Factors Affecting the In Vitro Embryo Production in Cattle Associated to Ovum Pick Up System
Production of embryos through the collection of immature oocytes by ovum pick up (OPU), and the following maturation, fertilization and culture in the in vitro laboratory, offers many benefits to optimize reproductive potential. Moreover, in vitro production of embryos (IVPE) with OPU oocytes supposes an alternative method with several advantages over the conventional hormonal treatment of superovulation and in vivo embryo recovery. However, currently, the IVPE is still not as efficient, or can produce embryos of similar quality to in vivo embryos, which has limited their widely application. This thesis was performed with the aim to optimize the IVPE technology in cattle, conditioned by the specific characteristics and deficiencies of the IVPE when OPU oocytes are used. To this end, five experiments were performed. Firstly, we studied the effect of the bovine oviductal fluid (bOF) on embryo development and quality (Experiment 1). The steps of the IVPE process at which the culture of a single or low number of oocytes/embryos could impair their further development to the blastocyst stage or/and their quality were studied in the Experiment 2. In the Experiment 3, we tested whether the development and quality of the embryos in vitro cultured in low number can be improved by the addition of a mixture of epidermal growth factor, insulin, transferrin, and selenium (EGF-ITS) or by the system of embryo culture known as ‘Well of Well’ (WOW). The protective role of the melatonin regarding the oxidative stress damage subsequent to the IVPE conditions or subsequent to the heat stress during oocyte maturation were assessed in the Experiment 4. Finally, in our last experiment (Experiment 5), we used oocytes collected by OPU to test the effect of sex-sorted sperm in the in vitro fertilization and their further embryo development and quality. The results of the Experiment 1 showed that a short bOF treatment in oocytes had no effect on the embryo development until blastocyst stage and neither on the quality attending their blastocyst morphology. However, the study of the genetic expression suggested that bOF treatment could be useful to mitigate some lacks on the lower quality blastocysts produced in vitro. Regarding the Experiment 2, we observed that the period of culture after the fertilization, especially between the days 3 and 8, seems to be the most important stage for embryo development on single and/or low number (5-10) of embryos culture. In the Experiment 3, the addition of EGF-ITS increased the quality of the embryos cultured in low number, whereas culture embryos in WOW improved significantly the development rates. In the fourth experiment (Experiment 4), the addition of melatonin (10-4 M) to heat-stressed bovine oocytes enhanced the further development to blastocyst stage. However, compared to the control, none of the melatonin concentration tested (10-12, 10-9 and 10-4 M) showed any significant effect when oocytes were matured under conventional in vitro conditions. Concerning the results of the Experiment 5, zygotes fertilized by sex-sorted sperm showed a lower cleavage rate in comparison with those fertilized with unsorted sperm, but similar blastocyst rate, as well as similar morphological quality and timing of blastocyst formation.