The recovery of good-quality cumulus-oocyte complexes (COCs) is essential to any laboratory involved in in vitro embryo production (IVEP), as well as in cloning or transgenesis. Overall, the high variability in the number and quality of COCs retrieved is one of the main bottlenecks impairing the worldwide propagation of IVEP in small ruminants (for a review, see Ref. [1]). In such species, laparoscopic ovum pick-up (LOPU) is the method of choice for obtaining oocytes from live females. It is a safe technique, that allows successive collections, although it demands hormonal protocols to improve the number of COCs recovered and their developmental capacity, with results relying on the FSH dose and regimen of administration adopted [2,3]. In order to optimize the ovarian superstimulation protocol in sheep, our group applied the “Day 0 protocol” (follicular wave synchronization) and evaluated four different treatments, varying only the pFSH dose and application regimen [4]. As a result, the use of the 80 mg pFSH dose provided the highest COCs quality, with an adequate gene expression pattern when applied in multiple (three) doses. Subsequently, it was demonstrated that the use of exogenous progesterone (P4) during the ovarian superstimulation protocol, compared to its analogue (medroxyprogesterone acetate), had positive effects on gene expression and oocyte competence [5]. Importantly to note is that a standard 12 h-interval from the last pFSH dose to the LOPU procedure (coasting time) was applied in both studies.

In cattle, the coasting time has been studied for about 25 years [[6], [7], [8]] as an alternative to improve IVEP results. In 1997, a group of researchers observed that the interval from FSH administration to slaughter, as well as from slaughter to oocyte retrieval, would impact the number of competent bovine COCs [9]. These authors speculated that the LH window – the period between luteolysis and the next LH surge – was shortened in superstimulated animals [10,11], which could affect the oocyte developmental competence. Later, it was reported that defining a “competence window” by adjusting the coasting time was extremely important for enhancing the oocyte competence and IVEP rates in cattle [7]. Even though the importance of defining the ideal coasting time in bovine species has been evident for many years, the literature is still incipient in sheep.

For reaching its developmental competence, the oocyte needs to accumulate inactive maternal transcripts, which will control all processes from follicle removal up to embryonic genome activation (for reviews, see Refs. [12,13]). Genes such as MATER, ZAR1, GDF9, BMP15, HAS2, and PTGS2, are indicators of oocyte competence and their expression analyses can better predict the oocyte quality [4,14,15]. Aiming for such prediction, the morphological evaluation in degrees is widely used worldwide, however, this classification is considered subjective and not a good quality predictor. The brilliant cresyl blue (BCB) test is an alternative predictor of oocyte quality that promotes the selection of a homogeneous and competent pool of oocytes in the final folliculogenesis period, when the follicle reaches its growth peak and there is full oocyte competence acquisition [2,16,17]. Another interesting possible predictor of oocyte quality is the chromatin condensation pattern of the germinal vesicle, which is remodeled during oocyte growth and is related to its development, seeming to coordinate the control of global transcription and define the developmental potential of a germinative cell [18]. Moreover, there is also a correlation between the oocyte quality and its size, as well as the follicle size, with the oocyte competence increases according to the follicle size [19].

Considering the above, we hypothesized that the coasting time may be beneficial, improving the COC quality in sheep, as in cattle [7]. Therefore, the present study aimed to evaluate in ewes the effect of different coasting times on: (1) follicular dynamics, follicle size, and COCs quantity; (2) COC or oocyte quality assessed by their morphology, BCB test, germinal vesicle chromatin condensation pattern evaluation, and oocyte diameter; and (3) gene expression analyses of COC competence markers.