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Theriogenology 77 (2012) 1679 –1685

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Superovulation of beef cattle with a split-single intramuscular

administration of Folltropin-V in two concentrations of hyaluronan
Andrés Tríbuloa,b,c, Dragan Rogand, Humberto Tríbuloa,b, Ricardo Tríbuloa,b,c,
Reuben J. Mapletofte, Gabriel A. Bóa,f,*
a
Instituto de Reproducción Animal Córdoba (IRAC), Zona Rural General Paz, (5145) Córdoba, Argentina
b
Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina
c
Facultad de Veterinaria, Universidad Nacional de La Rioja, Chamical, La Rioja, Argentina
d
Bioniche Life Sciences, Belleville, ON, Canada
e
Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada
f
Instituto de Ciencias Básicas, Medicina Veterinaria, Universidad Nacional de Villa, María, Villa del Rosario, Córdoba, Argentina
Received 16 May 2011; received in revised form 17 November 2011; accepted 9 December 2011

Abstract
Three experiments were designed to evaluate the superovulatory response of beef cows following two intramuscular (IM)
administrations 48 h apart of Folltropin-V diluted in reduced concentrations of hyaluronan (Split-single IM administrations;
Experiment 1—300 mg Folltropin-V on the first day and 100 mg 48 h later; Experiment 2—200 mg Folltropin-V on the first day
and 100 mg 48 h later). In Experiments 1 and 2, superovulatory response and ova embryo/embryo production did not differ
between donors receiving twice daily IM of Folltropin-V over 4 days and those given a Split-single IM administration of
Folltropin-V diluted in 10 mg/mL hyaluronan solution. Experiment 3 compared Split-single IM administration of Folltropin-V
diluted in two hyaluronan concentrations (5 or 10 mg/mL) with Folltropin-V diluted in saline and administered twice-daily over
4 days. Beef cows (17 Angus and 12 Simmental) were randomly assigned to one of three treatment groups to be superstimulated
three times in a cross-over design, so that all cows received all treatments. A total dose of 300 mg Folltropin-V was divided into
twice-daily IM over 4 days, or in two IM treatment 48 h apart (200 mg on first day and 100 mg 48 h later) in the hyaluronan
groups. Mean (⫾ SEM) numbers of transferable embryos did not differ among treatment groups (Control: 4.0 ⫾ 0.8; 10 mg/mL
hylauronan: 5.0 ⫾ 0.9; 5 mg/mL hyaluronan: 6.1 ⫾ 1.3). We concluded that the Split-single IM administration of Folltropin-V
diluted in either concentration of hyaluronan resulted in a comparable superovulatory response to the traditional twice-daily
protocol.
© 2012 Elsevier Inc. All rights reserved.

Keywords: Superovulation; Superstimulation; Folltropin-V; Hyaluronan; Ovarian follicle; Bovine

1. Introduction control emergence of the follicular wave [1,2] and

Although an increase in the number of transferable
embryos per treatment has been elusive, protocols that
* Corresponding author Tel./fax: 54 (0) 3543 493820.
E-mail address: gabrielbo@iracbiogen.com.ar (G.A. Bó).
ovulation [3,4] have allowed superstimulation of
groups of donors, regardless of the stage of the estrous
cycle, and permitted AI without the need to detect
estrus. However, the complexity of protocols can lead
to errors that may adversely affect results; therefore, the
need to administer FSH every 12 h is of concern [5].

0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2011.12.013
1680 A. Tríbulo et al. / Theriogenology 77 (2012) 1679 –1685
Traditional superstimulatory treatments consist of a
single intramuscular (IM) administration of equine cho-
rionic gonadotrophin (eCG) or twice daily IM injec-
tions of pituitary extracts containing FSH [6]. The eCG
is a complex glycoprotein with a long half-life (⬎40 h)
which represents a practical advantage, since a single
administration will induce ovarian superstimulation [7].
