159 Epididymal sperm and epididymosomes interaction before in vitro fertilization modulates sperm fertility potential in cattle

Sperm stored in cauda epididymis are quiescent until the moment of ejaculation. Although cauda epididymis sperm (Sp-Cau) are mature and able to fertilize the eggs, sperm interactions with epididymal epithelial cells may modulate this fertility potential. One of the main mechanisms of epididymal epithelial cells and sperm interaction can be mediated by small extracellular vesicles (EVs; i.e. epididymosomes) released by epithelial cells to luminal fluid. Here, we investigated the effect of a previous in vitro sperm-epididymosome interaction on the Sp-Cau fertility ability and in vitro embryo development in cattle. We established a suitable in vitro protocol to promote sperm-epididymosome interaction in a previous study. Bovine epididymal fluid was collected by retrograde perfusion from the epididymal cauda of three bulls. Sperm cells were isolated and cryopreserved using BoviFree® (Minitube) extender with 30 × 10⁶ Sp-Cau per 0.25-mL straw. A pool of epididymosomes was obtained from the epididymal fluid collected by retrograde perfusion from the epididymal cauda of five bulls and isolated by centrifugation (600g for 10 min, 4000g for 20 min, and 16 500g for 30 min) and ultracentrifugation at 119 700g for 70 min at 4°C twice. To investigate the impact on Sp-Cau fertility ability, grade I and II cumulus–oocyte complexes (COCs) were obtained from post-mortem bovine ovaries and matured for 22–24 h. Before the IVF, post-thawed Sp-Cau were selected by a Percoll® gradient and incubated with 1000 epididymosomes/sperm for 3 h in IVF medium at 38.5°C and 5% CO₂ in air (EVs group). The control group (Co group) was incubated under the same conditions in the IVF medium without epididymosomes. Following 3 h of incubation, sperm were centrifuged at 500g for 5 min and resuspended in IVF medium, and 1 × 10⁶ sperm mL⁻¹ were added to an IVF drop with 20–25 COCs for 6 h. Afterward, the presumed embryos were transferred to the in vitro culture step, and first cleavage, cleavage, and blastocyst rates were respectively evaluated at 28 h post-insemination (hpi), 96 hpi, and 168 hpi. The two groups (Co vs. EVs) were compared by Chi-square test. Statistical difference was considered when P < 0.05. Regarding the first cleavage (P = 0.41; Co: 49.0 ± 8.2% [71/145]; EVs: 53.7 ± 12.2% [80/149]) and cleavage (P = 0.07; Co: 60.7 ± 8.0% [88/145]; EVs: 70.5 ± 13. 8% [105/149]) rates, no differences were observed. However, blastocyst rates were higher (P = 0.03) for EVs (38.9 ± 7.3%; 58/149) compared with Co (26.6 ± 5.6%; 40/145). Thus, the previous interaction between SP-Cau and epididymosomes in vitro increased early embryo development in cattle. Sperm-epididymosome interaction can be used as a new tool to improve embryo in vitro production in cattle.

This study was supported by FAPESP 2021/08759-2, FAPESP 2019/23685-5, and CNPq 308014/2021-9.