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Theriogenology 77 (2012) 148 –154

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Ovum pick-up and in vitro embryo production (OPU-IVEP) in

Mediterranean Italian buffalo performed in different seasons
Serena Di Francescoa, Maria Virginia Suarez Novoab, Domenico Vecchioa,*,
Gianluca Negliaa, Lucia Bocciaa, Giuseppe Campanilea, Luigi Zicarellia,
Bianca Gasparrinia
a
DISCIZIA, Faculty of Veterinary Medicine, “Federico II” University, via F. Delpino, 1, 80147, Naples, Italy
b
Lisandro Alvarado University, Decanato de Ciencias Veterinarias, Cabudare, Venezuela
Received 29 April 2011; received in revised form 29 June 2011; accepted 21 July 2011

Abstract
This study was designed to evaluate the effect of season on in vivo oocyte recovery and embryo production in Mediterranean
Italian buffalo (Bubalus bubalis). For this purpose repeated transvaginal ultrasound-guided ovum pick up (OPU) was conducted
twice a week throughout autumn, mid-winter (transitional period) and spring-summer. The number and size of follicles was
determined before puncture. The recovered oocytes were first classified in morphological categories and then used for in vitro
embryo production (IVEP) according to standard procedures. The mean number of total follicles observed per session did not
differ among the three periods we examined (on average 4.6). Although season did not considerably affect the number of oocytes
recovered (on average 2.3/buffalo/session), the number of degenerated and abnormally expanded oocytes increased during
autumn. Furthermore, the percentage of abnormally expanded oocytes significantly increased during autumn (6.1%) compared
with both the transitional period and spring-summer (1.9 and 2.3%, respectively). Interestingly, the embryo output we recorded
at day 7, in terms of tight morulae-blastocysts was higher in autumn (30.9%) compared to the other two periods (13.3% and 10.3%,
respectively, in spring-summer and in the transitional period; P ⬍ 0.01). The results of this trial demonstrated that the
morphological features of the oocytes did not vary substantially among the considered periods, with the exception of degenerated
and abnormally expanded oocytes. On the other hand, the oocyte developmental competence improved in autumn compared to
spring-summer and the transitional period. This datum reflects buffalo reproductive pattern expressed in vivo at Italian latitudes.
© 2012 Elsevier Inc. All rights reserved.

Keywords: Buffalo; Ovum Pick up; Reproductive seasonality

1. Introduction buffalo have emerged as an increasingly important

The water buffalo (Bubalus bubalis) is a fundamen-
tal livestock species for developing countries in tropical
and sub-tropical environments and is also an important
production animal in developed countries [1,2]. In fact,
* Corresponding author. Tel.: ⫹390812536500; fax: ⫹3908129-
2981.
E-mail address: d.vecchio@unina.it (D. Vecchio).
source of high quality animal protein, both milk and
meat [2].
There is a worldwide increasing interest in large-
scale in vitro production of buffalo embryos to improve
genetic progress through the maternal lineage. Indeed,
the Ovum pick-up (OPU) and the in vitro embryo
production (IVEP) are currently the most competitive
technologies to produce transferable embryos (TE) over
the long term [3].

0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2011.07.028
 S. Di Francesco et al. / Theriogenology 77 (2012) 148 –154
Furthermore, OPU can be performed on a wide
variety of donors, such as non-cyclic animals, pregnant
cows, and subjects with non-patent oviducts or genital
tract infections [3]. It can also be carried out in animals
that do not respond to multiple ovulation procedures,
the last representing a high proportion in buffalo spe-
cies [4,5]. Therefore, this procedure is a valid alterna-
tive to multiple ovulation/embryo transfer (MOET)
programs [4] because of the low response to hormonal
stimulation, the poor embryo recovery [6], and the
impossibility to repeat continuously the MOET treat-
ments over the long-term. Indeed, multiple ovulations
can be induced only in cyclic animals, whereas it is
likely that buffalo cows enter seasonal anestrus.
