Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality.

Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.

Crossbreeding effect of double-muscled cattle on in vitro embryo development and quality.

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 ± 6 and 27 ± 4.3%, respectively) than for BB (69.8 ± 8.5 and 19.6 ± 3.1%, respectively) and HF (45.1 ± 10 and 12.3 ± 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 ± 7.8) than HF (40.4 ± 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts.