A Journal of Integrative Biology Changes in Testosterone or Temperature During the In Vitro Oocyte Culture Do not Alter the Sex Ratio of Bovine Embryos CARMEN DI ´ EZ 1Ã , PABLO BERMEJO-ALVAREZ 2 , BEATRIZ TRIGAL 1 , JOSE ´ NE ´ STOR CAAMAN ˜ O 1 , MARTA MUN ˜ OZ 1 , IRENE MOLINA 1 , ALFONSO GUTIE ´ RREZ-ADA ´ N 2 , SUSANA CARROCERA 1 , DAVID MARTI ´ N 1 , AND ENRIQUE GO ´ MEZ 1 1 SERIDA, A ´ rea de Gene´tica y Reproduccio´n Animal, Gijo´n, Spain 2 Dpto de Reproduccio´n Animal y Recursos Zoogene´ticos, INIA, Madrid, Spain ABSTRACT High follicular testosterone levels have been associated with a skew in the sex ratio in favor of males following in vitro fertilization, whereas egg incubation temperature has been found to influence sex ratio in some reptiles. The incubation temperature interferes with the aromatase activity, resulting in a sex determination mechanism thought to be lost in mammals. In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in vitro and to determine whether effects of sex and temperature are effectively decoupled in mammals. Bovine oocytes were in vitro matured for 22 hr in TCM199, PVA, FSH and LH after a 22 hr meiotic arrest in TCM199, PVA and roscovitine 25 mM. Matured oocytes were in vitro fertilized and cultured up to Day 3, and embryos having three or more cells were sexed. In the first experiment, testosterone (0, 30, 300 and 1,500 nM), present both during meiotic inhibition and subsequent in vitro maturation (IVM), did not affect development rates or embryonic sex ratio. In the second experiment, increasing incubation temperatures (38, 39 or 401C) during meiotic inhibition and subsequent IVM, reduced embryo development, but did not change the sex ratio. Under our experimental conditions, testosterone does not promote a preferential selection of Y-chromosome bearing spermatozoa by the oocyte, and temperature and sex ratio seems to be decoupled in mammals. J. Exp. Zool. 311A:448–452, 2009. r 2009 Wiley-Liss, Inc. How to cite this article: Dı ´ez C, Bermejo-Alvarez P, Trigal B, Caaman ˜o JN, Mun ˜oz M, Molina I, Gutie ´rrez-Ada ´n A, Carrocera S, Martı ´n D, Go ´mez E. 2009. Changes in testosterone or temperature during the in vitro oocyte culture do not alter the sex ratio of bovine embryos. J. Exp. Zool. 311A:448–452. The ability to preferentially produce either male or female offspring would be of enormous econo- mic benefit to the livestock industry. However, mechanisms for a satisfactory system of adaptive control of the sex ratio in mammals remain unexplained (Williams, ’79; Reiss, ’87; Hardy, ’97; Krackow, 2002; Jime´nez et al., 2003). In many reptile species, sexual differentiation of gonads is sensitive to temperature (temperature-dependent sex determination) during a critical period of embryonic development (thermosensitive period, TSP) (Lance, 2008). Experiments carried out with different models, including turtles, crocodilians and lizards, have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme that converts androgens to estrogens) in the ovary differentiation during TSP and in the ovary structure maintenance after TSP (Pieau et al., 2001). In some of these experiments, the occurrence of various degrees of gonadal inter- sexuality is related to weak differences in aroma- tase activity, suggesting that environmental temperature can affect the transcription level of the aromatase gene. Published online 21 April 2009 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jez.540 Received 6 February 2009; Revised 20 March 2009; Accepted 24 March 2009 Grant sponsor: CAJASTUR; Grant numbers: AGL2008-01530; RTA2008-0082; Grant sponsors: MICIN; Fondo Social Europeo. Ã Correspondence to: Carmen Dı ´ez, SERIDA, Camino de los Claveles 604, Somio´. 33205 Gijo´n, Spain. E-mail: mcdiez@serida.org r 2009 WILEY-LISS, INC. JOURNAL OF EXPERIMENTAL ZOOLOGY 311A:448–452 (2009)