Embryo transfer in cattle is performed with blastocysts produced in vivo or in vitro using defined media. However, outdated systems such as those that use serum and co-culture remain of interest for research purposes. Here, we investigated the effect of additional culture time on in vitro-produced embryos. Specifically, we compared embryos that formed a blastocoel at different times after fertilisation to those that stayed in culture for up to two additional days with respect to their development in vivo after temporary transfer to oestrus-synchronised recipients. A pre-transfer set (D6, D6+1, D6+2, D7, D7+1, D8) was examined using microarray analyses and correlated with a post-transfer set that included two different days of transfer (D6-T6, D6+2-T8, D7+1-T8, D8-T8). All surviving conceptuses reached primitive-streak stages and filamentous sizes similarly to in vivo (D18) or in vitro controls (D7/T7). The recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25%). With an intermediate survival rate (33%), the D6 embryos with two additional days in culture produced nine times more IFN-tau (IFNT) at D18 than the D6 embryos that were immediately transferred. At the end of culture, D6 and D6+2 embryos displayed the highest number of gene expression differences. Despite a mortality of 40–60%, no signature was detectable in any of the transferred groups that would account for the embryos’ fates. Initially reputed to be beneficial in producing more blastocysts, our culture system of B2 medium plus serum and co-culture generated blastocysts that were distinct from those developed in vivo (D7).
Isabelle Hue, Isabelle Dufort, Anaïs Vitorino Carvalho, Denis Laloe, Nathalie Peynot, Séverine Aude Degrelle, Christoph Viebahn, and Marc-André Sirard
Luiz Cordeiro, Hsiu-Lien Herbie Lin, Anaïs Vitorino Carvalho, Isabelle Grasseau, Rustem Uzbekov, and Elisabeth Blesbois
Male subfertility causes are very varied and sometimes related to post-gonadic maturation disruption, involving seminal plasma constituents. Among them, extracellular vesicles are involved in key exchanges with sperm in mammals. However, in birds, the existence of seminal extracellular vesicles is still debated. The aim of the present work was first to clarify the putative presence of extracellular vesicles in the seminal plasma of chickens, secondly to characterize their size and protein markers in animals showing different fertility, and finally to make preliminary evaluations of their interactions with sperm. We successfully isolated extracellular vesicles from seminal plasma of males showing the highest differences in semen quality and fertility by using ultracentrifugation protocol (pool of 3 ejaculates/rooster, n =3/condition). Size characterization performed by electron microscopy revealed a high proportion of small extracellular vesicles (probably exosomes) in chicken seminal plasma. Smaller extracellular vesicles appeared more abundant in fertile than in subfertile roosters, with a mean diameter of 65.12 and 77.18 nm, respectively. Different protein markers of extracellular vesicles were found by western blotting (n = 6/condition). Among them, HSP90A was significantly more abundant in fertile than in subfertile males. In co-incubation experiments (n = 3/condition), extracellular vesicles enriched seminal fractions of fertile males showed a higher capacity to be incorporated into fertile than into subfertile sperm. Sperm viability and motility were impacted by the presence of extracellular vesicles from fertile males. In conclusion, we successfully demonstrated the presence of extracellular vesicles in chicken seminal plasma, with differential size, protein markers and putative incorporation capacity according to male fertility status.