Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue Carolina M. Lucci a,b,* , Mirella A. Kacinskis a , Luiz Henrique R. Lopes a , Rodolfo Rumpf b , So ˆnia N. Ba ´o a a Lab. de Microscopia Electro ˆnica, Depto. de Biologia Celular, IB, Univ. de Brası ´lia, 70910-900 Brasilia, DF, Brazil b EMBRAPAGenetic Resources and Biotechnology, Brasilia, DF, Brazil Received 13 December 2002; accepted 5 June 2003 Abstract Cryopreservation of ovarian tissue is a new and promising technique for germ-line storage. The objective of this study was to evaluate the effect of four cryoprotectants (at two concentrations each) on the preservation of zebu bovine preantral follicles after ovarian cryostorage. Strips of ovarian cortex were cryopreserved using glycerol (GLY; 10 or 20%), ethylene glycol (EG), propanediol (PROH) or dimethylsulphoxide (DMSO; 1.5 or 3 M). In addition, a toxicity test was performed for each cryoprotectant by exposing the ovarian tissue to them without freezing. Tissues were analyzed by histology and transmission electron microscopy. Ovarian tissue frozen in either concentration of DMSO or PROH or in 10% GLY retained a higher percentage of morphologically normal follicles (73–88%) than tissue frozen in 20% GLYor in either concentration of EG (16–52%). In the toxicity test, exposure of tissues to DMSO, PROH or GLY resulted in higher percentages of normal follicles (80–97%) than exposure to EG (49%). Electron microscopy revealed damage to the ultrastructure of follicles frozen in 10% GLY, while follicles cryopreserved in DMSO and PROH at either con- centration exhibited normal ultrastructure. In conclusion, DMSO and PROH were the most effective cryoprotectants for zebu ovarian tissue, preserving the structural integrity of somatic and reproduc- tive cells within the ovary. # 2003 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Ovarian tissue; Preantral follicles; Cryoprotectants; Bovine Theriogenology 61 (2004) 1101–1114 * Corresponding author. Tel.: þ55-61-307-2424; fax: þ55-61-347-6533. E-mail address: cmlucci@unb.br (C.M. Lucci). 0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.06.004