The bovine oviductal environment and composition are negatively affected by elevated body energy reserves

Abstract

To analyze the effects of high body energy reserve (BER) within the oviductal environment and its composition, Nellore cows were fed two different nutritional plans to obtain animals with moderate BER (MBER) and high BER (HBER). After obtaining the groups with different BERs, all animals were subjected to oestrus synchronization and artificial insemination, and 120 hours after ovulation induction, the cows were slaughtered, the reproductive tract was removed, and the ipsilateral oviduct to the corpus luteum was collected and dissected. Analyses were performed only for animals that had an 8-cell embryo in the isthmus. After embryo identification, we evaluated the molecular profiles of extracellular vesicles from oviductal flushing (OF-EVs) and luminal epithelial cells (OV-Cell) and performed histomorphological analysis of oviductal tissue from the ampullary and isthmic oviductal regions. The HBER group presented higher concentrations of ampullary extracellular vesicles (AMP-EVs) and larger sizers of isthmic extracellular vesicles (IST-EVs). The miRNA profile of AMP-EVs showed that the differentially expressed miRNAs were predicted to regulate pathways associated with cell growth, migration, differentiation and metabolism, with the HBER group being more susceptible to insulin modulation. The MBER animals showed greater ampullary vascularization than the HBER animals did. Additionally, the miRNA profile and differential gene expression (DEG) data obtained for ampullary (AMP-Cell) and isthmic (IST-Cell) luminal epithelial cells revealed pathways related to insulin metabolism. Thus, elevated BER may lead to oviductal insulin resistance, affecting normal functioning and, probably, embryo metabolism during early development, thus impacting gestational rates in these animals.

Fig 1. Oviductal flushing extracellular vesicles characterization from cows with different body energy reserve.

A. Extracellular vesicles size and concentration in ampulla and isthmus analyzed by NTA. Mean ± standard error. P-value is on the right top of the figure. B. Transmission electron microscopy images show (white arrows) the extracellular vesicles presence in oviductal flushing. C. Flow cytometry representative results show positive events inside the gates created based on the unlabeled particles and negative control for each marker.

Fig 2. miRNAs expression in extracellular vesicles from ampullary flushing (AMP-EVs) from cows with different body energy reserve.

A. Venn diagram demonstrating the 82 common miRNAs between groups which 2 were up regulated in MBER group and 5 up regulated in HBER group. B. Relative expression of up regulated miRNAs in AMP-EVs in MBER group. C. Relative expression of up regulated miRNAs in AMP-EVs in HBER group. Mean ± standard error. P-value is on the right top of the figure.

Fig 3. Enrichment analysis performed in miRWalk 3.0 software of predicted pathways modulated by miRNAs differentially expressed in small extracellular vesicles from ampullary oviductal flushing (AMP-EVs).

A. The 10 predicted pathways with highest percent of genes predicted to be modulated by miRNAs up regulated in AMP-EVs in MBER group. B. The 10 predicted pathways with highest percent of genes predicted to be modulated by miRNAs up regulated in AMP-EVs in HBER group. The Y-axis in left represents the percent of genes (%) predicted to be modulated by miRNAs and the Y-axis in right shows the BH (BH < 0.05).

Fig 4. Oviductal histomorphological analysis to obtain

A. Collagen rate, by red and yellow colors segmentation of the image. B. Vascularization (blood vessels/mm²; white arrows). C. Luminal perimeter (micrometer; µm) from cows with different body energy reserve. D. Histomorphological analysis in ampullary region. E. Histomorphological analysis in isthmic region. Mean ± standard error. P-value is on the right top of the figure.

Fig 5. miRNAs expression in ampullary luminal epithelial cells (AMP-Cell) from cows with different body energy reserve.

A. Venn diagram demonstrating the 210 common miRNAs between groups and 10 exclusives miRNAs in HBER group. Of the 210 common miRNAs, 8 were up regulated in HBER group B. Relative expression of up regulated miRNAs in AMP-Cell in HBER group. Mean ± standard error. P-value is on the right top of the figure.

Fig 6. miRNAs expression in isthmic luminal epithelial cells (IST-Cell) from cows with different body energy reserve.

A. Venn diagram demonstrating the 242 common miRNAs between groups and 1 exclusive miRNAs in HBER group. Of the 243 common miRNAs, 14 were up regulated in HBER group B. Relative expression of up regulated miRNAs in IST-Cell in HBER group. Mean ± standard error. P-value is on the right top of the figure.

Fig 7. Enrichment analysis performed in miRWalk 3.0 software of predicted pathways modulated by miRNAs exclusives and differentially expressed in ampullary luminal epithelial cells (AMP-Cell) from HBER group.

A. The 10 predicted pathways with highest percent of genes predicted to be modulated by exclusives miRNAs in AMP-Cell in HBER group. B. The 10 predicted pathways with highest percent of genes predicted to be modulated by miRNAs up regulated AMP-Cells in HBER group. The Y-axis in left represents the percent of genes (%) predicted to be modulated by miRNAs and the Y-axis in right shows the BH (BH < 0.05).

Fig 8. Enrichment analysis performed in miRWalk 3.0 software of predicted pathways modulated by miRNAs differentially expressed in isthmic luminal epithelial cells (IST-Cell) from HBER group.

The 10 predicted pathways with highest percent of genes predicted to be modulated by miRNAs up regulated IST-Cells in HBER group. The Y-axis in left represents the percent of genes (%) predicted to be modulated by miRNAs and the Y-axis in right shows the BH (BH < 0.05).