OPU has been successfully carried out in buffalo by
Boni et al [7] and IVEP efficiency has greatly improved
throughout the years [8].
The application of OPU-IVEP technology to buffalo
can enhance the maternal contribution to genetic im-
provement [3], thus overcoming the reduced levels of
efficiency linked to the reproductive seasonality pattern
of this species. In fact, buffalo are a seasonally polye-
strous species, in which the increase of ovarian cyclic
activity and, subsequently, fertility coincides with de-
creasing daylight hours. It follows that buffalo are
short-day breeders, as their reproductive efficiency im-
proves with a negative photoperiod. A particularly crit-
ical period in buffalo bred at Italian latitudes is mid-
winter when the daily light begins to increase. This
corresponds to the so-called transitional period, in
which an increased AI failure is observed in this species
[9]. However, data regarding the effects of seasonal
shifts in the activity of the reproductive axis on the
quality and the developmental potential of buffalo
oocytes from live donors in temperate climates are
lacking.
Hence, the aim of the present study was to compare
the quality and developmental competence up to trans-
ferable embryos after IVEP of buffalo oocytes collected
from live donors, in three different periods of the year:
mid-winter, i.e., the transitional period (January, Feb-
ruary, and March), spring–summer (May, June, and
July) and autumn (September, October, and Novem-
ber).
2. Materials and methods
2.1. Reagents and media
If not otherwise stated, all chemicals and reagents
were purchased from Sigma (Sigma-Aldrich, Milan,
Italy).
149
2.2. Oocyte recovery
The study was conducted in a farm located in the
Campania region during three different periods of the
year: mid-winter, i.e., the transitional period (Janu-
ary, February, and March), spring–summer (May,
June, and July) and autumn (September, October,
and November). Ovum pick-up was carried out re-
spectively on 14, 9, and 9 healthy, multiparous and
lactating buffalo cows, over 18 puncture sessions per
each period. The donors were under controlled nu-
trition, barn-housed, and restrained in a chute at the
moment of the oocyte retrieval session. During the
study, buffalo cows did not show any behavioral
modification and the OPU treatment did not cause
any adverse effect.
Ovum pick up setting consisted of a portable ultra-
sound unit (SonoAce - PICO, Medison, Cypress, CA,
USA) with a convex 4MHz⬃9MHz probe (mod.
CD4⫺9/10EDN) and a metal guide, to fit on the top 18
gauge needles, allocated in a properly designed vaginal
guide (WTA Ltda., Cravinhos/SP, Brazil). A vacuum
pressure of 40 mm-Hg was constantly maintained by
using a suction unit (K-MAR-5100, Cook IVF Co.,
Australia) and the aspiration line was continuously
rinsed with 25 mM hepes buffered TCM 199 supple-
mented with 100 USP units ml⫺1 of heparin and 10%
Fetal calf serum (FCS) and 1% penicillin and strepto-
mycin complex (20000 IU and 20000 ␮g/ml, respec-
tively, Lonza, Milan, Italy) during follicular aspiration.
The 15 ml Falcon tubes (Becton & Dickinson Co.,
Lincoln Park, NJ, USA) for oocyte collection were
constantly maintained at 37 °C. All visible antral fol-
licles were punctured and classified in three categories,
according to their size: small (diameter ⬍ 0.5 cm),
medium (diameter between 0.5 and 1 cm) and large
(diameter ⬎ 1 cm).
Cumulus-oocytes complexes (COCs) were searched
immediately after follicular aspiration by using proper
filters (Emcon Technologies, Columbus, IN, USA) and
classified in 7 categories: Grade A (oocytes with more
than three layers of cumulus cells and homogenous
cytoplasm); Grade B (oocytes with at least 2 layers of
cumulus cells and homogenous cytoplasm); Grade C
(oocytes partially denuded, but still showing homoge-
nous cytoplasm); Grade D (degenerated oocytes with
irregular shrunken cytoplasm); Grade E (oocytes with
expanded cumulus and homogeneous cytoplasm); ab-
normally expanded (oocytes showing an abnormal ex-
pansion of cumulus cells that appeared dark and clus-
tered) and naked (totally denuded oocytes). Recovery
rate (calculated as the percentage of the total number of
150 S. Di Francesco et al. / Theriogenology 77 (2012) 148 –154
oocytes in relation to the total number of follicles) was
also recorded.
