Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture F.L.N. Aguiar a , F.O. Lunardi a , L.F. Lima a , R.M.P. Rocha a , J.B. Bruno a , D.M. Magalhães-Padilha b , F.W.S. Cibin c , A.P.R. Rodrigues a , M.O. Gastal d , E.L. Gastal d, * , J.R. Figueiredo a a Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), Faculty of Veterinary Medicine, State University of Ceará, Fortaleza, Ceará, Brazil b Biotechnology Graduate School, Potiguar University/Laureate International Universities, Natal, Rio Grande do Norte, Brazil c Federal University of Pampa, Uruguaiana, Rio Grande do Sul, Brazil d Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, Illinois, USA article info Article history: Received 1 June 2015 Received in revised form 9 November 2015 Accepted 14 November 2015 Keywords: Insulin In vitro culture Preantral follicle ROS Equine ovary abstract This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in a-MEM þ supplemented with 0 ng/mL, 10 ng/mL, or 10 mg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 mg/mL) for in vitro culture of equine preantral fol- licles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth. Ó 2016 Elsevier Inc. All rights reserved. 1. Introduction The progress of assisted reproductive biotechnologies in horses has been slower than in other domestic animals, mainly because of some technical barriers not present in other species and the deficient acceptance in many breed registries [1]. However, the high economic value of indi- vidual animals coupled with changing registry attitudes has resulted in a resurgence of interest and advances on horse-assisted reproductive techniques in recent years [1,2]. Studies using equine oocytes have been done with limited numbers of oocytes because of the failure of mares to respond to superovulatory regimes, and the scarce availability of horse abattoirs to collect ovaries for research projects. In this regard, the use of matured equine oocytes * Corresponding author. Tel.: þ1 618 453 1774; fax: þ1 618 453 5231. E-mail address: egastal@siu.edu (E.L. Gastal). Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.11.017 Theriogenology 85 (2016) 1063–1069