Results of the project

Consortium partner: Szent István University of Gödöllő

1. Improving the effectiveness of sperm freezing in domestic fowl and goose using non permeable osmoprotectants such as sucrose, trehalose and betaine, as well as ATP and cholesterol:

In the subtask related to the development of sperm freezing in poultry neither the various non-permeable osmoprotectants nor the different applications of ATP supplementation could improve significantly the survival rates of spermatozoa of the two mentioned species, although the sucrose and trehalose combinations seemed to be slightly more efficient. It needs further investigations.

2. Investigations on the correlation between the inseminated sperm concentration and the early embryo mortality in domestic hen and goose as well as guinea fowl:

In the subtask related to the correlation between the inseminated sperm concentration and the early embryo mortality we could not improve the fertility with extremely high sperm doses, nor increased the early embryo death in either poultry species examined. However, in the case of very low sperm concentrations the mortalities of the embryos being in the very early stage significantly increased. It was demonstrated that in the case of domestic goose and guinea fowl the demand of spermatozoa for satisfying fertility rates is around 10 times lower than that of the domestic fowl.

3. Characterization of the samples for in vitro gene bank:

Poultry populations of our gene bank were investigated based on microsatellite markers: in chicken breeds 11 specific markers were found from 17, in turkey 3 from 13, in goose populations 5 from 15, in duck breeds 8 from 14, finally in guinea fowl 2 unique markers from 11 were detected which are suitable for population identification.
Specific marker setups were established for the Hungarian native poultry breeds, which can help us to decide how many and what samples to preserve in the gene bank, so we could improve efficiency of in vitro gene conservation. Furthermore, these samples were successfully genotyped with the already optimized marker setups.

4. Storage possibilities of different embryonic cell types and examination of their tolerance against deep-freezing process:

The deep-freezing protocol of the early embryonic cells in case of old Hungarian chicken breeds; the Transylvanian Naked Neck chicken breeds; the Copper and Hungarian Bronze Turkey breeds; the Hungarian Frizzle Feathered Goose and the Hungarian landrace Guinea fowl was elaborated. We concluded there is no significant difference in cell surviving between either the breeds of chicken or the breeds of turkey. If the difference between the species was tested, it is concluded, that the fresh live cells ratio in turkey was significantly lower, than the other two species (p≤0.01). After adding the DMSO cryoprotectant, the geese live cell ratio was significantly higher than the chicken and the turkey (p≤0.01). However, examining the survival in the straws or in ampoules, there were no significant differences between the three species. Furthermore, the long-term storage of chicken primordial germ cells (PGCs) in tissue culture and the deep-freezing protocol were also elaborated. This was proved by immunostaining and transplantation of frozen-thawed PG cells.

5. Optimization of donor/recipient combination of day-old gonadal tissue transfer in chicken:

In this subtask the effective vitrification method of day-old gonadal tissue was elaborated. The frozen/thawed organs adhered, started to grow and formed gametes. The Yellow Hungarian/White Leghorn donor/recipient pair can be applied in the gene conservation. Based on genetic distance preservation of further indigenous breeds becomes possible. The possibility of immunological incompatibility of pairs was excluded. Successful transfers were made using frozen / thawed gonadal tissues. It was proved that the grafting of early gonadal tissues can be made under simply circumstances with high efficiency.
6. Genetic and spermatology studies on the improvement of effectiveness of rabbit sperm cryopreservation:
In the task aiming the deep freezing of the sperm of Hungarian giant rabbit the in-vitro vitality and suspected fertilizing capacity studies (motility, CASA, IVF, Kovács-Foote staining) has proved that the Basenfelder method modified by our research team is highly suitable for maintaining frozen sperm bank for long periods of time. After appropriate conditioning of the animals the sperm collection can be easily performed and the deep freezing of sperm can be successfully done using the methodological description attached to the project report. In terms of pathogen free freezing based on our results we suggest to use 1.5 % ESL milk instead of egg based diluent. The lyophilization of sperm was carried out and sperm injection with lyophilized sperm was also tested. For this reason an in vitro fertilization method was also developed.