Although genetic manipulation of farm animals is of great interest for animal production and the pharmaceutical industry, its efficiency remains far from satisfactory. Pronuclear injection, which is the most widely used technique for such modification, mainly in mice, remains limited for this species. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), days 9 and 12 of embryo culture, respectively. Exogenous DNA-positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer.

With the exception of the domestic cat, all felid species (Felidae) are currently threatened with extinction in their natural habitat. To develop effective and optimal wild cat conservation programmes with assisted reproductive technology (ART) it is necessary to combine advances from different disciplines of science, starting from the biology of the species, through research into the population and habitat, assisted reproductive technologies, establishment of gene banks, developing bioinformatic systems, and ending with biodiversity and endangered species management. In the last few years knowledge of felid reproduction has expanded considerably thanks to comparative studies utilising the domestic cat as a research model for endangered wild cats. Basic reproductive techniques utilised in both domestic cat breeding and rescuing wild felid populations that are threatened with extinction include semen collection and cryopreservation, artificial insemination, oocyte collection, in vitro maturation, in vitro fertilisation, somatic cloning, and embryo transfer. The main directions in which assisted reproductive technologies are being developed in wild cat conservation implementations and the contribution of Polish research centres in advancing these methods are presented.

Methods of increasing the efficiency of semen fertilizing capacity in vitro with application of hormonal and biophysical methods of influence, as well as the determination of metabolic criterion of spermium viability were studied in the conditions of the republic of Belarus. Research object was frozen-deiced sperm of cattle. Research results showed that entering of 50 mkg/ml of prostaglandin into capacitation media increased the breaking level on 2,3-3,1%, but at the same time there was the decreasing of embryo output at the pre-implantation stages. Increasing of estrophan concentration in media for capacitation up to 100 mkg/ml, and its addition into media for fertilization made it possible to increase the embryo output at the stage of morula-blastocyte up to 1,6-18,5%. Application of 8mg/ml of caffeine as a capacitation agent for the preparation of cattle sperm for fertilization in vitro made it possible to obtain embryo output of 16,7% with breaking level of 25,9%. Influence of directed polarized light on semen after its maturation was more efficient in comparison with the influence on it just after swim-up procedure; the output of pre-implantation embryos was 16,7% against 12,9%. Application of laser radiation made it possible to get 14,7 % of morula-blastocytes. Intensity of sperm breath (0,41-0,63 tg), intensity of lipid peroxidation (0,88-0,97 conditional units /10E6 cells), intracellular content of adenosine triphosphate (0,97-1,60 Nm/10E6 cells) and membrane potential (33-35 uV) in the conditions of matured in vitro oocyte made it possible to get 16,4-17,3% of pre-implantation embryos with the breaking level of 39,8-42,3%