Cumulus-oocyte complexes were washed twice in
Hepes-buffered TCM 199 with 10% FCS and then
allocated in the same medium supplemented with 0.3
mM cystine, 50 ␮M cysteamine [10] and 0.5 ␮g ml⫺1
FSH, 5 ␮g ml⫺1 LH, 1 ␮g ml⫺1 17-␤-estradiol [11].
The collected oocytes were then pooled and stored in
15 ml Falcon tubes in a portable incubator at 38.5 °C
and moved to the laboratory within 4 to 6 h for in vitro
embryo production [3].
2.3. In vitro Embryo Production (IVEP)
For IVM, COCs were individually transferred into
50 ␮l droplets (10 COCs/droplet) under mineral oil of
the final maturation medium, consisting of bicarbon-
ate– buffered TCM 199 with cystine, cysteamine, and
hormones in the same concentration previously de-
scribed. The droplets were incubated at 38.5 °C for 22 h
under controlled gas atmosphere of 5% CO2 in humid-
ified air.
IVF was carried out according to the method previ-
ously described by Parrish et al [12] the day after IVM.
Frozen-thawed sperm form a bull previously proven
suitable for IVF were prepared by Percoll density gra-
dient (Nidacon, Mölndal, Sweden). The pellet obtained
after centrifugation was resuspended to a final concen-
tration of 2 ⫻ 106 ml⫺1 in the fertilization medium, a
modified TALP supplemented with 0.2 mM ml-1 pen-
icillamine, 0.1 mM ml-1 hypotaurine, and 0.01 mM ml-1
heparin. Fifty ␮l fertilizing droplets (5 COCs/droplet)
covered by mineral oil were incubated under the same
gas atmosphere as for IVM.
After 20 –22 h of co-incubation with spermatozoa,
presumptive zygotes were cultured (IVC) in 20 ␮l
droplets (10 COCs/droplet) of synthetic oviduct fluid
(SOF) added with essential and non-essential amino
acids and bovine serum albumin (BSA) [13–14] for 7 d
in modular chamber with a gas atmosphere of 5% CO2,
7% O2, and 88% N2. At day 5 (day 0 ⫽ IVF day)
Table 1
Data on follicular and oocyte population among the different seasons.
Mid-winter
Mean⫾SEM
Total follicles n 4.76 ⫾ 0.27
Large follicles n (%) 0.81 ⫾ 0.08 (17.61 ⫾ 1.83)
Medium follicles n (%) 1.33 ⫾ 0.11 (27.86 ⫾ 1.79)
Small follicles n (%) 2.61 ⫾ 0.20 (54.53 ⫾ 2.64)
Total oocytes 2.31 ⫾ 0.11
Recovery rate (%) 53.55 ⫾ 3.44
cleavage rate was assessed and embryos were trans-
ferred into fresh droplets of the same medium for a
further 2 d of culture. The final embryo output, both in
terms of tight morulae-blastocysts (TMBL) and ad-
vanced blastocysts (fully developed blastocysts, ex-
panded blastocysts and hatched blastocysts) was eval-
uated at day 7 of culture.
3. Statistical analysis
The differences among periods in all the parameters
considered, both as numbers and percentages (ex-
pressed in mean ⫾ SEM) were analyzed by one way
ANOVA by using PASW Statistic 18.0, 2009 [15].
Differences among means were compared by Duncan
test.
4. Results
The results regarding the follicular population, the
number of oocyte recovered, as well as the recovery
rate are shown in Table 1.
The average number of total follicles aspirated did
not vary among seasons. Furthermore, both the number
and the percentage of follicles of different sizes were
similar in the three periods. Likewise, the average num-
ber of total COCs recovered per animal per session, as
well as the recovery rate (i.e., the percentage of total
oocytes out of the total number of follicles) did not
show any variation among seasons.
