Advances in swine embryo production and transfer

Laburpena

OBJECTIVES: The objective of the Thesis consists of the improvement of two biotechnologies in swine reproduction, in vitro embryo production (IVP) and embryo transfer (ET). The improvement of IVP has focused on the modification of current protocols, trying to reduce the problem of polyspermy during in vitro fertilization by modifying sperm cryopreservation protocols, the improvement of the culture conditions by using antioxidants such as coenzyme Q-10 or N-(2-mercaptopropionyl)-glycine (NMPG) and finally the development of a chemically defined culture medium for the porcine embryo production. Regarding ET, research has focused on the role of the recipient sows, studying on the one hand the immune environment at the uterine level by evaluating different cytokines at different periods of gestation. On the other hand, the effects of seminal plasma infusions prior to insemination and their impact on gestation have also been evaluated, assessing their possible use in ET programs. METHODOLOGY: Different experimental procedures have been conducted to address the objectives set out. Among the most used techniques are included the IVP process, divided in its three essential stages of in vitro oocyte maturation, in vitro fertilization of the oocytes and their subsequent in vitro culture until reaching the adequate preimplantational stage; as well as the technique for obtaining embryos from donor sows by surgical procedures. Through these two techniques, both in vitro produced embryos and in vivo derived embryos have been obtained, whose quality has been subsequently analyzed by different analytical techniques, such as differential staining of blastocysts, measurement of free radical and glutathione levels or by transcriptome analysis with techniques such as q-PCR or microarrays. These types of techniques have also been used to analyze the endometrium of donor and recipient sows, obtaining the samples through hysterectomies. RESULTS AND CONCLUSIONS: The results of the different experiments conducted have been compiled and published in a total of nine articles. The conclusions reached are the following: -High sperm dilution before freezing increases monospermy without affecting penetration rates, thereby increasing the efficiency of porcine IVP. -Exogenous supplementation of Coenzyme Q10 in maturation and in vitro culture media do not improve in vitro embryo production results, however, supplementation of in vitro culture medium with the antioxidant NMPG increases blastocyst production. -Chemically defined medium based on platelet factor 4 supplementation can effectively replace traditional undefined culture medium supplemented with bovine serum albumin with similar blastocyst production and transcriptomic profile. -Cytokine levels expressed in the endometrium of embryo-bearing sows show a uterine immune environment biased toward an anti-inflammatory profile compared to recipient sows 6 days after the onset of estrus. -Low endometrial levels of leukemia inhibitory factor during the implantation period in allogeneic pregnancies may lead to an unfavorable environment for embryo survival. -Seminal plasma infusions modify the transcriptome at the endometrial and embryonic level and promote the development and quality of the embryos by expressing genes related to embryonic development, implantation and/or maternal immunity, among others; making these infusions a promising tool for use in the swine industry in both artificial insemination and ET programs