Circulating extracellular vesicles across bovine gestation conserve characteristics independently of fetal age and sex.

INTRODUCTION

Extracellular vesicles (EVs) are released by cells into extracellular space and play a key role in intercellular communication. During pregnancy, embryo- and placenta-derived EVs are present in the maternal circulation, where they can serve as biomarkers of pregnancy status.

METHODS

The present study aimed to characterize the circulating EVs in pregnant cows carrying male or female calves. Crossbred Angus cows were subjected to Timed- Artificial Insemination (TAI) using the 7-Day CoSynch + CIDR protocol. Thirty days after TAI, pregnancy diagnoses were performed using gray mode ultrasonography, and thirty pregnant cows were randomly selected for the experiment. Blood samples were taken at days 45, 60, 90, 120, 150, 180, 210, 240, and 270 of gestation. Samples from each cow were centrifugated to separate the plasma, and 1 mL aliquots were stored at -80 °C. After parturition, calf sex was recorded, and samples from five male-carrying gestations (MCG) and five female-carrying gestations (FCG) were selected for EVs analysis.

RESULTS

EVs mean size and concentration were measured using nanoparticle flow cytometry and electron microscopy. Capillary wester analysis was used to corroborate the presence of EVs surface proteins CD63, CD9, CD81, and TSG101, confirming the presence of EVs in the isolates. The observed size ranged from 50 to 200 nm, with an average size of 87-105 nm, classifying the majority as small extracellular vesicles (sEVs). EVs size was not affected by fetal sex or gestation day. EVs concentration did not follow a clear pattern throughout pregnancy, and no significant effect of fetal sex or gestational day was observed.

CONCLUSION

Most circulating EVs found in the plasma of pregnant of beef cattle, regardless of the fetal sex, are primarily sEVs. No differences were observed between male- and female-carrying gestations in EVs size or concentration throughout gestation.