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The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na C /K C transporting a 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52 mM) and classified according to their cytoplasm colouration as grown BCBC (blue cytoplasm) and growing BCBK (colourless cytoplasm). Staining oocytes with 13 mM BCB during 60 min allows selection of (BCBC) the largest (123.66 mm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71G6.19S.E.M.) compared with non-stained BCBK oocytes (106.82 mm, 9% and 45.91G3.35 S.E.M. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCBC than in BCBK oocytes after in vitro maturation (3369 and 1565 AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCBC than in BCBK oocytes (1.479G0.09 and 1.184G0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

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Contents Sheep and goat production is an important economic activity in Spain with an increasing interest in milk production. Multiovulation and Embryo Transfer (MOET) and In vitro Embryo Production (IVEP) are assisted reproductive technologies aimed at increasing the genetic diffusion of females. In vitro embryo production is a multi‐step methodology comprising the following procedures: (i) In vitro Maturation (IVM) of oocytes recovered directly from the follicles, (ii) In vitro Fertilization (IVF) or co‐incubation of capacitated spermatozoa with in vitro matured oocytes and (iii) In vitro culture (IVC) of zygotes up to the blastocyst stage. In vitro embryo production from oocytes recovered from prepubertal females is called JIVET (Juvenile in vitro Embryo Transfer) and allows shortened generation intervals and increased genetic gain. Embryo production together with embryo cryoconservation would allow large‐scale embryo marketing, a pathogen‐free genetic movement and easier and cheaper germplasm commercial transactions. Commercial Embryo activity in small ruminants is low compared to cows in the European Union (data from the European Embryo Transfer Association) and in the world (data from the International Embryo Transfer Association). There is less IVEP research in small ruminants compared to other livestock species. The aim of this review was to provide an overview of the current status of IVEP of small ruminant with an emphasis on (i) description of the main methodologies currently used for IVM, IVF and IVC of embryos (ii) comparing procedures and outputs from JIVET and IVEP of adult females and (iii) the future research perspectives of this technology.