However, it is necessary to neutralize eCG with anti-
bodies at the time of AI to avoid the adverse effects of
large unovulated follicles at the time of embryo collec-
tion [8]. In contrast, the half-life of pituitary-derived
FSH is 5 h in the cow [9,10] and requires frequent
applications to induce superovulation [10 –12]. Twice
daily administration of FSH has been found to be more
efficacious than once daily [12–14]. Therefore, super-
stimulation requires attention to detail and increases the
possibility of failures because of mishandling and er-
rors in giving treatments. In addition, the traditional
protocol can cause stress, resulting in a decreased su-
perovulatory response [5]. Simplifying superstimula-
tory protocols in cattle may reduce donor handling
costs and improve response, particularly in less tracta-
ble animals.
A single subcutaneous injection of pituitary-derived
FSH has been shown to result in an equivalent super-
ovulatory response to the traditional 4-day treatment
protocol in beef cows in good body condition [5].
However, results were not repeatable in Holstein cows
that had less subcutaneous adipose tissue [15]. In one
study in Holstein cows, the single subcutaneous injection
of Folltropin-V was split into two, with 300 mg adminis-
tered on the first day, and the remaining 100 mg admin-
istered 48 h later, when prostaglandin is normally ad-
ministered [16]. Although superovulatory response did
not differ from the traditional 4-day protocol, numbers
of transferable embryos was numerically lower with the
Split-single protocol 16. As the single subcutaneous
injection of Folltropin-V was body condition- and user-
dependent, a more efficacious method of superstimula-
tion was of interest.
An alternative to induce superovulation with a single
injection of FSH would be to combine it with a biode-
gradable polymer that results in a sustainable slow
release over several days. A single IM injection of
Folltropin-V in a hyaluronan solution resulted in a
similar number of transferable embryos as the tradi-
tional twice-daily protocol [17,18]. However, the opti-
mum concentration of hyaluronan (20 mg/mL) was
viscous and difficult to mix with Folltropin-V [19]. A
50% reduction in concentration (10 mg/mL) was less
viscous and easier to mix, but a single IM injection
resulted in a lower superovulatory response, presum-
ably because a lesser depot effect. Mean follicle sizes
did not differ from the 20 mg/mL hyaluronan group on
the days of Folltropin-V injection and for the following
2 days, but were less at the time of estrus, 3 days later,
suggesting that several follicles did not reach an ovu-
latory size [19]. We speculated that an additional in-
jection of Folltropin-V in the more diluted hyaluronan
solution 48 h after initiating treatment would have
caused follicles to continue growth to an ovulatory size
as occurred in the 20 mg/mL hyaluronan group.
Three experiments evaluated superovulatory re-
sponses in beef cows treated with two IM injections (48
h apart) of Folltropin-V diluted in reduced concentra-
tions of hyaluronan (Split-single IM administration).
Specifically, the superovulatory response of beef cows
treated with a Split-single IM administration of Foll-
tropin-V diluted in a solution of 10 mg/mL hyaluronan
(Experiments 1, 2, and 3) or 5 mg/mL hyluronan (Ex-
periment 3) were compared with the traditional twice
daily IM treatment protocol in saline.
2. Materials and methods
2.1. Experiment 1
Cross-bred beef cows (n ⫽ 12) and Simmental do-
nor cows (n ⫽ 14), with a body condition score of 3 to
3.5 (1–5 scale) and 400 to 550 kg of body weight, were
randomly allocated to one of two treatment regimens in
two replicates (with an interval of 50 days). Cows in the
Control group received 400 mg NIH-FSH-P1 of Foll-
tropin-V (Bioniche Animal Health, Inc., Belleville,
ON, Canada) in a constant dose, twice daily IM sched-
ule over 4 days; cows in the Split-single administration
group received 400 mg Folltropin-V in a 10 mg/mL
hyaluronan solution (MAP-5, Bioniche Animal Health,
Inc.) divided into 300 mg given as an IM injection on
the first day, followed by a second IM injection of 100
mg Folltropin-V-48 h later. On day 0 (beginning of
experiment), all cows received 5 mg estradiol-17␤ (es-
tradiol-17␤, Río de Janeiro, Laboratorios Allignani
Hnos SRL, Argentina) plus 50 mg progesterone (Pro-
gesterona, Río de Janeiro) and an intravaginal device
impregnated with 1.56 g progesterone (Cue-Mate, Bi-
oniche Animal Health, Inc.). On day 4 (expected day of
follicle wave emergence), all cows were superstimu-
lated with Folltropin-V, according to the groups de-
scribed above. The multiple doses of Folltropin-V were
prepared as usual, with 20 mL of saline diluent injected
into the vial, whereas single doses were prepared by
injection of 10 mL of map-5 into the vial containing
 A. Tríbulo et al. / Theriogenology 77 (2012) 1679 –1685
Folltropin-V lyophilized powder, immediately before
administration; 7.5 mL of the map-5 preparation was
injected on Day 4 and 2.5 mL was injected on Day 6.
