Effect of the type of fixative and time of fixation on the morphology of equine preantral follicles

The objective of this study was to investigate the efficacy of tissue fixatives Bouin, Carnoy and formaldehyde in equine ovarian fragments. Ovaries (n=4) from mixed breeds mares were obtained at a local slaughterhouse and transported to the laboratory in a thermo-recipient at 20oC. Immediately after collection ovaries were washed with modified PBS solution (Cultilab®, Campinas-SP, Brazil) and divided into nine fragments with approximately 5x5x1 mm. Ovarian fragments were immersed in Bouin (B), Carnoy (C) or Formalin 10% (F) for 6, 12 or 24 hours. Each fragment was individually immersed 20 mL Falcon tubes containing 20 times the volume of fixative solution. After this period, they were kept in 70% ethanol for 24 hours. Each procedure was performed in four replicates. For histological analysis, samples were dehydrated in increasing concentrations of alcohol, diaphanized in xylene and embedded in paraffin. Five µm thick serial sections were obtaneid with a rotating microtome (Leica® type, Wetzlar, Germany) and stained with periodic acid-Schiff (PAS) and hematoxylin. A total of 540 slides with 1,620 sections were evaluated and 465 preantral follicles were counted and classified as normal or degenerated. Degeneration was determined by the presence of at least one of the following aspects: retraction of the cytoplasm, pyknotic nucleus, cytoplasmic vacuoles, displacement of granulosa cells and disruption of the basal membrane. The statistical model was logistic regression (p u003c0.05). The Carnoy fixative for 24 hours provided best integrity of ovarian follicles (53.3%; 32/60) compared to the others. Boiun for 24 hours was the worst treatment (19.1%; 9/47). The other treatments showed the following results; C12h 50% (30/60), C6 H 40% (24/60), F24h 37.8% (17/45), F12h 35.1% (13/37), F6h 32% (16/50), B12h 30.5% (18/59) and B6h 24.4% (11/45). Fixation with Carnoy for 24 hours provided best integrity of equine preantral follicles.