg) of young CLs (3.5 to 5 d old).
The concept that a reduced dose of PGF2␣ would im-
prove the reproductive performance, based on reduced
side effects, was not supported. Perhaps some of the
young CLs failed to respond to PGF2␣ [25], as observed
in the Synchrovine® protocol, thereby reducing the
number of ewes that were close to the time of ovulation
at TAI.
 J. Olivera-Muzante et al. / Theriogenology 76 (2011) 1501–1507 1505

Table 2
Reproductive performance in sheep obtained with two PGF2␣ (delprostenate) at two intervals and cervical insemination (fresh semen) at two
times (Experiment 2).
Conception Prolificacy Fecundity
Synchrovine® (n ⫽ 145) a
0.45 (65/145) a
1.09 (71/65) 0.49a (71/145)
Synchrovine®-48 (n ⫽ 145) 0.51a (74/145) 1.04a (77/74) 0.53a (77/145)
Synchrovine®8-42 (n ⫽ 147) 0.33b (48/147) 1.02a (49/48) 0.33b (49/147)
Synchrovine®8-48 (n ⫽ 146) 0.29b (42/146) 1.19b (50/42) 0.34b (50/146)
Synchrovine®, two PGF2␣ treatments 7 d apart and TAI 42 h after second PGF2␣; Synchrovine®-48, two PGF2␣ treatments 7 d apart and TAI
48 h after second PGF2␣; Synchrovine®8-42, two PGF2␣ treatments 8 d apart and TAI 42 h after second PGF2␣; Synchrovine®8-48, two PGF2␣
treatments 8 d apart and TAI 48 h after second PGF2␣; Conception, pregnant at 40 d after AI/inseminated ewes; Prolificacy: fetuses/pregnant ewe;
Fecundity, fetuses/inseminated ewe.
a,b
Within a column, rates without a common superscript differed (P ⬍ 0.05).

Variations in the time of insemination after the last
PGF2␣ had no significant effects (Experiment 2). As
observed previously with this TAI protocol using fresh
semen, pregnancy rates were similar after cervical in-
seminations both at 42 and 48 h [14,26], offering a
reasonable time-window for use of this protocol in the
field. However, reproductive performance was signifi-
cantly affected by altering the interval between doses of
PGF2␣ in the Synchrovine® protocol, with better re-
sults for treatments given 7 vs 8 d apart. Although
estrus response and ovulation was not determined in
this experiment, as in a previous study by Rubianes et
al [12], we speculate that the interval between treatment
and ovulation was more varied in ewes treated 8 d
apart. This was probably due to a higher proportion of
ovulations coming from the second follicular wave,
resulting in TAI occurring too early for optimal fertil-
ity.
Experiment 3 tested the effect of estrus detection,
with only ewes in estrus inseminated, and GnRH at
TAI. Firstly, AI of ewes showing estrus after PGF2␣
treatment did not improve the conception rate of this
new synchronization protocol using fresh semen. The
higher cumulative proportion of ewes that showed es-
trus between 25 and 48 h after the second PGF2␣ in the
Synchrovine®-DE group, and the prolonged lifespan of
fresh semen, may explain the similar conception rate
achieved in the Synchrovine® TAI group. Therefore,
we concluded that estrus detection is not required when
using this protocol with fresh semen, reinforcing the
advantage of TAI procedures. Secondly, giving a
GnRH analogue concurrent with TAI did not improve
the final reproductive outcomes of Synchrovine® pro-
tocol. Furthermore, the conception rate of this treatment
tended to be lower (P ⫽ 0.09), but the prolificacy was
higher (P ⫽ 0.07) than in the Synchrovine® group.
These results agreed with previous reports where, de-
spite the synchrony in ovulation achieved, giving
GnRH within 24 to 44 h after a P4-eCG treatment failed
to improve ewe fertility [27–29]. Other authors re-
ported a tendency to increase litter size as a result of
GnRH treatment immediately after the AI of ewes syn-
chronized with a P4-eCG-PGF2␣ [30], or 30 h after a
P4-PGF2␣ or P4-PGF2␣-eCG treatment [8]; this was
attributed to GnRH increasing ovulation rates [30 –32].