With regard to the incidence of the different mor-
phological oocytes categories (Table 2), although sea-
son did not considerably affect the percentage of good
quality oocytes (Grades A and B), it affected the rate of
degenerated and abnormally expanded oocytes. In fact,
the mean number of Grade D oocytes increased (P ⬍
0.05) during autumn compared to both the mid-winter
(transitional period) and spring–summer (Table 2). Fur-
thermore, both the mean number and the percentage of
Spring-summer Autumn
Mean⫾SEM Mean⫾SEM
4.68 ⫾ 0.22 4.24 ⫾ 0.20
0.63 ⫾ 0.08 (14.30 ⫾ 2.00) 0.54 ⫾ 0.08 (16.86. ⫾ 2.77)
1.21 ⫾ 0.12 (26.10 ⫾ 2.45) 1.25 ⫾ 0.11 (30.03 ⫾ 3.28)
2.84 ⫾ 0.22 (59.61 ⫾ 3.00) 2.45 ⫾ 0.21 (53.11 ⫾ 3.18)
2.24 ⫾ 0.11 2.23 ⫾ 0.18
49.25 ⫾ 3.54 57.43 ⫾ 5.00
 S. Di Francesco et al. / Theriogenology 77 (2012) 148 –154
Table 2
Distribution of different oocyte morphological categories among different seasons.
Mid-winter
N. (%) Mean⫾SEM
Total oocytes 2.3 ⫾ 0.1
Grade A⫹B COCs 0.8 ⫾ 0.1 (34.3 ⫾ 4.1)
Grade A 0.3 ⫾ 0.0 (11.9 ⫾ 1.9)
Grade B 0.5 ⫾ 0.1 (22.5 ⫾ 3.4)
Grade C 0.8 ⫾ 0.1 (33.2 ⫾ 3.5)
Grade D 0.2 ⫾ 0.0a (11.5 ⫾ 1.6)
Grade E 0.1 ⫾ 0.0 (3.4 ⫾ 1.2)
Abnormally expanded 0.1 ⫾ 0.0a (2.3 ⫾ 1.1)a
Naked 0.4 ⫾ 0.0 (15.2 ⫾ 1.4)
a,b
Values with different superscripts within rows are different; P ⬍ 0.05.
the abnormally expanded oocytes increased (P ⬍ 0.05)
during autumn compared to the other periods (Table 2).
Interestingly, while oocyte categories were not much
affected, the oocytes recovered during autumn showed a
significant improvement of the developmental compe-
tence compared to both spring–summer and the transi-
tional period. In fact, although the percentage of cleavage
did not vary significantly over the three seasons, it is
worth reporting that it was higher during autumn (65.6 ⫾
3.2) compared with the percentages recorded during the
transitional period (52.7 ⫾ 3.7) and spring–summer
(59.8 ⫾ 5.5). More interestingly, the percentages of
TMBL increased (P ⬍ 0.01) during autumn (30.9 ⫾ 3.5)
compared with mid-winter (10.3 ⫾ 1.6) and spring–sum-
mer (13.3 ⫾ 2.3), as shown in Figure 1. Similarly, the
percentages of advanced BL differed over the three sea-
sons, being higher (P ⬍ 0.01) during autumn than during
mid-winter and spring–summer (16.3 ⫾ 3.0, 4.7 ⫾ 1.3,
and 4.2 ⫾ 1.5, respectively, Fig. 1).
Interestingly, both the percentages of TMBL and ad-
vanced BL calculated out of the cleaved oocytes were
much higher (P ⬍ 0.001) during autumn (46.7 ⫾ 4.5 and
23.2 ⫾ 3.7, respectively) than during mid-winter (19.1 ⫾
2.8 and 8.6 ⫾ 2.2, respectively) and spring–summer
(22.0 ⫾ 3.8 and 6.4 ⫾ 2.2, respectively) periods.
