Divergent selection for litter size variability affects RNA cargo in oviductal extracellular vesicles related to embryonic development and survival

Selection for increased homogeneity of litter size has been proposed to improve resistance to stress and diseases in animals. Previous studies have shown that lines selected for decreased litter size variability (L lines) have higher litter sizes at parity than lines selected for increased litter size variability (H lines), possibly due to higher embryo survival in the oviduct. Thus, the study aimed at examining the effect of the line selection on the oviduct environment and its contribution to embryo development, particularly via extracellular vesicles (EVs). Oviductal EVs (oEVs) and their molecular cargo play essential roles in supporting early embryo development in different species, but little is so far known in rabbits. 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) from the two lines, resulting in 4 experimental groups: pregnant H line (H_P); pregnant L line (L_P); non-pregnant H line (H_NO); non-pregnant L line (L_NO). Oviductal EVs were isolated and characterized. 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. Furthermore, 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. This is the first study unveiling the differential oEV RNA cargo between lines selected for low versus high variation in litter size, and in each line, between P and NO does. The changes in protein-coding RNA and miRNA cargo might reflect the different maternal support to the early embryo development in the different lines.