Animal Reproduction Science 113 (2009) 51–59

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Effect of recombinant bovine somatotropin

(bST) on follicular population and on
in vitro buffalo embryo production
M.F. Sá Filho a, N.A.T. Carvalho a, L.U. Gimenes a, J.R. Torres-Júnior a,
L.F.T. Nasser a, H. Tonhati b, J.M. Garcia b, B. Gasparrini c,
L. Zicarelli c, P.S. Baruselli a,∗
a
Departamento de Repodução Animal, FMVZ-USP, São Paulo, SP, Brazil
b
Universidade Estadual Paulista, UNESP-Jaboticabal, Jaboticabal, SP, Brazil
c
DISCIZIA, Frederico II University, Napoli, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to evaluate the effect of bovine soma-
Received 19 March 2007 totropin (bST) on ovarian follicular population in buffalo heifers and
Received in revised form 18 May 2008
its influence on oocyte quality, recovery rates and in vitro embryo
Accepted 30 June 2008
production. We tested the hypothesis that bST treatment in buf-
Available online 6 July 2008
falo females submitted to an ovum pick-up (OPU) program would
improve the number of follicles recruited, oocyte quality and in
Keywords:
vitro embryo production. A total of 10 heifers were assigned into
Buffalo-OPU
IGF-I two treatment groups: group bST (n = 5; receiving 500 mg of bST in
In vitro embryo production regular intervals) and control group (n = 5; without additional treat-
Bubalus bubalis ment). Both groups were subjected to OPU sessions twice a week
(every 3 or 4 days), for a total of 10 sessions per female, although
due to procedural problems, only the first five OPU sessions pro-
duced embryos. The number of follicles and the diameters were
recorded at all OPU sessions. The harvested oocytes were counted
and classified according to their quality as either A, B, C, D or E,
with A and B considered good quality. Cleavage and blastocyst pro-
duction rates were evaluated 2 and 7 days after in vitro fertilization,
respectively. The bST treatment increased the total number of antral
follicles (>3 mm in diameter; 12.2 compared with 8.7; p < 0.05) and
of small antral follicles (<5 mm; 9.1 compared with 6.5; p < 0.05)
per OPU session. The bST also tended to increase the number of

∗ Corresponding author.
E-mail address: [email protected] (P.S. Baruselli).

0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2008.06.008
52 M.F. Sá Filho et al. / Animal Reproduction Science 113 (2009) 51–59

oocytes recovered per session (5.2 compared with 4.1; p = 0.07),

and enhanced the percentage of good quality oocytes (48.8% com-
pared with 40.6%; p = 0.07). bST showed no effect on cleavage
and blastocyst production rates (p > 0.05). The significant effects of
performing repeated OPU sessions were decreasing the follicular
population (p < 0.001) as well as the number of follicles aspirated
(p < 0.001), and oocytes recovered (p < 0.02). In conclusion, bST
treatment improves the follicular population, demonstrating its
possible application in buffalo donors submitted to OPU programs.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Buffalo respond less to multiple ovulation and embryo transfer procedures in comparison to cattle.
Buffalo produce slightly poorer ova and have fewer embryos recovered than cattle (Drost et al., 1983;
Baruselli et al., 2000; Carvalho et al., 2002). Pregnancy in buffalo has been achieved through the transfer
of fresh (Madan et al., 1991; Boni et al., 1997; Sá Filho et al., 2005), frozen (Galli et al., 1998) and
vitrified (Neglia et al., 2004; Sá Filho et al., 2005) buffalo embryos produced in vitro. Given each of
these considerations, utilizing ovum pick-up (OPU) technology in conjunction with in vitro embryo
production (IVP) presents the possibility of enhancing genetic progression through the female lineage
(Galli et al., 2001; Gasparrini, 2002; Neglia et al., 2003).
Although there has been interest in applying OPU and IVP techniques in buffalo females, certain
factors have limited the commercial use of OPU–IVP in buffalo. These include the lesser number of
primordial follicles present in the ovaries (Danell, 1987; Baruselli et al., 1997), lesser number of oocytes
recovered per OPU session (Gasparrini, 2002; Gasparrini et al., 2006), greater incidence of atretic
follicles (Le Van Ty et al., 1989) and oocytes, and lesser cleavage rates (Gasparrini, 2002; Gasparrini et al.,
2006). Therefore, the development of strategies that increase the ovarian follicular population, improve
the proportion of non-atretic follicles, and enhance oocyte quality is very important for improving the
OPU–IVP efficiency in buffalo.
In cattle, bovine somatotropin (bST) treatment has been applied during OPU–IVP programs to
improve follicular population prior to OPU. Positive results include enhancing oocyte quality and
embryo development capacity, improving in vitro oocyte maturation, and increasing fertilization
(Pavlok et al., 1996; Bols et al., 1998; Tripp et al., 2000; Roth et al., 2002). However, the precise effects
of the bST treatment in such programs are still not clearly defined.
The objective of the present study was to evaluate the effect of bST on follicular population in buffalo
heifers and its influence on oocyte quality, recovery rate and in vitro embryo production. The hypothesis
was that bST treatment in buffalo females submitted to ovum pick-up (OPU) programs would increase
the number of follicles susceptible to puncture, enhance oocyte recovery both in quality and quantity,
and consequently improve the in vitro embryo production.

