Collection Rates and Morphology of Equine Oocytes Obtained from Immature Follicles after Treatment with Equine Pituitary Extract (EPE) and Human Chorionic Gonadotropin Ieda D.P. Blanco, DVM, MSc, Liane G. Devito, DVM, MSc, Heder N. Ferreira, DVM, MSc, Gustavo H.M. Araujo, DVM, MSc, Claudia B. Fernandes, DVM, PhD, Marco A. Alvarenga, DVM, PhD, and Fernanda C. Landim-Alvarenga, DVM, PhD ABSTRACT This study examined the effect of treating mares with equine pituitary extract (EPE) in combination with human chorionic gonadotropin (hCG; EPE/hCG) on the recovery rate of immature oocytes by ovum pick-up (OPU) and on oocyte morphology. Ten mares were sub- jected to each of two treatments in a random sequence: superstimulated with EPE (25 mg, twice daily) and treated with hCG (2,500 IU) or control (no exogenous treat- ment). The cytoplasmic morphology of oocytes recovered was evaluated through transmission electron microscopy. Follicular fluid was collected at aspiration for progesterone analysis, which was performed by radioimmunoassay. The EPE/hCG did not increase the oocyte recovery rate from immature follicles when compared with the controls (15.5% and 16.7%, respectively). A significantly higher oocyte recovery rate per mare was observed (70% versus 50%). However, precocious granulosa cell expansion was observed with EPE/hCG treatment in contrast to the control (64.4% and 33% of follicles with expanded cumu- lus, respectively), and increased intrafollicular progester- one concentration was also seen (158.80 ng/mL versus 82.05 ng/mL). The ultrastructural analysis of oocytes from both groups showed morphologic features related to immaturity. Numerous vesicles containing cortical granules were found, distributed in clusters into the cyto- plasm, and junctional complexes were still seen between oocyte and granulosa cells. In conclusion, EPE/hCG treatment induced some follicular modifications, but the recovery rate was not increased. All oocytes examined presented signs of immaturity. Keywords: OPU; Equine; Oocytes; EPE; hCG INTRODUCTION Successful in vitro production of embryos depends on a number of factors, such as availability of healthy imma- ture oocytes, efficient and repeatable methods for in vitro maturation, physiologic sperm capacitation, and optimal in vitro embryo culture systems. Successful results obtained for in vitro production of bovine embryos is, in great part, related to the unlimited availability of abat- toir-collected bovine oocytes and the total freedom to con- duct research with such material. In contrast, for horses the availability of abattoir ovaries is much scarcer, which limits the rate of advancement of research using horse oocytes. 1 Ovum pick up has been used successfully in the equine to obtain oocytes for oocyte transfer. 2,3 Oocytes are usually collected from one preovulatory follicle per mare that has been treated with hCG to accelerate follicular maturation, resulting in high recovery rates. However, the aspiration of immature follicles has resulted in poor recovery rates. 4 The low recovery rate of oocytes from immature follicles seems to be linked with the presence of a thecal pad beneath the cumulus cells, which act as an anchor for the cumulus at- tachment. In preovulatory follicles (>35 mm in diameter), final oocyte maturation is accompanied by the expansion of the cumulus, which releases the oocyte from the follicular wall, increasing recovery rates after follicular aspiration. 5 The number of oocytes recovered in each OPU section should be higher with exogenous follicle stimulation. However, although the total number of oocytes recovered by each individual mare increased, the recovery rate per fol- licle was still low. 6,7 The expression of luteinizing hormone receptors (LHr) in immature follicles was restricted to the theca cells, ORIGINAL RESEARCH From the Department of Animal Reproduction and Veterinary Radiology, FMVZ-UNESP-Botucatu-SP-18618.000, Brazil. Reprint requests: Professor Fernanda da Cruz Landim e Alvarenga, Department of Animal Reproduction and Veterinary Radiology, F.M-Z.—UNESP—Botucatu—SP, Brazil - 18.618-000. 0737-0806/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2009.07.011 Journal of Equine Veterinary Science  Vol 29, No 8 (2009) 613