Finally, both the percentages of TMBL and ad-
vanced BL calculated out of the Grade A⫹B COCs,
i.e., the oocytes that are suitable for IVEP, were higher
(P ⬍ 0.01) during autumn (138.5 ⫾ 35.4 and 111.8 ⫾
30.5, respectively) than during mid-winter (57.2 ⫾ 14.0
and 45.5 ⫾ 9.5, respectively) and spring–summer
(50.8 ⫾ 9.7 and 41.9 ⫾ 9.7, respectively) periods.
5. Discussion
The present study examined whether season could
influence the follicular and oocyte population and,
151
Spring-summer Autumn
Mean⫾SEM Mean⫾SEM
2.2 ⫾ 0.1 2.4 ⫾ 0.1
0.6 ⫾ 0.1 (28.2 ⫾ 4.0) 0.6 ⫾ 0.1 (25.1 ⫾ 4.1)
0.2 ⫾ 0.0 (7.4 ⫾ 1.7) 0.2 ⫾ 0.1 (7.9 ⫾ 2.1)
0.4 ⫾ 0.1 (20.7 ⫾ 3.8) 0.4 ⫾ 0.1 (18.3 ⫾ 3.1)
0.7 ⫾ 0.1 (32.1 ⫾ 3.6) 0.7 ⫾ 0.1 (31.9 ⫾ 4.0)
0.3 ⫾ 0.1a (15.0 ⫾ 3.0) 0.4 ⫾ 0.1b (15.8 ⫾ 2.4)
0.1 ⫾ 0.0 (3.9 ⫾ 1.2) 0.1 ⫾ 0.0 (3.6 ⫾ 1.2)
0.1 ⫾ 0.0a (1.9 ⫾ 0.6)a 0.1 ⫾ 0.0b (6.1 ⫾ 1.5)b
0.5 ⫾ 0.1 (19.1 ⫾ 2.5) 0.4 ⫾ 0.1 (16.3 ⫾ 2.7)
more importantly, the oocyte developmental compe-
tence, and hence the overall efficiency of OPU in Med-
iterranean Italian buffalo. The results of this study dem-
onstrated an overwhelming seasonal effect, with a
significant improvement of the OPU-IVEP efficiency,
in terms of the final embryo output, during autumn, i.e.,
when the daily light hours decrease compared with both
mid-winter and spring–summer.
A first result of this study is that season did not
affect the follicular population in buffalo submitted to
OPU: in fact, the average number of total follicles, as
well as the incidence of follicles of different size (i.e.,
large, medium, and small) did not vary among the three
seasonal periods we considered. This result is in con-
trast with another study performed in Italy [16] in
which it was observed that both the number of follicles
and that of oocytes was higher in autumn–winter than
in spring–summer in buffalo that had undergone OPU
[16]. However, it is worth pointing out that the earlier
study was performed on the same animals for many
sessions and this decline could be explained by a func-
tional exhaustion of the ovary, resulting from repeated
punctures.
On the other hand, our results in terms of number of
follicles are in agreement with those reported in deep
anoestrous buffalo at the same latitude during 4 mo-
OPU carried out between autumn and the beginning of
the transitional period [7]. Furthermore, the average
number of follicles we observed was similar to that
reported by other authors in Murrah buffalo with re-
productive problems [17], cyclic Murrah buffalo [18]
and river buffalo [19].
The average number of oocytes recovered per period
was also not affected by season. It is worth remarking
that the number of total oocytes collected was low
(2.2–2.4), confirming that this is the major limitation of
the OPU technology in this species. In fact, the majority
152 S. Di Francesco et al. / Theriogenology 77 (2012) 148 –154
Fig. 1. Percentages of tight morulae-blastocysts (TMBL) and advanced blastocysts (adv BL) calculated out of total COCs, in different seasons in
relation to daylight hours.
of the authors reported similar or lower oocyte numbers
[18,20].