2. Materials and methods

2.1. Animals

Ten buffalo heifers (Bubalus bubalis), ages 20–30 months and weighing 418.1 ± 35.2 kg, were selected
from a commercial dairy farm (Santa Eliza Farm, Dourado-SP, Brazil). All females were previously
examined by ultrasonography and selected according to the following criteria: (1) had or had not
initiated onset of estrous cycle (all heifers should have a corpus luteum—CL), (2) ovarian diameter
(each ovary should have ≥2 cm of diameter) and (3) follicular population (all heifers should have ≥8
follicles per ovary).
The females were maintained at UNESP-Jaboticabal Campus (Jaboticabal-SP, Brazil) under grazing
conditions with mineral salt and water ad libitum, from July to August of 2004.
 M.F. Sá Filho et al. / Animal Reproduction Science 113 (2009) 51–59 53

Fig. 1. Experimental procedures of bST treatment during OPU–IVP program in buffalo heifers (Bubalus bubalis).

2.2. Experimental procedure

The heifers were homogenously assigned based on their follicular population (total number of fol-
licles present on the ovary at ultrasonographic examination) and body weight into two experimental
groups. Those in the bST group (n = 5) were treated with a subcutaneous injection of 500 mg of bST
(Lactotropin, Elanco Animal Health, Bruxelas, Belgum) on the day of pretreatment follicle synchroniza-
tion, 5 days before the first OPU session, and received two additional injections of bST 14 days apart (at
the third and seventh OPU sessions; Fig. 1). The control group (n = 5) did not undergo these treatments.
To synchronize the status of the follicular wave prior to the first follicular aspiration session (OPU),
all females were subjected to follicular ablation via puncture of all follicles ≥3 mm within each ovary 5
days before the first OPU session. OPU sessions were performed twice weekly, and whereas the entire
experiment lasted 5 weeks, resulting in 10 OPU sessions for each female.
An OPU session involved aspiration of all antral follicles ≥3 mm in diameter within each ovary,
using a 7.5-MHz micro-convex transducer (100 Falco, Pie Medical, Netherlands) with a disposable,
40 mm long, 19 g needle at 60–65 mmHg of negative vacuum pressure. Prior to each OPU session,
the number of follicles ≥3 mm was recorded, and from this, the average number of follicles per pair
of ovaries was determined. To further analyze the ovarian follicular population, the total number of
visible follicles was divided into three size categories: small diameter (3–5 mm), medium (5–10 mm)
and large (>10 mm).

2.3. Oocyte processing

The cumulus oocytes complexes (COC) recovered by OPU were searched immediately after the
aspiration of follicles and graded into five categories (A, B, C, D and E). Cumulus oocytes complexes
54 M.F. Sá Filho et al. / Animal Reproduction Science 113 (2009) 51–59

characterized by uniform cytoplasmatic appearance and enclosed with three layers of viable com-
pact granulosa cells were classified as Grade B, while Grade A contained more than three layers. The
cumulus oocytes complexes characterized by fewer than three layers or partially denuded were char-
acterized as Grade C. Grade D were distinguished by expanded granulosa cells, pyknotic or vacuolized
oocyte cytoplasm. COC with misshapen, partially absent oocyte cytoplasm or empty zona pelucida
were assigned as Grade E. Oocytes classified as A, B, C or D were considered suitable for in vitro pro-
duction, while A and B were deemed good quality, based on a classification created by Neglia et al.
(2003). The percentage of good quality oocytes was defined as the number of good quality oocytes out
of the total number of oocytes recovered.
Oocytes were washed four times in Hepes-buffered TCM-199 with 10% fetal calf serum (FCS) and
then placed in the same medium supplemented with 1 ␮g/ml FSH (FolltropinTM , Bioniche Animal
Health, Belleville, Ont., Canada), 50 ␮g/ml hCG (ProfasiTM , Serono, São Paulo, Brazil), 1 ␮g/ml estradiol,
0.20 mM of sodium pyruvate, 83.4 ␮g/ml amikacine, and 50 ␮M of cysteamine with 0.3 mM of cystine
(Gasparrini et al., 2006). The COC were placed for in vitro maturation into 50 ␮l droplets of TCM-199
medium under mineral oil and incubated under 5% of CO2 in air at 38.5 ◦ C and high humidity for
22–24 h.

