44 Potential of extracellular vesicles to improve capacitation of bull sperm in vitro

Cryopreservation induces damage to frozen–thawed (FT) sperm plasma membranes resulting in premature acrosome exocytosis. As a result, sperm longevity and viability after AI is reduced and fertilization capability impaired during IVF, leading to poor reproductive outcomes. The addition of seminal plasma (SP) or oviducal secretions to FT semen affects sperm physiology and function. The SP delays sperm capacitation thus improving longevity in the female reproductive tract; conversely, oviducal secretions enhance hyperactivation, capacitation, and acrosome reaction of sperm. It is believed that extracellular vesicles (EVs) play a physiological role in bull sperm function by aiding in post-testicular modification of sperm. So far, the role of oviducal fluid-derived EVs in inducing sperm capacitation is not investigated fully. For this we developed a three-dimensional oviducal organoid culture in vitro to generate oviduct specific EVs and investigate their role in bull sperm capacitation in vitro. The objective of this study was to demonstrate that EVs from the female (oviducts) and male (accessory sex glands in the bull) reproductive tract alter sperm physiology and fertility. We hypothesise that EVs collected from SP and in vivo-derived diestrus oviducal cells (DO) would delay capacitation, while EVs collected from in vivo-derived oestrus oviducal cells (EO), and in vitro oviducal organoids (OO) would enhance capacitation. Frozen–thawed sperm from 6 bulls of proven IVF fertility were placed into a capacitating medium (tyrode’s albumin lactate pyruvate) with 6 treatments: (1) no EVs (control), (2) SP, (3) DO, (4) EO, (5) OO or (6) dilauryl-phosphatidyl choline (PC12; + control), to induce acrosome reaction. Samples were incubated at 37°C and assessed at 0, 1, 2, and 4 h post-treatment for motility using microscopy/computer-assisted sperm analysis system (CASA), viability (PI), acrosome integrity (FITC-PNA), membrane fluidity (MC540), and tyrosine phosphorylation (pY20 antibody) using flow cytometry. Statistical analysis was performed for each assessment at each time point using ANOVA; differences were separated using SNK mean separation technique, identified by superscripts. SP EVs addition to FT bull sperm increased membrane fluidity at 1 h (55^b ± 2), while OO EVs decreased membrane fluidity at 1 h (44^c ± 2) compared to controls (52^bc ± 2 and 64^a ± 2; no EVs and PC12, respectively, P < 0.05). Addition of OO and EO EVs enhanced capacitation by increasing % of acrosome reacted sperm at 4 h (8^a ± 1 and 6^a ± 1; OO and EO resp, P < 0.05), compared to controls (5^a ± 1 and 6^a ± 1; no EVs and PC12 respectively, P < 0.05). The SP EVs decreased % of acrosome reacted sperm at 0 h post-treatment (2^b ± 1) compared to controls (5^a ± 1 and 10^a ± 1; no EVs and PC12 respectively, P < 0.05). The proportion of sperm that underwent tyrosine phosphorylation at 4 h after OO and EO EVs addition increased (67^a ± 3 and 55^ab ± 3, respectively) compared to no EVs control (51^b ± 3, P < 0.05). Excitingly, this study indicates in vivo-derived oviducal EVs addition to FT bull sperm enhanced capacitation, and SP EVs addition to FT bull sperm improved sperm longevity. In future studies, the addition of oviducal EVs to FT bull semen may improve IVF rates and addition of SP EVs to FT bull sperm may improve pregnancy rates after AI.