In the present study the incidence of good quality
oocytes (Grade A ⫹ B COCs) was also not affected by
season. Interestingly, among all the oocyte morpholog-
ical categories, only the degenerated and abnormally
expanded oocytes were affected by season, being un-
expectedly higher during autumn than during the other
two periods. A similar increase of degenerated oocytes
was found in autumn in a recent study performed by our
group to screen the oocyte population in ovaries col-
lected at the slaughterhouse in different seasons [21].
Interestingly, an increased incidence of expanded
oocytes was also recorded during the last period of a 9
mo-OPU trial [22] that coincided with autumn months
(September–December). It is worth specifying that in
the previous work, however, repeated punctures were
carried out for a long time on the same donors.
The intriguing increase of abnormally expanded
oocytes during autumn requires further investigations
because we do not currently know the biological mean-
ing of these oocytes. It is worth noting that oocytes of
this category are different from those we classified as
expanded because they show obvious signs of degen-
eration. Whether they are expanded oocytes that under-
went degeneration or they are oocytes in which the
cumulus expansion process was altered we still do not
know.
However, the increased incidence of both degener-
ated and abnormally expanded oocytes was unexpected
because OPU, by resetting the follicular population
every 3– 4 d, should avoid the dominance occurrence.
This can be related to the enhancement of buffalo
reproductive and cyclic activity during the months
characterized by decreasing light, which may result in
accelerated follicular turnover in autumn. Therefore,
we speculate that during autumn the oocytes grow at a
faster rate than in the other seasons. However, in con-
trast to this hypothesis, we did not observe any increase
in the incidence of medium and large follicles in au-
tumn compared with the other periods in the present
study.
The most interesting results of this study were ob-
tained by comparing the oocyte developmental compe-
tence in relation to season. The different parameters
considered for assessing IVEP efficiency were im-
proved in autumn. Although cleavage rate was not
statistically different among periods, the higher values
were recorded in autumn. More importantly, both the
percentages of the tight morulae-blastocyst (TMBL)
and of the more upgraded embryos (advanced BL) were
significantly higher in autumn compared to the other
two periods of the year (spring–summer and mid-win-
ter): this would represent a further confirmation of
buffalo sensitivity to photoperiod [23]. These results
suggest that the beginning of decreasing daylight has
actually improved the developmental competence of
the oocytes recovered, despite of their apparent mor-
phology.
Interestingly, the greater production of blastocysts
recorded during autumn is not a direct result of a higher
fertilization rate. In fact, eliminating the differences in
cleavage, by calculating the blastocyst rate on cleaved
oocytes, a superior developmental competence was still
recorded in autumn.
Furthermore, it is worth noting that in this study we
processed all the oocytes recovered because a strict
selection of the gametes would have resulted in a fur-
 S. Di Francesco et al. / Theriogenology 77 (2012) 148 –154
ther reduction of the germinal material. In fact, one of
the targets was to assess the feasibility of OPU in the
field that is strongly linked to the final embryo output.
Interestingly, when we calculated the percentage of
embryos on Grade A ⫹ B COCs, that are the only two
categories considered suitable for IVEP, the value in
autumn exceeded 100% whereas it was around 50% in
the other two periods. This result reflects well on the
efficiency, indicating that the oocyte developmental
competence improved regardless of the morphological
aspect. It follows that the assessment of oocyte quality,
made by observing just the morphology, cannot be
viewed as highly reliable for predicting competence.
This suggests that further investigation is required to
find non-invasive molecular biomarkers, to predict the
capacity of the oocytes to undergo normal embryo
development.
The overall results of the present study are different
from those obtained in a recent trial carried out in India
on local breed river buffalo [20]. These authors re-
ported a seasonal effect on IVEP efficiency that was
mainly due to the reduction of follicular population. In
fact, the oocyte developmental competence, indicated
by blastocyst production rate, was not affected by sea-
son, although the numbers of follicles observed and
punctured, oocytes recovered, blastocysts per animal
per session decreased during the unfavorable season.
