| Summary: | The general aim of this thesis was to optimise the current methodologies of
oocyte cryopreservation in order to obtain live offspring from cryopreserved
rabbit oocytes.
In chapter 1, meiotic spindle configuration, cortical granules (CGs) distribution
and oocyte developmental competence were evaluated after
cryopreservation with the current slow-freezing and vitrification procedures. The
meiotic spindle organisation was dramatically impaired regardless of the
method used. Nevertheless, altered CG distribution is more evident in vitrified
oocytes than in slow-frozen ones and the developmental rate to blastocyst
stage after parthenogenetic activation was only obtained using slow-freezing
method. From this chapter it may be concluded that both methodologies
equally affect oocyte structure. However, slow-freezing method seems to be
the recommended option for this species as a consequence of the sensitivity to
high levels of cryoprotectants in this species.
The aim of the following two chapters was the optimisation of cryopreservation
procedures using different strategies to modify the oocytes in order to make
them more cryoresistant.
In chapter 2, Taxol and Cytochalasin B were employed to stabilise the
cytoskeleton system during vitrification. The effect of these two molecules on
the meiotic spindle and chromosome configuration and development to
blastocyst stage after parthenogenesis activation were also evaluated. There
were no significant differences in the structural configuration between vitrified
groups. Regarding cleavage and blastocyst developmental rate, no statistical differences were found between vitrified-non-treated and Taxol-treated
oocytes, but no oocytes treated with Cytochalasin B reached this stage.
Therefore, structural configuration and blastocyst development were not
improved by this pre-treatment. Moreover, Cytochalasin B pre-treatment seems
to cause a deleterious effect on developmental ability to blastocyst stage of
these oocytes.
In chapter 3, oocytes were incubated with cholesterol-loaded methyl-ß-
cyclodextrin (CLC) to increase the membrane fluidity and stability and improve
their developmental ability after parthenogenetic activation or
intracytoplasmic sperm injection (ICSI). Cholesterol incorporation and its
presence after cryopreservation were evaluated using confocal microscopy.
Results showed that cholesterol was incorporated into the oocyte and
remained, albeit in a lesser amount after cryopreservation procedures.
However, no improvements on developmental competence were obtained
after parthenogenetic activation or intracytoplasmic sperm injection.
In the last three chapters of this thesis, the main objective was to develop a
reliable technique which would allow us to obtain live offspring from
cryopreserved oocytes. For that purpose, in vivo fertilisation using intraoviductal
oocyte transfer assisted by laparoscopy was considered a good alternative to
bypass the inadequacy of conventional in vitro fertilisation in rabbit.
In chapter 4, two recipient models (ovariectomised or oviduct ligated
immediately after transfer) were used to compare the ability of fresh oocytes to
fertilise in vivo. This first work showed that embryo recovery rates in all
transferred groups decreased significantly, but ligated oviduct recipients provided significantly higher results compared to ovariectomised ones. For that
reason, in the second experiment the ligated oviduct recipient model was used
to generate live births. Results obtained in this chapter suggested that it was
possible to obtain offspring from cryopreserved oocytes using this technique,
but this kind of animal models compromised the use of the reproductive tract in
a high percentage of females.
For that reason, chapter 5 was focused on the development of another type of
animal model as an alternative. First, the ability of cyanoacrylate tissue
adhesive to block the oviducts before the ovulation would take place was
evaluated. Then, in vivo fertilisation ability of fresh transferred oocytes after
blocking the oviduct with the adhesive was also assessed. Finally, slow frozen
oocytes were transferred to generate live birth. Results showed that
cyanoacrylate tissue adhesive was effective in blocking the oviduct, as no
embryos were recovered in the blocked oviduct six days after artificial
insemination (AI). Moreover, this method could fertilise fresh and also slowfrozen
oocytes with a higher live birth rate than the previous recipient models.
This study showed that successful production of live offspring using slow-frozen
oocytes in combination with in vivo fertilisation was possible, which suggested
that in vivo environment could help improve the results of oocyte
cryopreservation.
Thus, this method was employed in the last chapter of this thesis to generate
live offspring from vitrified rabbit oocytes for the first time. Results obtained
revealed that there were no differences in the rate of birth between vitrified
and slow-frozen transferred oocytes. Nevertheless, based on the results with fresh oocytes, further experiments are still needed if the efficiency of
cryopreservation procedures are to be improved. === Jiménez Trigos, ME. (2014). Generation of offspring from cryopreserved rabbit (Oryctolagus cuniculus) oocytes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37977 === TESIS
|
|---|
