2011 (SBTE) 25th Annual Meeting Proceedings - International ...

B. Gasparrini. 2011. Ovum pick-up and in vitro embryo production in buffalo species: an update.................................................................
jjjjjjjjjjj Acta Scientiae Veterinariae. 39(Suppl 1): s317 - s335.
to 50% [96]. Recently, we demonstrated that OPN
improves the efficiency of capacitation in vitro [72] and
that the supplementation of IVF medium with OPN
significantly enhances cleavage rate (71.6 vs 59%) and
blastocyst yields (29.9 vs 17.4%; [34]).
It is known that proteolytic enzymes appear to
have an essential role in multiple phases of mammalian
fertilization. We recently reported that the addition of
plasmin, the active enzyme of the plasminogen activation
system, to heparin-capacitated buffalo sperm increases
the percentage of acrosome reacted spermatozoa and
stimulates motility [115]. Our results suggest that plasmin
may play a role in events surrounding fertilization and
suggest to evaluate in further studies whether the addition
of plasmin during IVF improves the efficiency in buffalo.
A positive effect of cumulus cells at the time of
IVF has been observed in buffalo, as in cattle [118],
demonstrated by the higher cleavage rate and embryo
development obtained with cumulus-enclosed oocytes vs
oocytes that were freed of their cumulus investment [45].
Recently, we demonstrated that co-culture of buffalo
oocytes deprived of their cumulus at the end of IVM
with bovine intact COCs in a 1:1 ratio completely restores
their fertilizing capability and post-fertilization development
[28], suggesting that this approach can be used for
technologies requiring cumulus removal, such as oocyte
vitrification.
Another factor that may affect embryo
development is the duration of gamete co-incubation
during IVF. It has been suggested that prolonged gamete
co-incubation under the conditions of IVF, in which high
concentrations of spermatozoa are incubated in small
volumes of medium, results in the production of high levels
of hydrolytic enzymes [98] and free radicals [1] that
damage the oocytes. It was demonstrated that the optimal
sperm-oocyte co-incubation time for maximizing the
blastocyst yield in buffalo is 16 h [49]. Interestingly, the
lower blastocyst development recorded at the shorter
durations of sperm-oocyte co-incubation tested were
mainly due to the lower cleavage rates, as suggested by
the fact that the oocytes that had cleaved developed
further and as fast as those in the 16 h group. On the
contrary, extending gamete co-incubation to 20 h was
deleterious because, despite similar cleavage rates, the
blastocyst production was reduced. Furthermore,
increasing the sperm-oocyte incubation time to 20 h was
found to be correlated to a higher incidence of polyspermy.
It is worth pointing out that these results were obtained
using semen from a single bull previously tested for IVF.
Subsequently, it was demonstrated that marked
differences in the kinetics of sperm penetration exist
among buffalo bulls and that this parameter is correlated
to the blastocyst rate [99], and, hence, can be a useful
marker to predict the in vitro fertilizing ability of buffalo
bulls. The great variability in the penetration speed
suggests to insert this assessment in the preliminary
screening of bulls before their utilization in IVF programs.
It is worth noting that among the 6 bulls tested the bull
with the fastest penetration rate (64% penetration at 3 h
post-insemination with the maximum rate – 72-77% –
between 6 and 9 h) showed a significant increase in
polispermy as early as at 12 h post-insemination. This
finding strongly suggests to adapt the gametes coincubation
time during IVF in relation to the bull used.
As previously mentioned, the poor cleavage rate
may also be due to the lack of oocyte developmental
competence, normally acquired during the maturation
process. In order to investigate these aspects oocytes
were parthenogenetically activated. A significant
improvement of cleavage (71 % vs 56 %, respectively)
and blastocyst yield (33 vs 23 %, respectively) was
obtained with ethanol-induced activation vs IVF, indirectly
suggesting that buffalo oocytes had acquired the
developmental competence during IVM [46]. It has more
recently reported that activation with different methods N
give significantly higher cleavage and blastocyst rates
compared to IVF, suggesting that the problem has paternal
rather than maternal origin [78].
Nevertheless, it is worth pointing out that, after
many fruitless attempts to increase cleavage rate in this
species, the fertilization efficiency has at last improved,
reaching approximately 80 % of cleavage rate, by
enriching the IVM medium with thiol compounds. This
improvement has been proven to be related to enhanced
intracytoplasmic GSH levels [48]. This interesting finding
would indicate that the poor cleavage rate of this species
so far recorded was in part related to an inappropriate
maturation of the female gamete. It has been, in fact,
suggested that the GSH production is critical for the
acquisition of developmental competence of oocytes at a
cytoplasmic level and that the measurement of GSH at
the end of IVM can be a reliable indicator of the
cytoplasmic maturation [29].
V. IN VITRO CULTURE (IVC)
The development of in vitro culture systems for
buffalo embryos has followed that for other ruminant
s325