In another study, the male effect was used after a
short-interval, cloprostenol-based protocol to induce an
earlier preovulatory LH surge and ovulation, thus im-

Table 3
Reproductive response obtained with variations of Synchrovine® protocol using cervical insemination with fresh semen in sheep (Experiment 3).
Conception Prolificacy Fecundity
Synchrovine® (n ⫽ 111) ab
0.50 (55/111) a
1.13 (62/55) 0.56a (62/111)
Synchrovine®-DE (n ⫽ 102) 0.47ab (44/94) 1.18a (52/44) 0.55a (52/94)
Synchrovine®-GnRH (n ⫽ 104) 0.38a (40/104) 1.28ab(51/40) 0.49a (51/104)
Spontaneous Estrus (Control, n ⫽ 104) 0.59b (51/86) 1.39b (71/51) 0.83b (71/86)
Synchrovine®, two PGF2␣ treatments 7 d apart and TAI 42 h after second PGF2␣; Synchrovine®-DE, two PGF2␣ treatments 7 d apart and AI 12 h
after detected estrus; Synchrovine®-GnRH, two PGF2␣ treatments 7 d apart plus 8 ␮g GnRH (busereline acetate) at TAI (42 h after second
PGF2␣); Spontaneous Estrus, pre synchronized ewes with one dose of PGF2␣ 17 d in advance, ignoring the induced estrus, and AI 12 h after
detected spontaneous estrus; Conception, pregnant 30 d after AI/inseminated ewes; Prolificacy, fetuses/pregnant ewe; Fecundity, fetuses/
inseminated ewe.
a,b
Within a column, rates without a common superscript differed (P ⬍ 0.05).
1506 J. Olivera-Muzante et al. / Theriogenology 76 (2011) 1501–1507
proving fertility compared to a progesterone-based pro-
tocol [13]. Consequently, a GnRH dose was expected to
favor the Synchrovine® TAI protocol. Assuming that
ovulation in Synchrovine® protocol occurs approxi-
mately 60 h after the second PGF2␣ treatment [12,13],
and that the physiological LH-surge is ⬃24 h before
ovulation [33], the LH surge should be ⬃36 h after the
last PGF2␣. If the GnRH was given at TAI (42 h),
perhaps this promoted a second LH surge 2– 4 h later
[21,28]. In this regard, the GnRH was probably too late
to augment the endogenous LH surge to target a timed
ovulation. Although it is logistically easier to give
GnRH concurrent with TAI, earlier administration
should be considered in future trials.
Finally, the Synchrovine® protocol and the studied
alternatives had lower reproductive outcomes com-
pared to spontaneous estrus and AI based on estrus
detection. An environment dominated by lower proges-
terone concentration during the growth phase of the
ovulatory follicle, stimulating a faster growth rate, and
a larger follicular size, appeared to be a cause of the
lower ovulation rate, conception, and prolificacy
achieved with Synchrovine® protocol compared to
ewes AI in spontaneous estrus [15].
In conclusion, reproductive performance of the Syn-
chrovine® protocol was: a) better when using 160 ver-
sus 80 ␮g delprostenate; b) better when using a 7 d
interval compared to an 8 d interval between PGF2␣
treatments; c) similar with insemination at prefixed
time or AI 12 h after estrus detection; and d) not
improved by giving GnRH at TAI.
Acknowledgments
This work was supported by funds from Universidad
de la República (CSIC 600/6015) and MGAP-DILAVE
“M.C. Rubino”. We acknowledge “Coraje” Farm
(Dighiero-Triay family), “Piedra Mora” Farm (Filliol-
Barreiro family) and EEMAC (Facultad de Agronomía,
UdelaR, Paysandú, Uruguay) for allowing us to use
their facilities and animals. Thanks also to E. Campos
and S. Kmaid from Universal Lab and J. Amaro from
Dispert for donating delprostenate and testosterone, re-
spectively. We are also grateful to J.L. Alabart for
statistical analysis; to G. McCann for revising the man-
uscript; and to S. Forichi, M. Correa, A. Araujo, E.
Filliol, and G. Stoletniy for their support during the
trials. The authors do not have any financial or personal
relationships with other people or organizations that
could have inappropriately biased or influenced this
work.
References
[1] Menchaca A, Rubianes E. New treatments associated with
timed artificial insemination in small ruminants. Reprod Fert
Dev 2004;16:403–13.
