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.
embryo development. It has been demonstrated in other
species [43] that the GSH reservoir formed during IVM
is the only source of reducing power for the embryos
before genomic activation occurs. It has been previously
demonstrated that cysteamine supplementation during
IVM improves blastocyst yield in buffalo [51], by
increasing intracytoplasmic glutathione concentration [52],
without nevertheless affecting cleavage rate.
Subsequently, the addition of cystine, in the presence of
cysteamine, to the IVM medium [48] has been proven to
increase the proportion of oocytes showing normal
synchronous pronuclei post fertilization (81%), cleavage
rate (78%) and blastocyst yield (30%), via a further
increase of the GSH reservoir of the oocytes. Improved
blastocyst development has also been recorded by
enriching the IVM medium with other antioxidant factors,
such as taurine and melatonin [64].
It has been recently demonstrated [92] that
incorporation of mitogenic lectin in the IVM medium
improves maturation of buffalo oocytes, as indicated by
increased cumulus expansion and cleavage rate following
IVF, as well as by the up regulation of the transcripts
levels of genes involved in different physiological activities
like gap junction and cell communication protein (Cx43),
cumulus-expansion enabling factor and cell cycle protein
(GDF-9), basic growth factor (FGF-4) and cell membrane
protein (Fibronectin).
Among factors affecting mammalian embryo
development in vitro, the duration of IVM plays a critical
role, since an inappropriate timing of maturation results
in abnormal chromatin [37], oocyte aging [57] and
reduced development [73]. We have investigated both
the kinetics of oocyte maturation and the influence of the
duration of IVM on subsequent embryo development. In
this study the attainment of the MII stage commenced
after 18-19 h maturation and the majority of oocytes
completed nuclear maturation between 20 and 24 h;
furthermore, it was demonstrated that the duration of
IVM affects buffalo oocyte developmental competence,
with a progressive decrease of fertilization capability and
embryo development as the IVM duration increases from
18 to 30 h [49]. Therefore, the optimal time for IVF in
buffalo appears to be at 18 h post-IVM or, in any case,
not later than 24 h; in fact, delaying IVF over 24 h has
resulted in a significant deterioration of oocyte
developmental competence that could be predicted by
the poor morphological appearance of oocytes matured
for prolonged periods. An earlier aging of buffalo oocytes
had been previously hypothesized based on the anticipated
accomplishment of maturation, together with the
increased incidence of degenerated oocytes at increasing
times post-IVM [88]. The importance of oocyte aging in
this species is also confirmed by activation studies that
showed, in contrast to most other species, a deterioration
of post-parthenogenetic embryo development at
increasing times post-maturation [46]. This aspect is
important to be considered when OPU is carried out
because usually, to improve cost efficiency, all oocytes
batches collected the previous OPU day are fertilized in
one session.
IV. IN VITRO FERTILIZATION (IVF)
Fertilization has often been considered the most
critical step of the IVEP procedures in buffalo, as cleavage
rates lower than those obtained in other domestic species
have been widely reported [41,47,86]. In our earlier
studies, despite similar maturation rates (87% vs 94%
respectively in buffalo vs cattle) significantly lower
cleavage rates (65% vs 84%) were observed [86]. The
overall lower IVEP efficiency recorded in buffalo
compared to cattle (26 vs 34%, respectively) was mainly
related to the poor cleavage rate; in fact similar blastocyst
yields were obtained in buffalo and cattle (40%) when
the percentages were calculated in relation to the zygotes. N
Many factors may affect the in vitro fertilization
efficiency, such as the adequate in vitro environment for
gametes survival, the sperm viability and capability, the
appropriate time of insemination, the duration of gametes
co-incubation, the presence of cumulus cells and also the
acquisition of the oocyte developmental competence
during the complex process of cytoplasmic maturation.
In fact, it is likely that the fertilization failure is related to
inadequacies of the IVF system, but a previous
inappropriate maturation of the egg should not be ruled
out.
The media commonly utilized for buffalo IVF
are Tyrode’s modified medium (TALP) and Brackett
Oliphant (BO), supplemented by sperm motility inducing
factors, such as combined hypotaurine-penicillamine or
caffeine. However, significantly higher cleavage and
blastocysts rates have been obtained, in a direct
comparison trial, by using TALP medium, supplemented
with heparin, hypotaurine and penicillamine [47]. High
sperm motility is required to accomplish fertilization, and
this aspect is particularly important when frozen-thawed
sperm is employed. A preliminary selection of motile
s323