In the morning, and evening of Day 6, all cows received
150 ␮g D (⫹) cloprostenol (PGF, Bioprost, Biotay SA,
Argentina) and Cue-Mates were removed in the eve-
ning of Day 6. In the morning, of Day 8, cows received
12.5 mg pLH (lutropin-V, Bioniche Animal Health,
Inc.) and were inseminated 12 and 24 h later. Ova/
embryos were collected non-surgically on Day 15 and
evaluated following IETS recommendations [20].
2.2. Experiment 2
In Experiment 2, designed to confirm results of
Experiment 1 in another breed of beef cattle, Brangus
donor cows (n ⫽ 14) with a body condition score of 3
to 3.5 (1–5 scale) and 450 to 600-kg body weight, were
randomly allocated to one of two treatment groups in a
cross-over design (i.e., all cows received the 2 treat-
ments at an interval of 60 days and both groups were
represented equally in each replicate). Cows in the
Control group received 300 mg of Folltropin-V diluted
in 15 mL of saline diluent given in a twice-daily,
constant dose, IM injection schedule over 4 days, and
cows in the Split-single IM administration group re-
ceived 200 mg Folltropin-V diluted in 5 mL of a 10
mg/mL hyaluronan solution (MAP-5) on Day 4, fol-
lowed by 100 mg Folltropin-V diluted in 2.5 mL hya-
luronan, on Day 6. Estradiol-17␤, progesterone, PGF
and pLH administrations.
Cue-Mate insertion and removal, inseminations,
ova/embryo collections and evaluations were done as in
Experiment 1.
2.3. Experiment 3
To examine the use of a less concentrated solution of
hyaluronan, non-lactating Simmental (n ⫽ 12) and An-
gus donor cows (n ⫽ 17) with a body condition score
of 3 to 3.5 (1–5 scale) and 500 to 650 kg of body
weight, were assigned to one of three treatment groups
and superstimulated three times in a cross-over design
(i.e., all cows received the 3 treatments; at intervals of
30 days and the 3 treatment groups were equally rep-
resented in each replicate). Cows in the Control group
received 300 mg of Folltropin-V diluted in 15 mL of
saline diluent given in twice-daily constant dose IM
over 4 days; cows in the Split-single IM administration
groups received 300 mg Folltropin-V in 10 mg/mL
hyaluronan (MAP-5) or 5 mg/mL hyaluronan (MAP-5
[50 MG] Bioniche Animal Health, Inc.). In Split-single
IM administration groups, 10 mL of the hyaluronan
1681
solution was used to dissolve the 400-mg vial of Foll-
tropin-V lyophilized powder; 200 mg was administered
in 5 mL on Day 4 and 100 mg in 2.5 mL was admin-
istered on Day 6). Estradiol-17␤, progesterone, PGF
and pLH administrations, inseminations, ova/embryo
collections and evaluations were done as in Experiment
1; except that Cue-Mates were removed in the morning
of Day 7 rather than in the afternoon of Day 6, as in
Experiments 1 and 2.
2.4. Ultrasonography
In all experiments, cows were examined on Days 0,
4, and 8 for the presence of a CL and follicle size and
number. In addition, cows in Experiment 3 were exam-
ined on Day 10 to determine numbers of ovulations.
Superovulatory response was confirmed by counting
the number of CL by transrectal palpation on Day 15
[21].