2.4. Fertilization and embryo culture

The spermatozoa were prepared from frozen–thawed semen, obtained from a single commercial
standard bull that had been previously tested for IVP. Sperm were separated by Percoll gradient
(45/90%). The pellet obtained after centrifugation was re-suspended to a final concentration of
2 × 106 sptz/ml in the fertilization (IVF) medium, a modified TALP supplemented with 10 ␮g of heparin
with 160 ␮g of PHE as per Bavister (1995). Fertilizing droplets (10–20 COCs/100 ␮l droplet) covered
with mineral oil were incubated under the same gas atmosphere as for in vitro maturation for 16–18 h.
After the IVF period the putative zygotes were removed from the fertilization medium, stripped of
cumulus cells by gentle pipetting, washed twice in a Hepes-buffered synthetic oviduct fluid (SOF) ver-
sion of SOF medium with the addition of essential and non-essential amino acids. The culture medium
was composed of bicarbonate-buffered SOF supplemented with 2.5% of FCS and 5 mg/ml bovine serum
albumin. Presumptive zygotes were distributed into 50 ␮l droplets (20–25 per droplet), and the cul-
ture was carried out under the same atmosphere as for in vitro maturation and fertilization. On day
2 of embryo development (day 0 = day of insemination) cleavage rate was evaluated and “feeding”
was performed: 50% of the medium was removed and was replaced with the same volume of fresh
medium. On days 6 and 7 embryo production was evaluated. The cleavage and blastocyst produc-
tion rates were defined, respectively, as the number of zygotes with two or more cells on day 2 and
the number showing blastocyst formation on day 7, post-fertilization, divided by the total number of
oocytes cultured.

2.5. Statistical analysis

Data were analyzed using the SAS System for Windows (Statistical Analyses System, 2000) program.
As necessary, data were normalized using Guided Data Analysis; those not suitable for being normalized
were tested using Wilcoxon non-parametric test.
The bST treatment and the OPU sessions were considered independent variables. The follicular
population (average number of follicles per ovary pair) and number of aspirated follicles (average
number of aspirated follicles per female) were considered dependent variables and were analyzed
by ANOVA for repeated measures using the MIXED procedure. The effects of donor, treatment, OPU
session and treatment by OPU session on interactions were analyzed within this statistical model. The
quantity of oocytes recovered, number of follicles in each category, number of good quality oocytes, and
number of embryos produced per buffalo per OPU session were analyzed by ANOVA using the SAS GLM
procedure. Donor, treatment, OPU session and treatment by OPU session were utilized in the statistical
model to determine interactions. Means were compared using Duncan’s test. The percentage of good
quality oocytes, cleavage and blastocyst production rates were analyzed by Wilcoxon two-sample test.
 M.F. Sá Filho et al. / Animal Reproduction Science 113 (2009) 51–59 55

Fig. 2. Effect of bovine somatotropin (bST) treatment on follicular population (average number of follicles per pair of ovaries) of
buffalo heifers submitted to ten ovum pick-up (OPU) sessions twice a week (* is the effect of treatment on follicular population;
p < 0.05).

Differences with p < 0.05 were deemed significant, whereas those with 0.05 < p < 0.10 were consid-
ered as a tendency for the dependent variables evaluated.

3. Results

Follicular population was influenced by OPU session (p < 0.0001) and by the treatment (p = 0.04;
Fig. 2). The bST group produced a greater number of total antral follicles per OPU session (12.2 ± 0.86)
than did the control group (8.7 ± 0.86; Table 1). Follicular population decreased per OPU session in
both experimental groups (log 10 (follicular population) = 1.03–0.01 × session; p = 0.02).
Treatment and OPU session influenced the number of small follicles. The bST group had more small
follicles (9.13 ± 3.9) than did the control group 6.45 ± 2.7 (p = 0.0004). Treatment did not affect the
number of medium (p = 0.35) or large (p = 0.29) follicles. Also, OPU session did not alter the number of
medium (p = 0.83) or large (p = 0.48) follicles (Table 1 and Fig. 3).
The number of aspirated follicles was influenced by treatment (p = 0.0008) and OPU ses-
sion (p < 0.0001), but there was no OPU session by treatment interaction (p = 0.45; Table 1).

