Physiological approach for the optimization of the in vitro embryo production system in swine

Zusammenfassung

The objectives of this PhD Thesis are approaching the IVF and CE conditions to those recorded in the sow's reproductive tract for the improvement of the in vitro production of porcine embryos. To this end, four experiments have been carried out. The first two consisted of the modifications in the culture media used during IVF and CE by adding either specific proteins or natural fluids present in the oviduct and uterus of sows. The last two experiments consisted of obtaining in vivo, inside the reproductive organs of the female pig, the reference values for physical-chemical parameters such as oxygen and temperature, and transfer them to in vitro conditions. In the first experiment, porcine oocytes matured in vitro were incubated in a medium with heparin, ezrin, HSP-70-1A or HSP-90?. These same groups were used during IVF since heparin or proteins were added to the IVF medium with the oocytes and spermatozoa. In the control groups, gametes were incubated in a medium without heparin nor proteins. The results showed that heparin and each of the tested proteins hardened the ZP of the porcine oocytes, by increasing its resistance to enzymatic digestion. The combination of heparin with each of the proteins increased such hardening effect, except for HSP-90?, where the the ZP's resistance was reduced. While promising, since the effect of heparin and each of the proteins added into the IVF medium drafted the polyspermy rates, IVF outcome was not improved because of the reduction in the penetration rates. In the second experiment, two sperm selection methods were compared. One of the widely used procedures in swine, the density gradient centrifugation, was compared with a new procedure, which made the sperm swam up in a tube containing culture media with or without porcine oviductal fluid (POF). On the other hand, two IVEP systems were compared. A new system containing POF and porcine uterine fluid (PUF) within the culture media during IVF and EC was compared with a system where those biofluids were not added to the culture media. The results indicated that the use of swim-up as sperm selection procedure for the production of porcine embryos reached a 40% yield, indicating an improvement with regard to the best results obtained in pigs up to date (30-35%). Furthermore, that value increased in a 5% when the reproductive fluids were added to the different culture media, even surpassing the value of the yield given for the bovine species. Likewise, the addition of the fluids to the media improved the quality of the porcine in vitro embryos produced, which achieved a mean number of cells similar to porcine embryos collected in vivo. In the third and fourth experiments, a series of measurements of oxygen and temperature were made in the reproductive tract of sows and gilts at different phases of the estrous cycle, using minimally invasive surgery techniques and new probes. The values obtained were then transferred to IVF and CE procedures, with the purpose of improving the production of porcine embryos in vitro. The third experiment revealed the amount of oxygen within the reproductive organs of sow (7%), being higher in gilts treated with hormones (10%). Two profiles for oxygen measurements were observed: a flat profile, where the oxygen variations were minimal with respect to the average, and a wavy profile, which showed small variations (± 2) with respect to the average. The wavy profile was associated with the uterus. The use of hypoxia conditions (7% oxygen) during IVF followed by EC improved porcine IVEP outcome and the quality of the porcine blastocysts obtained. In the fourth experiment was demonstrated the existence of a temperature gradient between the oviduct (37.0 ºC) and uterus (38.7 ºC) for the first time. Transferring this gradient during IVF and CE improved the production of in vitro embryos in the porcine species through the reduction of the polyspermy rates. In addition, the use of a high temperature (39.5 ºC) during IVF exerted the opposite effect to the use of a low temperature (37.0 ºC), since its use increased the polyspermy rates, and a greater fragmentation of the embryos obtained at high temperature was observed. On the other hand, obtaining the transition temperature for capacitated boar sperm (37.0 ºC), as well as the incubation of these spermatozoa at different temperatures (37.0 ºC, 38.5 ºC and 39.5 ºC), indicated an important role of temperature in the remodelling of the lipids contained in the sperm membrane and its implication in IVF.