CSIRO PUBLISHING www.publish.csiro.au/journals/rfd Reproduction, Fertility and Development, 2009, 21, 901–908 In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment Giovanni Giuseppe Leoni A,D , Sara Succu A , Valentina Satta A , Mereu Paolo A , Luisa Bogliolo B , Daniela Bebbere C , Antonio Spezzigu C , Manuela Madeddu C , Fiammetta Berlinguer C , Sergio Ledda B and Salvatore Naitana C A Department of Physiological, Biochemical and Cellular Sciences,V.Vienna 2, 07100 Sassari, Italy. B Department of Veterinary Pathology and Clinic, V. Vienna 2, 07100 Sassari, Italy. C Department of Animal Biology, University of Sassari,V.Vienna 2, 07100 Sassari, Italy. D Corresponding author. Email: gioleoni@uniss.it Abstract. This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus–oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU).After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean ± s.e.m., 89.67 ± 5.74 and 26.69 ± 3.66, respectively; P < 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P < 0.01) and IVF (23.4% v. 9.1%; P < 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P < 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P < 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P < 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90β and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups. Introduction Use of prepubertal animals has been established as improv- ing selection by reducing the generation interval (Kelly et al. 2005; Morton 2008). A major factor that limits the applica- tion of this technique is the lower developmental competence of prepubertal oocytes compared with their adult counterparts (calf: Armstrong et al. 1992; goat: Martino et al. 1994; lamb: Ledda et al. 1996). Several studies have demonstrated that pre- pubertal goat oocytes have structural and functional limits that suggest a delay in the oocyte growing phase. The presence of severe cytoplasm organelle abnormalities, such as an altered distribution of cortical granules and mitochondria (Velilla et al. 2004, 2006) and microtubule and microfilament disorganisa- tion (Velilla et al. 2005) supports this hypothesis. These factors could be responsible for the low fertilisation rates and high polyspermic incidence observed in in vitro-matured and fer- tilised prepubertal goat oocytes (Izquierdo et al. 1998; Urdaneta et al. 2004). Furthermore, prepubertal goat oocytes present alter- ations in key molecule concentrations and activity such as total RNA content, p34 (cdc2) and cyclin B1 expression and matu- ration promoting factor (MPF) activity (Anguita et al. 2007a, 2008). These results suggest that prepubertal oocytes undergo incomplete maturation due to cytoplasmic (Leoni et al. 2007a) or nuclear deficiencies (Ptak et al. 2006) that would determine the low blastocyst output and quality. A large amount of data is available on the structure and functional properties of the prepubertal goat oocyte. On the other hand, data on the biology of the in vitro-derived embryos is lacking. The ability to evaluate the developmental poten- tial of the blastocyst provides a means for ensuring a high success rate for assisted reproductive technologies (ART) in small ruminants (Cognié et al. 2004; Morton 2008). Several researchers have highlighted that subjective microscopic eval- uation of embryo morphology is not directly related to the identification of embryos with a higher developmental com- petence (Maddox-Hyttell et al. 2003; van Soom et al. 2003; Guerif et al. 2007). Additional evaluation criteria such as speed of the first cell cycles (Fenwick et al. 2002), timing of cleavage © CSIRO 2009 10.1071/RD09015 1031-3613/09/070901