Table 1
Effect of bST treatment on follicular population and OPU efficiency in buffalo heifers (Bubalus bubalis) submitted to ten repeated
OPU sessions twice a weeka

Parameter bST (n = 5) Control (n = 5) p-Value

Treatment Session Treatment

× session

Follicular population (total number of follicles)a 12.2 ± 0.06 8.7 ± 0.04 0.04 <0.0001 0.12
Number of large follicles (>10 mm)a 0.2 ± 0.1 0.2 ± 0.1 0.29 0.48 0.58
Number of medium follicles (5–10 mm)a 1.6 ± 0.2 1.9 ± 0.2 0.35 0.83 0.16
Number of small follicles (<5 mm)a 9.1 ± 0.6 6.5 ± 0.4 0.0004 0.04 0.18
Number of aspired folliclesa 9.1 ± 0.6 6.8 ± 0.3 0.0008 <0.0001 0.45
Number of oocyte recovereda 5.2 ± 0.5 4.1 ± 0.5 0.07 0.02 0.10
Oocyte recovery rate (%)a 54.5 57.7 0.54 0.71 0.28
Number of good quality oocytea 2.44 ± 0.3 1.92 ± 0.3 0.15 0.34 0.27
Good quality oocyte (%)a 48.8 40.7 0.07 0.60 0.51
Cleavage rate (%)b 46.2 41.7 0.80 – –
Embryo production (%)b 19.7 26.0 0.38 – –
Number blastocyst produced per buffalo per sessionb 1.3 ± 0.6 1.2 ± 0.2 0.72 – –
a
Values (mean ± S.E.M.) correspond of the mean for each group per OPU session.
b
Data only from the first five OPU sessions.
56 M.F. Sá Filho et al. / Animal Reproduction Science 113 (2009) 51–59

Fig. 3. Effect of bovine somatotropin (bST) treatment on follicular categories (small, medium and large) of buffalo heifers
submitted to 10 OPU sessions twice a week. Summary of total number of small, medium and large follicles obtained in 10
buffalo heifers during 10 OPU session.

Aspirated follicles decreased per OPU session in both experimental groups (log 10 (follicular popu-
lation) = 0.88–0.01 × session; p = 0.05).
Significant differences were observed decreasing the number of oocytes recovered by OPU session
(p = 0.02). In addition, treatment (p = 0.07), OPU session and treatment interaction (p = 0.10) tended to
exert differences (Table 1).
Recovery rate was not impacted by treatment (p = 0.54), OPU session (p = 0.71) or OPU session by
treatment interaction (p = 0.28). The recovery rates were 54.5 ± 6.2% and 57.7 ± 6.1% in the bST and
control group, respectively (Table 1).
The number of good quality oocytes did not vary by treatment (p = 0.15) and by OPU session
(p = 0.34). Additionally, 48.8% (126/258) of oocytes recovered from animals treated with bST and 40.6%
(84/207) from control animals were classified as good quality (p = 0.07). However, OPU session (p = 0.60)
and OPU session by treatment interaction (p = 0.51) did not affect the percentage of good quality oocytes
(Table 1).
Due to procedural problems, only the first five OPU sessions produced embryos; thus, the remaining
sessions were excluded from analysis. Treatment did not appear to affect cleavage rate (p = 0.80), blas-
tocyst production rate (p = 0.38), or the number of embryos produced per buffalo per session (p = 0.72;
Table 1).

4. Discussion

Treatment with bST causes an increase in the follicular population in Bos taurus (Gong et al., 1991,
1993a) and Bos indicus (Buratini et al., 2000). In the present study, bST treatment successfully increased
the follicular population, demonstrating its potential for enhancing the efficiency of OPU programs in
buffalo. These findings are consistent with previous studies in which bST was utilized during OPU
programs (Bols et al., 1998; Tripp et al., 2000).
The mechanism by which bST promotes follicular population growth is not entirely elucidated.
While some authors argue about the direct effect of growth hormones (Hull and Harvey, 2001), others
point to alterations in IGF-1 and/or insulin plasmatic levels (Gong et al., 1993; Buratini et al., 2000;
Gong, 2002).
IGF-I has been cited as a major mediator of incremental growth in the follicular population following
bST treatment (Lucy, 2000). When granulosa cells were incubated with the addition of IGF-I and/or
insulin, it was observed that IGF-I amplified the FSH and LH effects, resulting in a significant positive
 M.F. Sá Filho et al. / Animal Reproduction Science 113 (2009) 51–59 57