This suggests that, in addition to photoperiod, other
factors also contribute to impair reproductive efficiency
at different latitudes. Apart from differences in meteo-
rological parameters, it is worth noting that in our
country animals are under constant nutrition throughout
the year.
The results of this trial are also in agreement with a
previous work carried out using abattoir-derived ova-
ries to evaluate the effect of season at our latitudes [21]
with few differences that are worth highlighting. When
the oocyte source was abattoir-derived ovaries, a lower
incidence of small oocytes was also observed in both
autumn and winter compared with spring, whereas no
variation was recorded with OPU-derived oocytes in
the present study. Furthermore, in contrast to this study,
no increase of abnormally expanded oocytes was found
with abattoir-derived oocytes during autumn. However,
the variation in these oocyte categories did not affect at
all the IVEP efficiency.
The developmental competence in both studies im-
proved in autumn compared to spring, as demonstrated
by higher blastocyst yields. More interestingly, in this
study an evident difference was also shown between
embryo yields recorded in autumn vs. mid-winter.
153
This is very important because during the transi-
tional period a higher incidence of embryonic mortality
is observed in buffalo after AI [2]. Furthermore, we
demonstrated that during autumn months pregnancy
rate at 45 d is significantly enhanced compared to the
transitional period, as a result of the reduced incidence
of embryonic mortality [9]. Therefore, the lower em-
bryo yields recorded in mid-winter compared with au-
tumn, suggest that oocyte competence could play a role
in the AI failures observed when daylight hours start to
increase. In order to better elucidate this aspect, a fur-
ther trial is ongoing to investigate the embryo viability
and hence the ability to sustain development to term
after transferring the embryos obtained in different pe-
riods of the year.
In conclusion, it was demonstrated that season af-
fects oocyte developmental competence in buffalo bred
in temperate climate, as indicated by higher embryo
yields recorded in autumn compared with mid-winter
and spring–summer. These results strongly suggest re-
stricting the gametes collection during autumn when
planning OPU trials at our latitudes, in order to save
resources and make the benefit/costs ratio more favor-
able.
Acknowledgments
The work was supported by Programma di Ricerca
scientifica di Rilevante Interesse Nazionale 2008, Strat-
egies aimed to improve the efficiency of reproductive
biotechnologies in buffalo species.
References
[1] Zicarelli L. Anaestro e induzione dell’estro in bufale acicliche.
Agricoltura e Ricerca 1994;153:55– 81.
[2] Campanile G, Baruselli PS, Neglia G, Vecchio D, Gasparrini B,
Gimenes LU, Zicarelli L, D’Occhio MJ. Ovarian function in the
buffalo and implications for embryo development and assisted
reproduction. Anim Reprod Sci 2010;121:1–11.
[3] Gasparrini B. In vitro embryo production in buffalo species:
state of the art. Theriogenology 2002;57:237–56.
[4] Boni R, Di Palo R, Barbieri V, Zicarelli L. Ovum pick-up in deep
anestrus buffaloes. Proc IV World Buffalo Congress 1994;3:480–2.
[5] Gasparrini B. Test-tube fertilization in buffalo. Pre-Congress
Courses in Biotechnology of the Buffalo Reproduction. Com-
parison with the Cow. Proceedings of the 9th World Buffalo
Congress Buenos Aires, Revista Veterinaria 2010, pp. 80 –98.
[6] Zicarelli L. Superovulatory response in buffaloes bred in Italy.
Third Course on Biotechnology of Reproduction in buffaloes
proceedings Caserta, Italy 1997, pp. 167–188.
[7] Boni R, Roviello S, Zicarelli L. Repeated Ovum Pick-up in
Italian Mediterranean buffalo cows. Theriogenology 1996;46:
899 –909.
154 S. Di Francesco et al. / Theriogenology 77 (2012) 148 –154
[8] Neglia G, Gasparrini B, Caracciolo di Brienza V, Di Palo R,
Campanile G, Presicce GA, Zicarelli L. Bovine and buffalo in
vitro embryo production using oocytes derived from abattoir
ovaries or collected by transvaginal follicle aspiration. Theriog-
enology 2003;59:1123–30.