2.5. Statistical analyses
In Experiment 1, superovulatory response (i.e.,
mean number of CL) and ova/embryo data were first
analyzed for heterogeneity of variance and normality,
and when variances were found to be heterogeneous,
data were transformed by square root and compared by
anova, using the software Infostat [22]. In Experiments
2 and 3, superovulatory response (i.e., number of CL)
and ova/embryo data were also transformed by square
root (i.e., variances were heterogeneous) and analyzed
by anova for mixed models, using treatment as a fixed
variable and cow (i.d.) as a random variable [22]. In
Experiment 3, when the effect of treatment was found
to be significant, differences between treatment groups
were determined by the method of least significant
difference. Proportions were compared by ␹2 test (Ex-
periments 1, 2, and 3).
An anova for mixed models was also used to analyze
the mean number of follicles 3 to 5 mm in diameter, 6
to 8 mm in diameter and ⱖ 9 mm in diameter over
treatment days in Experiments 1, 2, and 3, using treat-
ment and day (and their interaction) as fixed variables
and cow (i.d.) as a random variable [22].
3. Results
The results of Experiment 1 are summarized in Ta-
ble 1. There was no effect of replicate and data were
combined into a complete data set. There were no
differences between groups in the number of CL, total
ova/embryos, fertilized ova or transferable embryos
(IETS Grades 1, 2, and 3). One cow in the Split-single
1682 A. Tríbulo et al. / Theriogenology 77 (2012) 1679 –1685

Table 1
Superovulatory response (mean ⫾ SEM) of beef cows treated with 400 mg Folltropin-V divided into twice daily IM injections over 4 d
(Control) or diluted in MAP-5 (10 mg/mL hyaluronan) and given by a Split-single IM administration (i.e., 2 IM injections 48 h apart).
Treatment n CL Ova-embryos Fertilized ova Transferable embryos
Control 13 15.7 ⫾ 2.1 5.3 ⫾ 1.3 4.1 ⫾ 0.9 2.5 ⫾ 0.8
Split-single 13 15.4 ⫾ 1.9 9.8 ⫾ 2.8 5.2 ⫾ 2.2 3.7 ⫾ 1.3
Means did not differ (P ⬎ 0.2).

group had ⱕ 2 CL at the time of ova/embryo collection,
whereas no cows in the Control group had ⱕ 2 CL at
the time of ova/embryo collection. Furthermore, 4 out
of 13 (31.0%) cows in the Control group and 6 out of
13 (46.1%) cows in the Split-single group did not
produce any transferable embryos (P ⫽ 0.42). There
was a day effect (P ⬍ 0.01), but no treatment effect
(P ⫽ 0.6) or day by treatment interaction (P ⫽ 0.8) on the
mean number of follicles 3 to 5 mm in diameter, 6 to 8
mm in diameter or ⱖ 9 mm in diameter over treatment
days. The mean (⫾ SEM) number of follicles 3 to 5 mm
in diameter on the day of Folltropin-V injection (i.e., Day
4) was 13.6 ⫾ 2.0 and 14.2 ⫾ 1.6 (P ⫽ 0.12) and the
mean number of follicles ⱖ 9 mm in diameter on Day 8
was 19.4 ⫾ 3.2 and 21.1 ⫾ 2.8 (P ⫽ 0.76) in the Control
and Split-single groups, respectively.
In Experiment 2, superovulatory response and em-
bryo data are summarized in Table 2. There were no
differences between groups in the number of CL and
total ova/embryos, but the number of fertilized ova was
higher (P ⬍ 0.05) and number of transferable embryos
tended to be higher (P ⬍ 0.07) in the Control group. In
this experiment all cows had more than two CL at the
time of ova/embryo collection and only one cow in
each treatment group did not produce any transferable
embryos (P ⫽ 0.9). There was a day effect (P ⬍ 0.01)
but no treatment effect (P ⬎ 0.7) or day by treatment
interaction (P ⫽ 0.9) on the mean number of follicles 3
to 5 mm in diameter, 6 to 8 mm in diameter and ⱖ 9
mm in diameter over treatment days. The number of
follicles 3 to 5 mm in diameter on Day 4 was 16.4 ⫾
1.4 vs. 19.1 ⫾ 1.9 (P ⫽ 0.25) and the number of
follicles ⱖ 9 mm in diameter on Day 8 was 28.1 ⫾ 3.3
vs. 26.5 ⫾ 3.9(P ⫽ 0.52) in the Control and Split-single
groups, respectively.
