183 Effects of feeding nonstructural carbohydrates on the metabolic function of oocytes and granulosa cells in older mares

Feed components can affect metabolism and function of cells within the ovarian follicle. We have previously demonstrated that mare oocyte metabolic function is associated with developmental competence and can be impaired by advanced maternal aging. Diet supplementation to old mares with ingredients that promote cell metabolism was beneficial for oocyte metabolism and quality. Nonstructural carbohydrates, in the form of grains and sugars, are common additives to the equine diet. However, potential effects of such feed additives on metabolism of cells in the follicle have not been previously investigated in mares. We hypothesized that feeding grains and sugars alters metabolic function of oocytes and granulosa cells from preovulatory follicles of older mares. Older light-horse reproductively sound mares were randomly grouped as CON (control, fed only forage throughout the study, n = 6, 22.5 ± 1.5 years) or NSC (fed forage supplemented with grains and sugar, n = 7, 22 ± 1.6 years). Grain was fed daily in equal parts of corn, oats, and barley with dry molasses at 1.8 kg and 227 g, respectively, until Week 8 (Week 0 = start of study) and at 2.7 kg and 340 g until Week 14. Aspirates were collected from preovulatory follicles ~24 h after induction of follicular maturation with a GnRH agonist (0.5 mg of histrelin). Initial samples were collected while all mares were on a forage-only diet (Pre) and again after 12–14 weeks on the different feed regimens (Post). Oocyte metabolism was analyzed using a microsensor, and granulosa cells were analyzed using an Oxygraph-2k high-resolution respirometer (Oroboros Instruments). After completion of microsensor assays, oocytes were stained for cytosolic (H2DCFDA) and mitochondrial reactive oxygen species (ROS; MitoSOX Red, Thermo Fisher Scientific) and imaged with inverted fluorescence microscopy. Tukey-pairwise comparisons were conducted to observe differences between groups (CON and NSC) and time points (Pre and Post). Oocyte oxygen consumption rate, indicative of aerobic metabolism, increased (P < 0.05) from Pre to Post for NSC, but not CON. Fluorescence microscopy analyses of oocyte mitochondrial and cytosolic ROS after microsensor measurements were not different between groups or times. Release of ROS from granulosa cells increased in both groups during the course of the study. However, ROS release relative to the oxygen consumption rate under basal conditions was higher (P < 0.05) from Pre to Post only for NSC. Short-term feeding of grain and sugar was associated with alterations in ovarian cell metabolic function. Although the extent that fertility could be affected is still to be determined, these findings underscore the biological importance of dietary components in modulating ovarian cell metabolism and potentially influencing reproductive outcomes in aged mares.

The study was funded by the Cecil and Irene Hylton Foundation, the Foundation for the Horse Young Investigator grant, and the Abney Foundation Scholarship.