High body energy reserve influences extracellular vesicles miRNA contents within the ovarian follicle

Abstract

Aiming to evaluate the effects of increased body energy reserve (BER) in Nellore cows' reproductive efficiency, cows were fed with different nutritional plans to obtain animals with high BER (HBER; Ad libitum diet) and moderate BER (MBER: cows fed 70% of HBER group ingestion). To evaluate the BER, cows were weekly weighted and evaluated for subcutaneous fat thickness and insulin serum concentration along the experimental period. At the end of the experimental period, animals were submitted to estrous synchronization and artificial insemination. Animals were slaughtered approximately 120 h after ovulation induction and the reproductive tracts were collected for embryo recovery and samples collection. Cumulus-oocyte-complexes (COC) and follicular fluid were collected from 3-6 mm in diameter ovarian follicles to perform miRNA analysis of cumulus cells (CC) and extracellular vesicles from follicular fluid (EV FF). As expected, differences were observed among MBER and HBER groups for body weight, fat thickness, and insulin serum concentration. HBER animals showed lower ovulation and embryo recovery rates compared to MBER animals. Different miRNAs were found among CC and EV FF within groups, suggesting that the BER may influence follicular communication. This suggests that small follicles (3-6 mm diameter) are already under BER effects, which may be greater on later stages of follicular development.

Fig 1. Schematic representation of experimental period.

A. Timeline of feedlot period showing days of adaptation to electronic feeder (D-34 to D-1); adaptation diet (D0 to D21) and final diet (D22 to Slaughter (D67); S). During the feeding period the animals were submitted to: Subcutaneous fat thickness analysis of Biceps femoris (D-34, D22 and D65) and Longissimus thoracis (D30, D51 and D65); blood collection for Insulin serum concentration (D-34, D22 and D65); and weekly weighing average daily gain of each animal. B. Two nutritional plans were defined for cows with similar body conformations from a same herd in order to obtain cows with moderate body energy reserve (MBER) and cows with high body energy reserve (HBER) at the end of the experimental period. C. At the end of the experimental period, the animals were submitted to an estrous synchronization protocol (EB: estradiol benzoate, PGF2α: prostaglandin- 2α; P4: progesterone; GnRH: gonadotropin-releasing hormone), dominant follicle (DF) measure, artificial insemination (FTAI) and blood collection for reproductive hormones concentration (Day scheduled for ovulation; D0 OV and S).

Fig 2. Body weight and subcutaneous fat ultrasound of Nellore cows during the experimental period.

A. Evolution of body weight of cows with different energy reserves. B. Analysis of subcutaneous fat thickness of Biceps femoris and Longissimus thoracis measured by carcass ultrasound of cows with different energy reserves. Mean standard error for body energy reserve, time and body energy reserve and time interaction. P-values are on the right top of the Figs. *Beginning and prevalence of statistical differences (P<0.05).

Fig 3. Serum insulin and reproductive hormones levels of Nellore cows during the experimental period.

A. Insulin concentration. B. E2 concentration. C. P4 concentration. Standard error of the mean for body energy reserve, time and body energy reserve and time interaction. P-values are on the right top of the Figures. *Statistical difference (P<0.05). D0 OV: day scheduled for ovulation. S: day of slaughter.

Fig 4. Relative expression of bta-miR-489 which is higher in the follicular fluid extracellular vesicles in MBER group.

Mean ± standard error of the mean. P-value is in bold on the right top of the figure.

Fig 5. Total numbers of miRNAs detected in cumulus cells (CC) and follicular fluid extracellular vesicles (EV FF) from 3–6 mm follicles from cows with different body energy reserve.

A. Venn diagram demonstrating a total of 79 miRNAs commonly detected between CC and EV FF for MBER group, which 2 were up regulated in CC samples and 60 up regulated in EV FF and, 6 exclusively detected miRNAs in EV FF but not in CC. B. Venn diagram demonstrating a total of 46 miRNAs commonly detected between CC and EV FF for HBER group, only 1 was up regulated in CC samples and 21 up regulated in EVs FF and, a total of 42 exclusively detected miRNAs in EV FF but not in CC.

Fig 6. Enrichment analysis performed in miRWalk 3.0 software of predicted pathways modulated by exclusives or up-regulated miRNAs of cumulus cells (CC) and follicular fluid extracellular vesicles (EV FF) from 3–6 mm follicles from cows with moderated body energy reserve (MBER).

A. The 10 predicted pathways with highest percent of genes predicted to be modulated by exclusives miRNAs in EV FF compared to CC in MBER group. B. The predicted pathways modulated by miRNAs up regulated in CC compared to EV FF in MBER 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 adjusted P-value (Adjusted P-value < 0.05).

Fig 7. Enrichment analysis performed in miRWalk 3.0 software of predicted pathways modulated by exclusives or up-regulated miRNAs of cumulus cells (CC) and follicular fluid extracellular vesicles (EV FF) from 3–6 mm follicles from cows with high body energy reserve (HBER).

A. The predicted pathways modulated by exclusives miRNAs in EV FF compared to CC in MBER group. B. The 10 predicted pathways with highest percent of genes predicted to be modulated by miRNAs up regulated in EV FF compared to CC 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 adjusted P-value (Adjusted P-value < 0.05).