Cryopreservation of gametes and embryos is an important part of assisted reproductive
technologies, since it can be used to preserve genetic material for a longer time. The introduction of
vitrification resulted in a promising technique for oocyte and embryo cryopreservation. However, the
developmental competence of the oocytes and embryos is compromised after warming. An overview
of the difficulties encountered in the vitrification process and a description of the different variables
influencing the vitrification process and the cryotolerance of oocytes and embryos is given in Chapter
The general aim of this thesis was to increase the effectiveness of vitrification for oocytes and
embryos in cattle and horses (Chapter 2). To this purpose, we investigated two variables influencing
the vitrification: (1) the presence of cumulus cells surrounding the oocyte during vitrification and
their influence on the vitrification protocol and (2) the effect of maturation in the presence of serum
on the vitrification of oocytes and blastocysts.
In chapter 3, specific attention was given to the role of cumulus cells surrounding mature bovine
oocytes during their vitrification. It is already known that cumulus cells play a fundamental role
during maturation, fertilization and blastocyst development. However, they might decrease the
exchange of water and cryoprotectants (CPAs), which leads to an inadequate protection of the
oocyte during vitrification. Our results showed that the presence of multiple layers of cumulus cells
surrounding the oocytes compromised their survival after vitrification. On the other hand, we
observed that total removal of cumulus cells led to similar survival rates in vitrified denuded oocytes
compared to their fresh counterparts.
In bovine, it is known that removal of cumulus cells decreases the fertilization rates of the oocytes.
Therefore, in our study, we used three different approaches in order to distinguish ‘vitrifiability’ from
‘fertilizability’. Firstly, vitrified denuded oocytes were fertilized in the presence of intact fresh
cumulus complex oocytes (COCs). Secondly, we used corona radiata (CR) oocytes as a compromise
between COCs and denuded oocytes. Finally, vitrified oocytes were activated parthenogenetically.
Our results showed that removal of cumulus cells before vitrification reduced the fertilization in
vitrified denuded oocytes. Interestingly, this situation could be reverted when vitrified denuded
oocytes were fertilized in the presence of fresh COCs. Secondly, we observed that the mere act of
removing only the outer cumulus cells (CR oocytes) was already reducing the fertilization of fresh
oocytes compared to the ones surrounded by multiple layers of cumulus cells. Such an effect was not detected in vitrified CR oocytes, which showed similar fertilization and embryonic development
compared to vitrified COCs. Surprisingly, vitrified CR oocytes showed similar capacity to become
blastocysts compared to fresh COCs after parthenogenetic activation.
Together, these results clearly show that the presence of cumulus cells might reduce the entrance of
CPAs, compromising the vitrification of mature bovine oocytes and therefore it is advisable to
remove some of the external layers of cumulus cells.
If cumulus cells reduce the exchange of CPAs, the use of high concentrations of CPAs could help
speeding up their entry into the cells. Therefore, in Chapter 4, two protocols were evaluated (high
concentrations of CPAs for a short time of exposure and lower concentrations of CPAs for a long time
of exposure) for the vitrification of immature equine oocytes surrounded by some (CR) or multiple
layers of cumulus cells (COCs). In our study, we observed a low maturation competence when COCs
were vitrified in high concentrations of CPAs for a short time of exposure. Fortunately, this situation
could be reverted with the use of CR oocytes or with the exposure of COCs to CPAs for a longer
period of time. These findings support our observation in Chapter 3, where the presence of multiple
layers of cumulus cells surrounding the oocytes prevent the movement of CPAs and water, implying
the need for a longer time of exposure for a correct protection.
Consequently, COCs were discarded from our following studies and only CR oocytes were used to
investigate the effect of the two different protocols on the spindle configuration and the embryonic
development of vitrified immature equine oocytes (Chapter 4). Our data showed that both
vitrification protocols resulted in higher rates of aberrant spindle configuration in vitrified oocytes
compared to fresh ones in the horse. More interestingly, we observed that blastocyst development
only occurred in oocytes vitrified in high concentrations of CPAs for a short time of exposure.
Pregnancies were established after transfer of such blastocysts, and a healthy male foal was born on
May 12, 2017, a wordl’s first. The longer time of exposure to CPAs used in the other protocol might
lead to toxic effects, which impaired the further embryonic development of vitrified oocytes.
These findings strongly indicate that the presence of cumulus cells might be adjusted with other
variables, such as the concentration of CPAs and time of exposure, which are critical for the
development of a successful vitrification protocol.
In Chapter 5, another variable, which influences the vitrification of oocytes and embryos, was
evaluated. It is known that the presence of serum during culture reduces the cryotolerance of
oocytes and blastocysts. In a first part of our study, we investigated the vitrification of bovine oocytes
matured in two conditions: in presence of serum, glutamine and pyruvate or in absence of serum but
with epidermal growth factor (EGF, Chapter 5.1). Our results showed that fertilization is not
influenced by the maturation condition, but it was drastically decreased after vitrification. When
embryonic development was studied in a second experiment, we observed how the presence of
serum reduced the cleavage rate of vitrified oocytes.
Vitrification is considered a stressful process that leads to hemichannels opening in cells. This
produces the loss of different molecules and ions that are important for cell survival. It has been
demonstrated that the inclusion of a mimetic connexin peptide closes these hemichannels, improving
cells cryopreservation. In analogy with the previous study, we further investigated if such an effect
also occurred in vitrified blastocysts resulting from oocytes matured in serum or EGF conditions, and
we included a mimetic peptide in order to improve the vitrification of blastocysts (Chapter 5.2). We
observed that vitrification induces opening of the hemichannels in blastocysts derived from oocytes
matured in the presence of serum. Although opening of hemichannels is related to increased
apoptotic cell rates, we could not observe such an effect when hemichannels were opened.
Interestingly, the inclusion of the mimetic peptide closed hemichannels during vitrification, improving
the embryonic development of vitrified blastocysts matured in the serum condition. On the other
hand, vitrification did not result in hemichannels opening in blastocysts matured in EGF condition.
Apparently, EGF might have an effect on hemichannels by modifying their activity. Additional
experiments are required to confirm this effect and to investigate the opening of hemichannels
under different conditions.
Finally, in chapter 6, the main results of this thesis are summarized and discussed. Research was
focussed on the optimization of the vitrification conditions for bovine mature oocytes, equine
immature oocytes and bovine blastocysts. Our results indisputably demonstrated that the presence
of cumulus cells impaired the vitrification of oocytes in both cattle and horses and that their effect
needs to be addressed with other variables such as the concentration and time of exposure to CPAs.
Therefore, partial removal of the cumulus cells (CR) is advised for the vitrification of bovine mature
oocytes in order to allow penetration of CPAs, while maintaining fertilization potential. In the horse,
blastocyst development was achieved in immature CRs vitrified by short exposure to high
concentrations of CPAs. Finally, vitrification induced opening of hemichannels in bovine blastocysts
matured in the presence of serum, impairing their competence. This could be alleviated by the use of
a mimetic peptide or by replacing serum by EGF during maturation.