The domestic cat (Felis catus) is considered as a practical and valuable model to study assisted reproductive technologies (ART). Techniques that can be considered as ART are artificial insemination, in vitro embryo production consisting of in vitro maturation, in vitro fertilization
or intracytoplasmic sperm injection and in vitro culture, embryo transfer, cryopreservation of sperm, oocytes and embryos which would provide an accessible supply for research, somatic
cell nuclear transfer and sexing of sperm and embryos. These techniques are not only important for basic fundamental research and the improvement of reproductive performances in domestic cats, but also for preserving biodiversity in wild felids. Within the Felidae family, almost 90% of feline species are included in the International Union for Conservation of Nature (IUCN) red list, and due to ongoing habitat destruction, poaching, geographic isolation and depletion of prey, close to 50% of wild felids are listed as vulnerable, endangered or threatened by extinction somewhere in their natural range. The first step was to preserve and protect species living in their natural environments, however, conserving endangered wildlife only in their natural environment is unrealistic, and it is necessary to combine with intensive ART management in zoos and breeding centers. In vitro embryo production (IVP), embryo transfer (ET), artificial insemination (AI) and cryopreservation of
sperm, oocytes and embryos are important ART techniques for optimizing captive breeding programs of felid species. Sperm cryopreservation and AI have already been successfully used
in some felid species, however, technologies aiming at preserving genetics of females have not yet been fully developed. Cryopreservation is a helpful tool to exchange genetics between wild and captive populations, by reducing the need to transport stress-sensitive animals between institutions and by allowing reproduction between different generations in time, even after death of the animal. Since gametes of endangered felids are not readily available for research, the domestic cat is used as a model to develop ART in Felidae as described in Chapter 1.
The general aim of the present thesis was to optimize cryopreservation of feline oocytes and spermatozoa as a model for the cryopreservation of gametes of endangered wild felids, and potentially also as a model for human reproduction. In Chapter 2, the specific aims of the thesis are described.
First, we had to improve oocyte collection and maturation methodssince maturation rates for individual cats were ranging from 0% to 80%. A possible explanation for these highly variable maturation rates is that the ovaries that have been used came from young and old, fat or anorectic, pregnant or pre-pubertal cats, cats in heat or in anoestrus; all these parameters can affect the maturation rate of the obtained oocytes. Since many of the ovaries used for reproductive research are derived from stray cats, their age is often unknown, so we needed to find an age-related parameter which can predict the success of IVM in cats. In Chapter 3,
we hypothesized that AMH could be a possible parameter. We demonstrated for the first time in cats that an increase in age is significantly associated with a decrease in AMH levels and
that there is a significantly lower maturation rates in oocytes derived from cat in the age group between 0 and 3 months of age compared to the maturation rates of oocytes from older cats.
In the domestic cat, the development of embryos after vitrification of immature cat oocytes (at the germinal vesicle stage) has been described and a pregnancy has been established after vitrification of immature oocytes, but developmental competence of the oocytes after
warming still remains low. In order to improve the vitrification outcome, we hypothesized that by closing of connexin 37 and 43 by using Gap26 during vitrification and warming, we could
improve the maturation and developmental capacity of immature cat oocytes. By using Gap26, we prevent that closed hemichannels would open, which could occur under certain stress conditions, potentially also during vitrification and warming. The results from Chapter 4 showed that the addition of Gap26 to vitrification and warming media can improve maturation rates of vitrified immature cat oocytes and the oocytes still have the potential to become a blastocyst after parthenogenetic activation.
For male felids, collection and freezing of semen is useful for artificial insemination, in vitro fertilization, diagnostic purposes, research and in case of endangered wild animals for creation
of gene banks. Cryopreserving semen of wild felids is especially important for preserving valuable genetic material to increase genetic variation and to avoid inbreeding within small
populations. In caracals there are no data on semen collection by urethral catheterization nor cryopreservation of semen. In Chapter 5, we investigated if urethral catheterization is a
possible method to collect semen in a caracal (Caracal caracal) and we examined the resilience of caracal semen to cryopreservation under field conditions. The described results
show for the first time that it is possible to collect caracal semen by urethral catheterization, and that it is possible to cryopreserve caracal semen with a protocol, which was originally designed for domestic cats, in in field conditions which indicates the potential to use the combination of urethral catheterization and cryopreservation to preserve sperm of caracals and possibly also other wild endangered felids in a low cost and animal friendly manner.
In Chapter 6, the general discussion and the conclusions of this thesis are presented.
- We can conclude that we have established a parameter that can predict whether the oocytes of a particular cat will be more likely to mature or not. Age and AMH levels are
related to the maturation capacity of feline oocytes. Unfortunately, this implies that further research with oocytes retrieved from cats in Belgium is going to be a major
challenge when only prepubertal oocytes are available.
- We have also found that connexin 43 was present in cat immature cumulus-oocytecomplexes which made it possible to improve a vitrification protocol for immature cat
oocytes by closing connexin 43 by using Gap26 during vitrification and warming so that maturation and developmental capacity improves. In this research, the domestic cat
can be used as a model for wild felids. This method can be implemented in protocols to vitrify oocytes of wild felids, and so establish a frozen zoo creating a pool of oocytes
of valuable felids threatened with extinction.
- Also male gametes have to be preserved in this frozen zoo giving researchers the opportunity to develop an embryo at any given time and place whenever a suitable recipient queen is available. We have performed a case study which indicated that
urethral catheterization is a possible method to collect semen in a caracal (Caracal caracal) and investigated that caracal semen is resistant to cryopreservation in field conditions.