Mejora de la eficiencia de tecnologías embrionarias porcinas

Laburpena

Objectives: The general objective of this doctoral thesis is the optimization of two porcine embryonic technologies, in vitro embryo production and embryo transfer, through the study and evaluation of different factors that directly affect their efficiency and applicability. For this porpuse, in a first phase we focused on the study of mineral oil (MO) used to cover the drops of culture media in in vitro embryo production systems, which is also applicable to the culture of in vivo produced embryos. In a second phase, we studied factors that affect the efficiency of embryo transfer, including 1) the effectiveness of the surgical and non-surgical transfer of vitrified embryos; 2) the adequacy of a chemically defined medium with a stable pH for vitrification and embryo warming; 3) the development of an embryo storage procedure in liquid state and in the absence of CO2; and 4) the effects of surgical embryo collection and non-surgical embryo transfer on the reproductive future of the donor and recipient sows, respectively. Methods: Already established methods for the in vitro embryo production and vitrification were used. For embryonic conservation in a liquid state, embryos in the morula stage were washed 10 times and immediately preserved at different temperatures and in different media, according to the experimental design, for a period of up to 72 h. After the storage period, embryos were cultured conventionally to evaluate their in vitro development potential or transferred to recipient sows. To collect embryos surgically and for surgical and nonsurgical transfers, techniques developed in our laboratory in previous years have been used. Conclusions: 1.- Despite its pronounced impact on progesterone concentration in the maturation medium, the use of mineral oil overlay during maturation, fertilization and embryo culture does not affect the time-course of maturation or the developmental competence of pig oocytes in vitro. 2.- The use of a peroxidized mineral oil overlay dramatically decreases embryo production outcomes. This decrease is associated with the higher peroxide values of the oil but cannot be explained by the levels of hydrogen peroxide and reactive oxygen species transferred from the oil to the culture media. 3.- The type of oil cover used during maturation, fertilization and embryo culture affects porcine embryo production, which can be due to the differences detected in volatile organic compounds composition and compounds transfer into incubation medium between oils. 4.- Farrowing rate and litter size following a nonsurgical deep uterine embryo transfer procedure increase in function of a larger number of transferred vitrified embryos, with fertility equalizing that obtained with the invasive surgical approach. 5.- Vitrification and warming procedures using a chemically defined, pH-stable medium as a basal medium ensure an excellent in vitro developmental potential and an adequate reproductive performance after surgical embryo transfer of embryos vitrified at the morula and blastocyst stages. 6.- Morulae stored at 37ºC in a semi-defined medium containing bovine serum albumin maintain their in vitro viability and developmental competence for up to 72 h. Additionally, these embryos were able to develop into normal fetuses by day 38 of pregnancy. 7.- Surgical embryo collection procedure compromises the reproductive future of donor sows. By contrast, the nonsurgical deep uterine embryo transfer approach does not affect the reproductive potential of the recipients after reintroduction to the breeding stock of the farm.