In Experiment 3, superovulatory response and em-
bryo data are summarized in Table 3. Although the
mean numbers of CL and total ova/embryos were
higher in the Split-single groups (i.e., 10 mg/mL and 5
mg/mL hyaluronan), there were no significant differ-
ences among groups in the number of fertilized ova and
transferable embryos. Two cows in the Control group
and one cow in each Split-single group had ⱕ 2 CL at
the time of ova/embryo collection (P ⫽ 0.8). Further-
more, 7 out of 29 (24.1%) cows in the each of the
Control and 10 mg/mL hylauronan groups produced no
transferable embryos, whereas 4 of 29 (13.8%) cows in
the 5 mg/mL hyluronan group produced no transferable
embryos (P ⫽ 0.5).
There was a day effect (P ⬍ 0.01), but no treatment
effect (P ⫽ 0.4) or day by treatment interaction (P ⫽
0.6) on the mean number of follicles 3 to 5 mm in
diameter, 6 to 8 mm in diameter and ⱖ9 mm in diam-
eter over treatment days. The number of follicles 3 to 5
mm in diameter on Day 4 were 17.8 ⫾ 1.0, 17.3 ⫾ 0.9
and 16.7 ⫾ 1.1 (P ⫽ 0.7) and the number of follicles
ⱖ9 mm in diameter on Day 8 were 17.2 ⫾ 1.4, 15.9 ⫾
1.5 and 17.5 ⫾ 1.6 (P ⫽ 0.7) in the Control, 10 mg/mL
and 5 mg/mL hyaluronan groups, respectively.
4. Discussion
Overall, a Split-single IM administration of Folltro-
pin-V diluted in hyaluronan was highly efficacious in
the superstimulation of beef donors; results, both in

Table 2
Superovulatory response (mean ⫾ SEM) of Brangus cows treated with 300 mg Folltropin-V given by twice daily intramuscular (IM)
injections over 4 days (Control) or diluted in MAP-5 (10 mg/mL hyaluronan) and given by a Split-single IM administration (i.e., 2 IM
injections 48 h apart).
Treatment N CL Ova-embryos Fertilized ova Transferable embryos
Control 14 19.7 ⫾ 1.4 23.3 ⫾ 3.5 19.4 ⫾ 3.6 a 13.5 ⫾ 3.2
Split-single 14 20.2 ⫾ 1.3 17.3 ⫾ 2.3 10.1 ⫾ 1.6b 6.9 ⫾ 1.3
ab
Within a column, means without a common superscript differed (P ⬍ 0.05).
 A. Tríbulo et al. / Theriogenology 77 (2012) 1679 –1685
Table 3
Superovulatory response (mean ⫾ SEM) of Simmental and Angus cows treated with 300 mg Folltropin-V given by twice daily IM injections
over 4 d (Control) or diluted in MAP-5 (10 mg/mL hyaluronan) or MAP-5 (50 MG; 5 mg/mL hyaluronan) and given by a Split-single IM
injection (i.e., 2 IM injections 48 h apart).
Treatment n CL
Control 29 12.0 ⫾ 1.3a
Split-single MAP-5 29 15.1 ⫾ 1.0b
Split-single MAP-5 [50 MG] 29 15.3 ⫾ 1.1b
a,b
Within a column, means without a common superscript differed (P ⬍ 0.05).
terms of ova/embryo production and numbers of trans-
ferable embryos, which did not differ from the tradi-
tional approach of superstimulation with twice-daily
IM injections in saline in two of three experiments.
Furthermore, in the third experiment, the numbers of
CL and ova/embryos in the split-single injection groups
were higher than in the Control group.
It is disconcerting that 7 of 29 (24.1%) cows in the
Control and 10 mg/mL hyaluronan groups in Experi-
ment 3 did not produce any transferable embryos,
whereas only 4 of 29 (13.8%) cows in the 5 mg/mL
hyaluronan group did not produce transferable em-
bryos. Although not significant, this result coupled with
results in Brangus donors (Experiment 2) in which the
split-single injection in 10 mg/mL hyaluronan was less
efficacious than the twice daily treatment protocol, sug-
gests that the 10 mg/mL hyaluronan solution may not
be as efficacious as the 5 mg/mL hyaluronan solution in
inducing superstimulation with a Split-single IM ad-
ministration of Folltropin-V. The reason for this slight
difference is not clear, but it is possible that Folltro-
pin-V does not mix as well in the 10 mg/mL solution of
hyaluronan, which could account for the less consistent
superovulatory responses in animals treated with this
preparation.
