Stress preconditioning of semen before cryopreservation improves fertility and increases the number of offspring born: a prospective randomised study using a porcine model.

The aim of the present study was to investigate the effect of applying sublethal stress treatment at room temperature, before cryopreservation (hydrostatic pressure (HP): 40MPa, 80min) of 34 boar ejaculate samples, on post-thawed motility and sow fertility. Sows (n=102) were randomly allocated into equal groups inseminated with HP-treated or untreated frozen-thawed semen. Sows were inseminated twice, 10h apart, with 6×10(9) spermatozoa per dose without oestrus synchronisation. Rates of non-return of oestrus and pregnancy, and total numbers of piglets and live piglets were significantly higher (P<0.05) in the HP-treated group. There was also a numerical, albeit non-significant (P>0.05), improvement in the farrowing rate in the HP-treated group. Although the number of live piglets per litter decreased approximately 15% in both groups by 42 days after farrowing, but this remained significantly higher in the HP-treated group. Although total and progressive sperm motility were significantly (P<0.001) higher in the HP-treated group, there were no significant differences (P>0.05) in these parameters between pregnant and non-pregnant sows in either group; thus motility can indicate, but not predict, improved fertility. In conclusion, HP treatment, with sperm cryopreservation, increases in vitro sperm motility and improves reproductive performance without adversely affecting the health of the piglets.

Increased stress tolerance of matured pig oocytes after high hydrostatic pressure treatment.

The present paper describes a method which uses high hydrostatic pressure as a pre-treatment to in vitro matured porcine oocytes to improve their survival rates in the subsequent processes including cryopreservation, parthenogenetic activation and embryo culture. In Experiment I oocytes were treated with different pressure impulses in the range of 20-80 MPa (200-800 times greater than atmospheric pressure) for 30-120 min at 24 degrees C. For parthenogenetic activation a single dc of 12.5 kV/cm was used, to test shock tolerance of the treated vs. control oocytes and also compare their developmental competence evaluated with continued in vitro development. The upper limit of pressure tolerance was found in the 40 MPa range. In Experiment II oocytes pre-treated with pressures in the 20-40 MPa range were vitrified with the Cryotop method, and parthenogenetically activated subsequently with combined electric (single dc of 1.25 kV/cm) and chemical treatment after warming. According to our investigations performed with a total of 1980 oocytes and 3-5 replicates, pressure treatment increased cleavage and blastocyst rates after activation and cleavage rates after vitrification followed by activation. Our results indicate that the sublethal pressure treatment may induce specific responses in oocytes increasing their resistance and developmental competence. The mechanism that may lie behind the observations is the sublethal stress-induced post-transcriptional activation of shock proteins in the oocytes. Further investigations are needed to reveal the biophysical and molecular biological background of the findings and to optimize the protocol of pressure pre-treatment in both animal- and human-assisted reproductive technology (ART) to increase the efficiency of in vitro procedures.

Survival of mouse blastocysts after low-temperature preservation under high pressure.

Cryoinjuries are almost inevitable during the freezing of embryos. The present study examines the possibility of using high hydrostatic pressure to reduce substantially the freezing point of the embryo-holding solution, in order to preserve embryos at subzero temperatures, thus avoiding all the disadvantages of freezing. The pressure of 210 MPa lowers the phase transition temperature of water to -21 degrees C. According to the results of this study, embryos can survive in high hydrostatic pressure environment at room temperature; the time embryos spend under pressure without significant loss in their survival could be lengthened by gradual decompression. Pressurisation at 0 degrees C significantly reduced the survival capacity of the embryos; gradual decompression had no beneficial effect on survival at that stage. Based on the findings, the use of the phenomena is not applicable in this form, since pressure and low temperature together proved to be lethal to the embryos in these experiments. The application of hydrostatic pressure in embryo cryopreservation requires more detailed research, although the experience gained in this study can be applied usefully in different circumstances.