53 Investigating the improvement of bull in vitro fertility through seminal plasma extracellular vesicles

Seminal plasma extracellular vesicles (EVs) affect the biological function of sperm by enhancing their motility and fertilizing capability by transferring essential proteins, lipids, and RNAs, which potentially affects embryo development and offspring health. Using EVs derived from known high in vitro fertility (HF) bulls might allow for tailored IVF treatments to address specific fertility issues and improve success rates for known low in vitro fertility (LF) bulls. The main aim of this study was to test the possibility of adding seminal plasma-derived EVs from known HF bulls to our IVF system without impairing embryo development. The second aim was to see if adding seminal plasma-derived EVs from known HF bulls affects the fertilization capacity of known LF bulls in our IVF system. Oocytes were retrieved from abattoir-derived ovaries. All media were purchased from Stroebech Media. IVM and culture were carried out using the manufacturer’s standard protocols. EVs from known HF bull seminal plasma were isolated with a size exclusion chromatography benchtop column. Concentration and size of EVs were determined using nanoparticle tracking analysis. During IVF, EVs in three different concentrations (A, 200 × 10⁶ EVs mL⁻¹; B, 400 × 10⁶ EVs mL⁻¹; C, 600 × 10⁶ EVs mL⁻¹) were added to 100 µL IVF medium and co-cultured with sperm and oocytes for 24 h under mineral oil. As a control, PBS without Ca²⁺ and Mg²⁺ was added to 100 µL of IVF medium. Blastocyst formation and the morphology and kinetics of blastocysts were recorded on Day 7 (D7) and Day 8 (D8) after fertilization. Morphological and kinetic scores were calculated similarly to Gebreyesus et al. (2024 Anim. Reprod. Sci. 263, 107436). The statistical analysis was carried out using the GraphPad Prism 10 software. Blastocyst rate, kinetic, and morphological scores of different treatment groups were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons test with a significance value of P ≤ 0.05. On D7, the highest blastocyst morphological score was recorded in group C (3.0), which was 1.1 points higher than the control group morphological score (1.9). No differences between blastocyst rates and developmental kinetics were recorded. On D8, the difference in blastocyst rates (%) between group C and the control group was 19.8 ± 2.6 and 9.4 ± 0.9, respectively, but insignificant with a P ≥ 0.05. The morphological score of D8 in group C was the highest (2.35), 0.55 points higher than the control group score (1.8). In conclusion, adding seminal plasma-derived EVs to our IVF system did not hinder embryo development. The preliminary results suggest improvement in embryo morphology and blastocyst rates when adding 600 ×10⁶ EVs mL⁻¹ seminal plasma-derived EVs from known HF bulls to IVF medium to improve the fertility capacity of known LF bulls. More research is needed to confirm and validate the benefits of adding seminal plasma-derived EVs from HF bulls to improve LF bull IVF outcomes.

This study was supported by the Estonian Research Council grant PRG1665.