In an earlier study, Tríbulo, et al. [19] reported that
a solution of 20 mg/mL hyaluronan was quite viscous,
and that following mixing with Folltropin-V, it was not
possible to extract all the mixture from the vial. This
resulted in a numerically lower superovulatory re-
sponse following a single IM injection, which may
have been because of a lower amount of Folltropin-V
actually being administered. This conclusion was sup-
ported by follicle growth profiles. Although numbers of
3 to 5-mm follicles did not differ at the time of initiat-
ing gonadotropin treatment, the numbers of follicles
reaching ⱖ 9 mm in diameter was lower in the single
IM injection group. In that experiment, problems asso-
ciated with the mixing were overcome by mixing the
Folltropin-V diluted in a small volume of saline with
the 20 mg/mL hyaluronan solution in the injection
syringe. However, this was considered unduly compli-
1683
Ova-embryos Fertilized ova Transferable embryos
10.2 ⫾ 1.8a 6.7 ⫾ 1.3 4.0 ⫾ 0.8
14.4 ⫾ 2.0b 8.9 ⫾ 1.4 5.0 ⫾ 0.9
14.3 ⫾ 2.1b 9.3 ⫾ 1.9 6.1 ⫾ 1.3
cated for use in the field, especially if applied by non-
professionals.
It was decided that a more repeatable method of
mixing Folltropin-V with a slow-release formulation
had to be developed, and it was therefore that a lesser
concentration of hyaluronan (10 mg/mL) was investi-
gated. The 10 mg/mL hyaluronan solution was much
less viscous and easier to extract from the vial, but a
single IM administration failed to sustain follicle
growth to an ovulatory size [19]. Follicle diameter
profiles over the treatment period suggested that Foll-
tropin-V diluted in 10 mg/mL hyaluronan induced a
superstimulatory response, but follicle growth seemed
to decrease after 2 days (i.e., after Day 6 in the treat-
ment schedule) and it appeared that fewer follicles
reached an ovulatory size than in the 20 mg/mL hya-
luronan group, presumably because of inadequate con-
centrations of circulating FSH during the second half of
the treatment protocol [19]. A similar observation was
reported following the single subcutaneous administra-
tion of Folltropin-V in saline in Holstein cows [16] and
following a single IM administration of Folltropin in
saline in beef cows [23].
Circulating concentrations of FSH following injec-
tion depend on absorption from the site of administra-
tion and metabolic clearance from the circulation.
Based on metabolic clearance rates [10], it would be
expected that FSH would be cleared from the circula-
tion at the same rate, irrespective of its concentration
and method of administration. Rapid absorption of the
pituitary extract should result in rapid FSH clearance.
Therefore, follicles exposed to high concentrations of
FSH for a very short interval would appear to be unable
to sustain growth until ovulation. Preliminary results
collected as part of a study published previously [5]
indicated that following a single IM administration of
Folltropin-V in saline (or subcutaneous injection in lean
animals), blood FSH concentrations were higher, and
subsequently decreased more rapidly than in cows in
high body condition that received a single subcutaneous
injection, presumably because of slower absorption in
the cows with high body condition. It has also been
1684 A. Tríbulo et al. / Theriogenology 77 (2012) 1679 –1685
shown that the addition of FSH to a solution of poly-
vinylpyrrolidone [24] aluminum hydroxide gel [25] or
polyehyleneglycol [26], resulted in slower absorption
and more prolonged, elevated blood concentrations
than when FSH was administered in a saline vehicle.
We concluded that mixing of Folltropin-V with a so-
lution of hyaluronan also reduced the rate of absorp-
tion, resulting in sustained elevated blood FSH concen-
trations. Therefore, it would seem that characteristics
affecting absorption rate rather than clearance of FSH
would be the limiting factor in inducing superovulation
with a single administration of Folltropin-V.
