Effect of fixative solutions in equine ovarian tissue

Histological processing is a classical methodology used for an efficient assessment of ovarian tissue quality. Although it is still controversial which is the most appropriate fixative solution for equine ovarian tissue, the choice of an appropriate fixative is critical to evaluate the improvement of assisted reproductive techniques for fertility preservation. This study aimed to evaluate immediate histological processing, based on an ethanol 70% fixative solution for a short-time exposure (ST-EtOH, 45 min), compared with two classically used fixative solutions [Carnoy (CAR) and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h) on equine ovarian tissue. The end points evaluated were morphology (normal and abnormal) and classes (primordial and developing) of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters. Ovaries (n = 6) from ovariectomized young mares (2–8 years) were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1,444 histological slides. High heterogeneity in the number of follicles per individual mare was observed. Equine ovarian fragments fixed in both CAR treatments had a greater (P = 0.014) percentage of morphologically normal primordial and total (primordial and developing classes combined) follicles compared toST-EtOH and both PFA treatments. Although the follicular diameter, regardless of follicular class, was smaller (P = 0.001) in both CAR treatments compared to the other treatments, the nuclear diameter and the nucleus-follicle ratio were greater (P = 0.001) in both CAR treatments compared to the other treatments. A lower (P < 0.001) percentage of morphologically normal developing follicles was observed in ST-EtOH compared to all other treatments. Both CAR treatments had greater (P < 0.001) stromal cell densities than the ST-EtOH and PFA 6h fixatives. In conclusion, Carnoy's solution is the recommended fixative, rather than ST-EtOH and PFA fixatives, for studies requiring more reliable outcomes concerning cellular morphology of equine ovarian tissue. The ST-EtOH fixative proved to be an acceptable alternative to fix equine ovarian tissue when a quick histological evaluation is required for small samples prior to more complicated, time-consuming, and expensive techniques. PFA can be a reasonable fixative for morphological evaluation of equine ovarian tissue, offering the advantage of being suitable for immunohistochemistry techniques since Carnoy's solution is not recommended for this purpose. More studies are required to further understand the effects of different fixative solutions on the morphological ultrastructure and morphometry parameters in different cellular populations of equine ovarian tissue.