Effects of feeding exogenous vitamins and microelements on follicular development and molecular function of granulosa cells in donkeys

Abstract

In mammalian ovarian physiology, vitamins and microelements are essential for follicular development, ovulation, and the synthesis and secretion of hormones and growth factors. However. the molecular mechanisms by which they influence maturation and growth of follicles in donkeys are still unknown. Here, we investigated how vitamins and microelements affected the development of follicles and the proliferation of ovarian granulosa cells (GCs). For this experiment, 40 jennies (average body weight: 292.946±33.543 kg, age: 3∼4 years) were randomly assigned into 4 groups (n=10) and fed 50% (A), 75% (B), 100% (C), and 150% (D) of a mixture of vitamins A, D and E and microelements once a day (dose 5 ml) for two consecutive months. After that, ultrasonography was applied to record the change in follicular dynamics in three estrous cycles. Using a transvaginal ultrasound-guided aspiration device to obtain follicular fluid (containing GCs) from the follicles (Ф=30mm-35mm), multi-omics analysis revealed its molecular mechanisms and physiological functions. The results showed that the time required for growing follicles to reach ovulation (Ф>40mm) was shortened in the vitamin D group compared to the three other groups(P<0.05). Of note, the follicles (Ф=30mm-35mm) in the vitamin C group had less discrete diameters and developed more evenly and had significantly more twin ovulations than the other three groups (P<0.05). The omics analysis showed that945 metabolites, including organic acids and derivatives (24.44%), lipids and lipid-like molecules (24.34%), organic heterocyclic compounds (13.44%), and benzenoids (12.06%) were identified in follicular fluid. The results also revealed 232, 207, 210 and 202 differential cumulative metabolites (DAMs) in group A vs. group B, group B vs. group C, group C vs. group D and group A vs. group C, respectively. The DAMs were most significantly enriched in protein digestion and absorption, ABC transporters, amino acid biosynthesis, mineral absorption, aspartate-arginine biosynthesis, and ovarian steroid synthesis process. Meanwhile, nine DEGs (INSR, CYP19A1, ALOX5, PLA2G4A, ADCY6, CYP1B1, PRKACB, CYP17A1, and PLA2G4F) associated with the secretory function of GCs were involved in the ovarian steroidogenesis pathway, and they were significantly correlated with estradiol (E2), cholesterol, progesterone (P4), and testosterone (T)(P<0.05). Furthermore, STEAP2, CYBRD1, ATP1B3, ATP1A1, SLC26A6, SLC5A1, SLC6A19, SLC40A1, and TRPV6 were closely associated with the mineral absorption of GCs, and they had a positive correlation with glycine and L-methionine (P<0.05). In conclusion, exogenous supplementation of jennies with vitamins A, D and E and micronutrients can contribute to follicular growth and ovulation and this was associated with the interaction and expression of key genes related to -metabolites in ovarian steroidogenesis and mineral absorption pathways.