When compared to the traditional twice-daily IM
injection protocol over 4 days, the Split-single IM ad-
ministration of Folltropin-V in hyaluronan (either 10
mg/mL or 5 mg/mL) was very efficacious. Although
not directly compared, the split-injection protocol
would appear to as efficacious as the single injection
protocol in the more concentrated hyaluronan solution
(20 mg/mL). However, the lower concentrations of
hyaluronan should ensure more repeatable mixing of
the polymer with Folltropin-V. In addition, superstimu-
lation was induced with the Single-split administration
protocol with no additional animal handling over the
single administration protocol, which should reduce the
likelihood of animal stress associated with handling.
The Single-split administration protocol was designed
to be applied when the animal had to be handled for
other treatments e.g., administration of prostaglandin.
Experiment 3 was considered a fairly rigorous test of
the Split-single IM administration of Folltropin-V in
hyaluronan in the induction of superovulation in cattle.
Not only did it support follicle growth profiles that were
comparable to the traditional twice daily IM regimen,
but there were no differences in the number of trans-
ferable embryos. The follicle growth profiles also did
not differ among groups, suggesting that the Split-
single IM administration of Folltropin-V in either a 10
mg/mL or 5 mg/mL hyaluronan solution provided suf-
ficient FSH to support follicle growth through to an
ovulatory size. Although the optimal dose of Folltro-
pin-V to be administered by Split-single IM adminis-
tration was not determined, it would appear to be sim-
ilar to that administered by twice-daily injections, as
has been shown previously with the single subcutane-
ous injection of Folltropin-V [5] or a single IM injec-
tion of Folltropin-V in a 20 mg/mL hyaluronan solution
[17–19].
In summary, superstimulation of beef cows of vari-
ous breeds with a Split-single IM administration of
Folltropin-V diluted in a 5 or 10 mg/mL solution of
hyaluronan resulted in a superovulatory response that
was comparable to the traditional twice-daily IM injec-
tion treatment protocol. The reduced concentrations of
hyaluronan were very easy to mix with lyophilized
Folltropin-V powder within the bottle and it appeared
that the entire contents were recovered following aspi-
ration. The Split-single IM administration of Folltro-
pin-V was designed to be administered at a time when
donors are normally handled during superstimulatory
treatments, reducing handling and labor; this protocol
may be particularly useful in the superstimulation of
animals in which handling stress is an impediment to
success.
References
[1] Bó GA, Adams GP, Pierson RA, Mapletoft RJ. Exogenous
Control Follicular Waves Emergence Cattle. Theriogenology
1995;43:31– 40.
[2] Bó GA, Baruselli PS, Moreno D, Cutaia L, Caccia M, Tríbulo
R, et al. The control of follicular wave development for self-
appointed embryo transfer programs in cattle. Theriogenology
2002;57:53–72.
[3] Baruselli PS, de Sá Filho MF, Martins CM, Nasser LF,
Nogueira MF, Barros CM, et al. Superovulation and embryo
transfer in Bos indicus cattle. Theriogenology 2006;65:77– 88.
[4] Bó GA, Baruselli PS, Chesta PM, Martins CM. The timing of
ovulation and insemination schedules in superstimulated cattle.
Theriogenology 2006;65:89 –101.
[5] Bó. Ga., Hockley DK, Nasser LF, Mapletoft RJ. Superovulatory
response to a single subcutaneous injection of a porcine pitu-
itary extract in beef cattle. Theriogenology 1994;42:963–75.
[6] Mapletoft RJ, Bennett-Steward K, Adams GP. Recent ad-
vances in the superovulation of cattle. Reprod Nut Dev
1992;42:601–11.
[7] Schams D, Menzer D, Schalenberger E, Hoffman B, Hahn J,
Hahn R. Some studies of the pregnant mare serum gonadotro-
phin PMSG and on endocrine responses after application for
superovulation in cattle. In: Sreenan JM, editor. Control of
reproduction in the cow. The Hague: Martinus-Nijhoff; 1977, p.
122– 42.
[8] Dieleman SJ, Bevers MM, Vos PLAM, De Los FAM, PMSG. /
anti PMSG in cattle: a simple and efficient superovulatory
treatment? Theriogenology 1993;39:25– 42.
