Supplementation of IVM media with equine follicular fluid of large follicles increases blastocyst yield in equine ICSI programs.

Despite advances in the use of intracytoplasmic sperm injection (ICSI) for the in vitro production of equine embryos, oocyte maturation and competence to sustain pre-implantation embryo development remain significant challenges. This study evaluated the supplementation of the in vitro maturation (IVM) medium with equine follicular fluid (eFF), obtained from young mares (2-15 years old) with oocyte-to-blastocyst conversion rates >22%, as a strategy to improve oocyte developmental competence. In the first experiment, IVM medium was supplemented with 10% eFF collected from ovarian follicles of different diameters: 10-15 mm, 15-20 mm, or 20-30 mm. Oocyte maturation was assessed, and mature oocytes were subjected to ICSI to evaluate embryo development to the blastocyst stage. In a second experiment, embryos produced in a commercial ICSI program from equine oocytes matured in IVM medium supplemented with eFF from large follicles (20-30 mm) or without supplementation (control) were transferred to recipient mares. Pregnancy was assessed by ultrasonography on Days 15 and 30 after embryo transfer to confirm gestation and determine pregnancy rates. Supplementation of IVM medium with eFF from small (10-15 mm) or medium-sized (15-20 mm) follicles had no effect on oocyte quality or embryo development. However, supplementation with eFF from large (20-30 mm) follicles resulted in higher blastocyst rate per matured oocyte (33.9% vs. 23.2%; p = 0.021) compared with controls. Pregnancy rates on Days 15 and 30 post-transfer were similar between groups. These results indicate that the supplementation of the maturation medium with eFF from 20 to 30 mm follicles is a promising strategy for improving the in vitro production of equine embryos in commercial ICSI programs.