[2] Martin GB, Kadokawa H. “Clean, green and ethical” animal
production. Case study: reproductive efficiency in small rumi-
nants. J Reprod Develop 2006;52:145–52.
[3] Colas G. The use of progestagen SC9880 as an aid for artificial
insemination in ewes. Ann Biol Anim Bioph 1975;15:317–27.
[4] Gordon I. Fixed-time sheep artificial insemination. In: Con-
trolled Breeding in Farm Animals. Gordon I (Ed.), Oxford.
Pergamon Press, 1983, pp. 197–208.
[5] Roy F, Combes B, Vaiman D, Cribiu EP, Pobel T, Deletang F,
Combarnous Y, Guillou F, Maurel MC. Humoral immune re-
sponse to equine chorionic gonadotropin in ewes: association
with major histocompatibility complex and interference with
subsequent fertility. Biol Reprod 1999;61:209 –18.
[6] Roy F, Maurel MC, Combes B, Vaiman D, Cribiu EP, Lantier
I, Pobel T, Delétang F, Combarnous Y, Guillou F. The negative
effect of repeated equine chorionic gonadotropin treatment on
subsequent fertility in Alpine goats is due to a humoral immune
response involving the major histocompatibility complex. Biol
Reprod 1999;60:805–13.
[7] Viñoles C, Forsberg M, Banchero G, Rubianes E. Effect of long
term and short-term progestagen treatment on follicular devel-
opment and pregnancy rate in cyclic ewes. Theriogenology
2001;55:993–1004.
[8] Martemucci G, D’Alessandro AG. Synchronization of oestrus
and ovulation by short time combined FGA, PGF2␣, GnRH,
eCG treatments for natural service or AI fixed-time. Anim
Reprod Sci 2011;123:32–9.
[9] González-Bulnes A, Veiga-Lopez A, García P, García-García
RM, Ariznavarreta C, Sanchez MA, Tresguerres JFA, Cocero
MJ, Flores JM. Effects of progestagens and prostaglandin ana-
logues on ovarian function and embryo viability in sheep. The-
riogenology 2005;63:2523–34.
[10] Loubser PG, van Niekerk CH. Oestrous synchronization in
sheep with progesterone-impregnated (MAP) intravaginal
sponges and a prostaglandin analogue. Theriogenology 1981;
15:547–52.
[11] Evans G, Maxwell WMC. Preparation of Females for Insemi-
nation. In: Salamon=s Artificial Insemination of Sheep and
Goats. (Butterworth Press & Co. Ltd. Australia), 1987, pp.
55–74.
[12] Rubianes E, Menchaca A, Carbajal B. Response of the 1 to
5-day aged ovine corpus luteum to Prostaglandin F2␣. Anim
Reprod Sci 2003;78:47–55.
[13] Contreras-Solís I, Vásquez B, Díaz T, Letelier C, López Se-
bastián A, González-Bulnes A. Efficiency of estrous synchro-
nization in tropical sheep by combining short-interval clopros-
tenol-based protocols and “male effect”. Theriogenology 2009;
71:1018 –25.
[14] Olivera-Muzante J, Fierro S, López V, Gil J. Comparison of
prostaglandin- and progesterone-based protocols for timed arti-
ficial insemination in sheep. Theriogenology 2011;75:1232– 8.
[15] Fierro S, Olivera J, Gil J, Viñoles C. Effects of prostaglandin
administration on follicular dynamics, conception, prolificacy
and fecundity in sheep. Theriogenology 2011, online http://
dx.doi.org/10.1016/j.theriogenology.2011.03.016.
 J. Olivera-Muzante et al. / Theriogenology 76 (2011) 1501–1507
[16] Hearnshaw H, Restall BJ, Nancarrow CD, Mattner PE. Syn-
chronization of oestrus in cattle, sheep and goats using a pros-
taglandin analogue. Proc Aust Soc Anim Prod 1974;10:242–5.
[17] Contreras-Solís I, Vásquez B, Díaz T, Letelier C, López Se-
bastián A, González-Bulnes A. Ovarian and endocrine re-
sponses in tropical sheep treated with reduced doses of clopro-
stenol. Anim Reprod Sci 2009;114:384 –92.