[9] Laster DB. Disappearance and uptake of (125 I)FSH in the rat,
rabbit, ewe and cow. J Reprod Fertil 1972;30:407–15.
[10] Demoustier MM, Beckers JF, Van Der Zwalmen P, Closset J,
Gillard JL, Ectors F. Determination of porcine plasma follitro-
pin levels during superovulation treatment in cows. Theriog-
enology 1988;30:379 – 86.
[11] Bellows RA, Anderson DC, Short RE. Dose-response relation-
ships in synchronized beef heifers treated with follicle stimu-
lating hormone. J Anim Sci 1969;28:638 – 44.
[12] Monniaux D, Chupin D, Saumande J. Superovulatory responses
of cattle. Theriogenology 1983;19:55– 81.
[13] Looney CR, Boutte BW, Archbald LF, Godke RA. Comparison
of once daily and twice daily FSH injections for superovulating
beef cattle. Theriogenology 1981;15:13–22.
 A. Tríbulo et al. / Theriogenology 77 (2012) 1679 –1685
[14] Walsh JH, Mantovani R, Duby RT, Overstrom EW, Dobrinsky
JR, Enright WJ, et al. The effects of once or twice daily
injections of pFSH on superovulatory response in heifers. The-
riogenology 1993;40:313–21.
[15] Hockley DK, Bo GA, Palasz AT. Del Campo, M.R., Mapletoft
RJ. Superovulation with a single subcutaneous injection of
Folltropin in the cow: effect of dose and site of injection.
Theriogenology 1992;37:224.
[16] Lovie M, Garcia A, Hackett A, Mapletoft RJ. The effect of dose
schedule and route of administration on superovulatory re-
sponse to Folltropin in Holstein cows. Theriogenology 1994;
41:241.
[17] Rogan D, Tríbulo A, Tríbulo H, Tríbulo R, Carballo D, Tríbulo
P, et al. Dose titration for superstimulation of Brangus and
Bonsmara donors with Folltropin-V by a single intramuscular
injection. Reprod Fertil Dev 2010;22:365.
[18] Tríbulo A, Tríbulo H, Tríbulo R, Carballo D, Tríbulo P, Rogan
D, et al. Bó GA. Superovulation of Angus donors with a single
intramuscular injection of Folltropin-V. Reprod Fertil Dev
2010;22:367.
[19] Tríbulo A, Rogan D, Tríbulo H, Tríbulo R, Alasino RV, Bel-
tramo D, et al. Superstimulation in beef cattle with a single
intramuscular injection of Folltropin-V. Anim Reprod Sci 2011;
129:7–13.
[20] Wright J. Photomicrographic illustration of embryo codes.
Manual of the International Embryo Transfer Society, Savory
1685
IL (3rd ed). International Embryo Transfer Society; 1998, p.
167–70
[21] Robertson L, Cattoni IC, Shand RI, Jeffcoate IA. A critical
evaluation of ultrasonic monitoring of superovulation in cattle.
Br Vet 1993;149:77– 484.
[22] InfoStat. InfoStat/Profesional, versión 2008p. Grupo InfoStat/
FCA. Universidad Nacional de Córdoba. Ed. Brujas, Córdoba,
Argentina 2008.
[23] Bo GA, Hockley D, Tríbulo H, Jofre F, Tríbulo R, Busso N, et
al. The effect of dose schedule and route of administration on
superovulatory response to folltropin in the cow. Theriogenol-
ogy 1991;35:186.
[24] Takedomi T, Aoyagi Y, Konishi M, Kishi H, Taya K, Watanabe
G, et al. Superovulation in Holstein heifers by a single injection
of porcine FSH dissolved in polyvinylpyrrolidone. Theriogenol-
ogy 1993;39:327.
[25] Kimura K, Hirako M, Iwata H, Aoki M, Kawaguchi M, Seki
M. Successful superovulation of cattle by a single adminis-
tration of FSH in aluminum hydroxide gel. Theriogenology
2007;68:633–39.
[26] Choi SH, Park YS, Cho SR, Kang TY, Sin SH, Kang SS, et
al. Superovulation response and quality of embryos recov-
ered from cattle after a single subcutaneous injection of FSH
dissolved in polyethylene glycol (in Korean). J Emb Trans
2002;17:67–77.