effect on mitogenic activity and steroidogenesis in a dose-dependent manner (Gong et al., 1993a,b;
Spicer et al., 2002). This positive effect is due either to an increase in gonadotropin receptors on
follicle cells or heightened activity of these receptors (Lucy, 2000). Thus, the increase in the follicular
population after bST treatment in present study could be explained by a possible increase in the IGF-I
plasmatic concentrations in the treated females.
The number of oocytes recovered tended to be greater in bST-treated animals (21%, p = 0.07). These
results are not in agreement with Bols et al. (1998) or Tripp et al. (2000) for cattle. In these studies,
no increase in oocyte numbers occurred following bST treatment. This difference can be attributed
to the absence of bST effect on CCO recovery rate in buffaloes. Bols et al. (1998) discussed that bST
promotes an asynchrony between follicular growth and development of CCO, causing a retainment of
CCO due to the follicular wall collapses after the OPU. In buffalo, a lesser number of cumulus cells and
the more fragile intercell connections (Gasparrini, 2002; Neglia et al., 2003) could reduce the difficult
on removing the CCO from the follicle during the follicular aspiration.
The percentage of good quality oocytes (A and B categories) tended to be augmented by the bST
treatment. These results are not in agreement with some previous studies (Bols et al., 1998; Tripp et
al., 2000) which did not find any difference in morphologic COC quality between oocytes recovered
from bST treated and control animals. However, Roth et al. (2002), working with dairy cows during
autumn, did note an increase in the percentage of good quality oocytes (72% bST compared with 26%
control).
The bST treatment did not alter the oocyte recovery rate, a result inconsistent with Tripp et al.
(2000) who identified a 14% decrease in the recovery rate among bovine females treated with bST
(30%) relative to control animals (44%). One plausible explanation for this divergence could be the
difference in aspiration intervals utilized: twice a week in the current study versus once a week (Tripp
et al., 2000). There is some evidence that bST treatment in cattle can trigger premature regression
of the dominant follicle reducing its dominance period by 24–48 h (Lucy et al., 1993). Additionally,
Bols et al. (1998) suggest that bST treatment could instigate an asynchrony between follicular and
COC development, which could make removing the oocyte from the follicle during aspiration more
difficult. Thus, the impact of bST treatment on oocyte recovery rates could, in fact, depend on the
follicular aspiration interval.
The blastocyst production rate and the number of blastocysts produced per buffalo per session did
not vary by treatment. This result is consistent with previous studies with cattle (Bols et al., 1998; Tripp
et al., 2000). However, it is important to emphasize that, considering the fewer follicular aspiration
sessions in the current study that proved successful in producing embryos, further investigation is
required to confirm these results.
In the present study, one significant effect of OPU sessions was reduction in the number of follicles
available for puncture and of oocytes recovered. There was approximately a 27% reduction in the
number of follicles available for aspiration in the last five OPU sessions compared with the first five.
This could be explained by the relatively short inter-aspiration interval (twice a week), decreasing the
period for any ovary injury that might have occurred during the previous session to heal. It has also been
described that the decrease in the number of follicles available for aspiration is a response to repetitive
mechanical injury which is responsible to promote degenerative alterations in the ovarian stroma as
well as an increase on local inflammatory mediators that can be detrimental to steroidogenesis and
oocyte metabolism (Espey, 1994; Petyim et al., 2001; Viana, 2002). Although all of these effects of
the short inter-aspiration interval, several investigations found no differences in females submitted
to the same OPU schedule (Gibbons et al., 1994; Boni et al., 1996; Garcia and Salaheddine, 1998). This
conflicting result could be due to: (1) peculiarities in ovarian physiology among buffaloes, especially
those raised in tropical conditions; (2) variations in responses to inflammation and/or scars due to
previous aspirations and (3) the operator’s precision in avoiding puncture of the medular portion
of the ovary, which, because it is well-vascularized, increases the risk for hemorrhages and fibroses
following OPU sessions.
Viana (2002) suggested that the risk of ovarian lesion due to follicular aspiration is proportional
to the sessions to which the donors are submitted, and more specifically, to the number of structures
recovered per session and the number of needle punctures on the ovary. In the present study, only
buffalo with a superior number of follicles (>16 follicles per female) compared to those described in
58 M.F. Sá Filho et al. / Animal Reproduction Science 113 (2009) 51–59

the literature (Baruselli et al., 1997; Manik et al., 2002) were used. The greater number of punctures
needed to accommodate this larger follicular population could have led to more ovarian lesions.
In conclusion, bST treatment improves the follicular population demonstrating its possible appli-
cation in buffalo donors submitted to OPU programs.

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