[18] Hawk HW. Uterine motility and sperm transport in the estrous
ewes after prostaglandin induced regression of corpora lutea. J
Anim Sci 1973;37:1380 –5.
[19] Prasad A, Kalyan NK, Bachlaus NK, Arora FC, Pandley RS.
Biochemical changes in the cervical mucus of buffalo after
induction of oestrous with prostaglandin F2 alpha and clopro-
stenol. J Reprod Fert 1981;62:583–7.
[20] Pope WF, Cárdenas H. Sensitivity of sheep to exogenous pros-
taglandin F2 early in the estrous cycle. Small Rum Res 2004;
55:245– 8.
[21] Rubianes E, Beard A, Dierschke DJ, Bartlewski P, Adams GP,
Rawlings NC. Endocrine and ultrasound evaluation of the re-
sponse to PGF2␣ and GnRH given at different stages of the
luteal phase in cyclic ewes. Theriogenology 1997;48:1093–104.
[22] Lee CN, Maurice E, Ax RL, Pennington JA, Hoffman WF,
Brown MD. Efficacy of gonadotrophin-releasing hormone ad-
ministration at the time of artificial insemination of heifers and
postpartum and repeat breeder dairy cows. Am J Vet Res 1983;
44:2160 –3.
[23] Nakao T, Narila S, Tanaka K, Horn H, Shirakawa J, Noshiro H,
Saga N, Tsunoda H, Kawata K. Improvement of the first-service
pregnancy rate in cows with gonadotrophin-releasing hormone
analog. Theriogenology 1983;20:111–9.
[24] Russell AJF, Doney JM, Jun RG. Subjective assessment of body
fat in live sheep. J Agric Sci 1969;72:451– 4.
[25] Silva PJ, Juengel JL, Rollyson MK, Niswender GD. Prostaglan-
din metabolism in the ovine corpus luteum: catabolism of pros-
1507
taglandin F2␣ (PGF2␣) coincides with resistance of the corpus
luteum to PGF2␣. Biol Reprod 2000;63:1229 –36.
[26] Menchaca A, Miller V, Gil J, Pinczak A, Laca M, Rubianes E.
Prostaglandin F2␣ treatment associated with Timed Artificial
Insemination in ewes. Reprod Dom Anim 2004;39:352–5.
[27] Walker SK, Smith DH, Ancell P, Seamark RF. Time of ovula-
tion in the South Australian Merino ewe following synchroni-
zation of oestrous. 2. Efficacy of GnRH treatment and its rele-
vance to insemination programs utilizing frozen- thawed semen.
Theriogenology 1989;31:555– 64.
[28] Eppleston J, Evans G, Roberts EM. Effect of time of PMSG and
GnRH on the time of ovulation, LH secretion and reproductive
performance after intrauterine insemination with frozen ram
semen. Anim Reprod Sci 1991;26:227–37.
[29] Reyna J, Thomson PC, Evans G, Maxwell WM. Synchrony of
ovulation and follicular dynamics in merino ewes treated with
GnRH in the breeding and non-breeding seasons. Reprod Do-
mest Anim 2007;42:410 –7.
[30] Türk G, Gür S, Sönmez M, Bozkurt T, Aksu EH, Aksoy H.
Effect of exogenous GnRH at the time of artificial insemination
on reproductive performance of Awassi ewes synchronized with
progestagen-PMSG-PGF2alpha combination. Reprod Domest
Anim 2008;43:308 –13.
[31] Nancarrow CD, Murray D, Boland MP, Sutton R, Hazelton IG.
Effect of gonadotrophin releasing hormone in the production of
single-cell embryos for pronuclear injection of foreign genes.
In: Lindsay DR, Pearce DT, editors. Reproduction in Sheep
Austr Acad Sci; Camberra, 1984, pp. 286 – 8.
[32] Khan TH, Hastie PM, Beck NF, Khalid M. hCG treatment on
day of mating improves embryo viability and fertility in ewe
lambs. Anim Reprod Sci 2003;76:81–9.
[33] Cumming IA, Buckmaster JM, Blockey MA de B, Goding JR,
Winfield CG, Baxter RW. Constancy of interval between lutei-
nising hormone release and ovulation in the ewe. Biol Reprod
1973;2:24 –9.