[9] Neglia G, Vecchio D, Di Palo R, Rossi P, Di Russo C, Campanile G.
Embryonic Mortality in Artificially Inseminated Buffaloes during the
Breeding Season. Proc of the 9th World Buffalo Congress, Buenos
Aires. Revista Veterinaria 2010, pp. 887–888.
[10] Gasparrini B, Neglia G, Di Palo R, Campanile G, Zicarelli L.
Effect of cysteamine during in vitro maturation on buffalo
embryo development. Theriogenology 2000;54:1537– 42.
[11] Caracciolo di Brienza V, Neglia G, Masola N, Gasparrini B, Di
Palo R, Campanile G. In vitro embryo production in chemically
defined media. Proc. 1° Congresso Nazionale sull’Allevamento
del Bufalo 3⫺5 ottobre, Eboli (SA), Italy. 2001, pp 341–344.
[12] Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser
ES, Eyestone WH, First NL. Bovine in vitro fertilization with
frozen-thawed semen. Theriogenology 1986;25:591– 600.
[13] Tervit HR, Whittingham DG, Rowson LE. Successful culture in
vitro of sheep and cattle ova. J Reprod Fertil 1972;30:493–7.
[14] Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced Rates of
Cleavage and Development for Sheep Zygotes Cultured to the
Blastocyst Stage In vitro in the Absence of Serum and Somatic
Cells: Amino acids, Vitamins, and Culturing Embryos in Groups
Stimulate Development. Biol Reprod 1994;50:390 – 400.
[15] PASW Statistic 18.0. 2009.User guide. SPSS Inc., Chicago, IL.
[16] Di Palo R, Neglia G, Campanile G, Presicce GA, Spadetta M,
Caracciolo di Brienza V, Gasparrini B, Zicarelli L. Seasonal
effect on follicle production and oocyte recovery by ovum
pick-up in the Mediterranean Italian Buffalo (Bubalus bubalis).
Theriogenology 2001;55:404.
[17] Manik RS, Chauhan MS, Singla SK, Palta P. Transvaginal
ultrasound guided aspiration of follicles from Indian buffaloes
with reproductive problems. Vet Rec 2002;150:22–24.
[18] Gupta V, Manik RS, Chauhan MS, Singla SK, Akshey YS,
Palta P. Repeated ultrasound-guided transvaginal oocyte re-
trieval from cyclic Murrah buffaloes (Bubalus bubalis): oocyte
recovery and quality. Anim Reprod Sci 2006;91:89 –96.
[19] Manjunatha BM, Gupta PSP, Ravindra J.P, Devaraj M, Nandi S.
In vitro embryo development and blastocyst hatching rates fol-
lowing vitrification of river buffalo embryos produced from
oocytes recovered from slaughterhouse ovaries or live animals
by ovum pick-up. Anim Reprod Sci 2008;104:419 –26.
[20] Manjunatha BM, Ravindra JP, Gupta PS, Devaraj M, Nandi S.
Effect of breeding season on in vivo oocyte recovery and em-
bryo production in non-descriptive Indian river buffaloes (Bub-
alus bubalis). Anim Reprod Sci 2009;111:376 – 83.
[21] Di Francesco S, Boccia L, Campanile G, Di Palo R, Vecchio D,
Neglia G, Zicarelli L, Gasparrini B. The effect of season on
oocyte quality and developmental competence in Italian Medi-
terranean buffaloes (Bubalus bubalis). Anim Reprod Sci 2011;
123:48 –53.
[22] Neglia G, Gasparrini B, Vecchio D, Boccia L, Varricchio E, Di
Palo R, Zicarelli L, Campanile G. Long term effect of Ovum
Pick-up in buffalo species. Anim Reprod Sci 2011;123:180 –
186.
[23] Zicarelli L. Reproductive seasonality in buffalo. In: Proceedings
of the Third Course on Biotechnology of Reproduction in Buf-
faloes (Issue II). 1997, pp. 29 –52.