03765naa a2200301 a 450000100080000000500110000800800410001902200140006002400380007410000170011224501640012926000090029350009060030252018960120865300220310465300090312665300270313565300350316270000170319770000190321470000140323370000190324770000180326670000210328470000190330570000170332477301220334110643652023-11-10       2023    bl uuuu     u00u1 u    #d  a1642-431X7 a10.1016/j.repbio.2023.1008142DOI1 aBORGES, M.A.  aEthanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.h[electronic resource]  c2023  aArticle history: Received 28 February 2023; Received in revised form 24 September 2023; Accepted 27 September 2023; Available online 25 October 2023. -- Correspondence author: Gastal, E.L.; Animal Science, School of Agricultural Sciences, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, United States; email:egastal@siu.edu  -- FUNDING: This research was supported by grants from the Brazilian Criollo Horse Breeder Association (ABCCC) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS; grant no. 21/2551-0002239-6). A.S. Varela Junior, C.D. Corcini, and B.R. Curcio had research productivity fellowships from the National Council for Scientific and Technological Development (CNPq). M.A. Borges and L. Gheno received DS and MS scholarships, respectively, from the Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil. --  aABSTRACT.- The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares 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 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted. © 2023 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn  aFixative solution  aMare  aOvarian tissue quality  aPreantral follicle morphometry1 aCURCIO, B.R.1 aGASTAL, G.D.A.1 aGHENO, L.1 aJUNIOR, A.S.V.1 aCORCINI, C.D.1 aNOGUEIRA, C.E.W.1 aAGUIAR, F.L.N.1 aGASTAL, E.L.  tReproductive Biology, December 2023, Volume 23, Issue 4, Article 100814. https://doi.org/10.1016/j.repbio.2023.100814