Divergent selection for litter size variability modifies the RNA cargo in oviductal extracellular vesicles favouring embryonic development and survival in the line selected for litter size homogeneity

Extracellular vesicles (EVs) are being pointed as key components of the oviductal fluid. It has been shown that oviductal EVs (oEVs) and their molecular cargo play essential roles in supporting gamete maturation, fertilization, and early embryo development in different species. However, little is known about oEVs in rabbits. In this species, like in other farm animals, genetic selection has considerably increased their productivity, but with a negative effect on reproduction and animal welfare. Selection for increased homogeneity of litter size has been proposed to improve resistance to stress and diseases in animals, but the effect of this selection on the oviductal environment is not well-known. We hypothesize that oEVs with their RNA cargo act as multi-signal messengers in the embryo-maternal crosstalk, are affected in lines with high litter size variability (H) compared to low litter size variability (L). From two lines divergently selected for litter size variability, oviductal fluid was collected by flushing oviducts from pregnant (with embryos at 72 h p.c., P) and control does (non-ovulated/non-pregnant, NO) in 4 experimental groups: pregnant heterogeneous litter size line (H_P); pregnant homogeneous litter size line (L_P); non-pregnant heterogeneous line (H_NO); non-pregnant homogeneous line (L_NO). Oviductal EVs were isolated and characterized to confirm their presence in oviducts of does of all experimental groups. RNA cargo of oEVs was analyzed by RNA-sequencing, revealing a high number of differential abundant (DA) genes between P vs. NO does in both lines (1223 DA genes in H line and 1519 in L line, FDR <0.1%). Additionally, 27 and 25 miRNAs were found as DA between P vs. NO in H and L lines, respectively. Interestingly, functional enrichment analysis of DA genes and predicted target genes of identified miRNAs revealed biological terms such as embryo development, epithelium morphogenesis and differentiation, and cilium movement, which were only associated to L line for P and NO groups. Additionally, the comparison between H and L lines identified 169 DA RNAs in NO does, but without significant differences in P does. For miRNAs, no differences were detected in H vs. L lines in P or NP does. In conclusion, this is the first study unveiling the differential oEV RNA cargo between P and NO does. Besides, the study pointed to interesting differences between lines selected for variance in litter size, which might reflect the different maternal support to the early embryo development in the different lines.