Seminal Plasma-Derived Exosome Preserves the Quality Parameters of the Post-Thaw Semen of Bulls with Low Freezeability

<b><i>Introduction:</i></b> Sperm cryopreservation is a useful storage technique in artificial insemination. Nanoparticles and nanovesicles such as exosomes are widely used in sperm cryopreservation procedures to alleviate cold-induced injury inflicted during sperm freezing. <b><i>Objective:</i></b> The objective of the present study was to examine the impact of varying concentrations of exosomes derived from seminal plasma added to a freezing extender on the quality of post-thawed bull sperm. <b><i>Methods:</i></b> Five Holstein bulls were chosen based on their samples having less than 30% progressive motility. After exosome extraction, semen samples from bulls (<i>n</i> = 5) with progressive sperm motility ≤30% were collected, diluted with different exosome concentrations (0, 25, 50, and 100 μg/mL), and aspirated into 0.5 mL straws. After the freeze-thaw process, sperm total and progressive motility, viability, morphology, plasma membrane integrity, mitochondrial activity, and apoptosis status were assessed. Furthermore, the expression levels of annexin (ANX1), dystrophy-associated Fer-1-like protein (DYSF), fibronectin 1 (FN1), and reactive oxygen species modulator 1 (ROMO1) were evaluated via real-time polymerase chain reaction (PCR). <b><i>Results:</i></b> Adding different concentrations of exosomes (25, 50, and 150 μg/mL) significantly increased the progressive motility, viability, and membrane integrity of sperm compared with the control group (<i>p</i> < 0.05). For the apoptosis index, treatment with 100 μg/mL exosomes significantly increased the percentage of live cells (<i>p</i> < 0.05), while the percentage of necrotic cells decreased significantly (<i>p</i> < 0.05) compared with 25 μg/mL exosome. The results of quantitative PCR showed that the expression levels of ANX1 were significantly (<i>p</i> < 0.05) upregulated at 50 μg/mL exosome, and the expression of ROMO1, FN1, and DYSF were downregulated upon treatment with different exosome concentrations. <b><i>Conclusions:</i></b> In conclusion, supplementing the freezing diluent with exosome-derived seminal plasma could preserve the quality parameters of the post-thaw semen of the bull with low freezeability and could be used as a helpful method for reproductive programs.