Study of Porcine Sperm Functionality during Interaction with Male and Female Reproductive Fluids, and its Implication in Fertilization

Abstract

The porcine industry aims to improve its productivity by enhancing assisted reproductive techniques, such as artificial insemination (AI), and gaining a better understanding of the physiological processes from ejaculation to semen deposition in the female reproductive tract. The ejaculate is emitted in various fractions (pre-sperm, sperm-rich, intermediate, and post-spermatic fractions) that differ in sperm concentrations, volume, and seminal plasma (SP) composition. The sperm-rich fraction is commonly used in the preparations of seminal doses for AI, while the inclusion of the post-sperm fraction is a subject of debate. Therefore, in Chapter 1, the effect of including cumulative ejaculate fractions in seminal doses was investigated regarding their preservation, reproductive performance, and offspring health after artificial insemination (AI). Three types of seminal doses (2000x106 spermatozoa/60 ml) were prepared: 1) F1: sperm-rich fraction; 2) F2: F1 + intermediate fraction; 3) F3: F2 + post-sperm fraction. The doses were stored (16°C, 3 days), and sperm quality was analyzed before their use for AI in multiparous sows. The results of sperm quality parameters and fertility did not show significant differences between the groups. Subsequently, in Chapter 2, the experimental groups (F1, F2, F3) were stored at 16°C for 5 days. The energetic metabolism of undiluted ejaculate and after the preparation of seminal doses (day 1) was analyzed, and sperm quality parameters were evaluated on day 1, 3, and 5. No differences were observed between the groups. On day 5, thermal stress conditions (38.5 °C, 300 minutes) and uterine environment simulation (incubation with uterine fluid, UF, at 38.5 °C for 180 minutes) were applied to mimic the conditions to which spermatozoa are exposed in the female reproductive tract. In both tests, groups F2 and F3 maintained better quality than F1. In Chapter 3, sperm quality parameters were analyzed by incubating spermatozoa with UF in the presence or absence of SP at 38.5 °C for 180 minutes. It was observed that UF has a detrimental effect on sperm functionality (motility and acrosome status) after 2 hours of incubation, and SP mitigates this adverse effect. Therefore, it is crucial to understand the changes that occur in spermatozoa during these interactions. In Chapter 4, a proteomic analysis of spermatozoa incubated with female reproductive fluids (UF and oviductal fluid, OF) in the presence or absence of SP was conducted. The results revealed a higher number of proteins in spermatozoa incubated with a single fluid (SP, UF, or OF) compared to when they were incubated with two fluids (SP+UF and SP+OF), indicating changes in the sperm proteome, likely due to steric hindrance. In addition to the interaction with fluids, sperm undergo a selection process during their journey towards the fertilization site. Therefore, in Chapter 5, the effect of SP and/or UF on sperm fertilizing capacity was analyzed through in vitro fertilization, as well as the energetic metabolism before and after an in vitro selection process. Following selection, spermatozoa incubated with UF exhibited higher penetration and efficiency, along with increased metabolic activity. Furthermore, it was observed that UF influences the metabolomic, lipidomic, and proteomic profiles of spermatozoa, enabling the identification of potential fertility biomarkers. In conclusion, the inclusion of all ejaculate fractions does not impact dose storage or reproductive performance. Thus, the use of the entire ejaculate can be considered for AI. Additionally, the complex interactions between spermatozoa and reproductive fluids provide valuable insights into the mechanisms involved in sperm functionality.