Non-surgical embryo recovery (NSER) is an effective non-invasive technique for embryo recovery procedures in small ruminants. In different sheep breeds, the technique achieves a successful rate ranging from 70 to 95 % [[1], [2], [3]]. This represents an important alternative to surgical embryo collection methods (laparotomy and laparoscopy), which may prove crucial for a consumer market increasingly worried about animal welfare in the production chain. Furthermore, the use of this technique allows for the reduction of intervals between collections and anatomical changes – such as adhesions – which surgical embryo collection requires. Notably, the success of the NSER procedure depends on the previous induction of cervical relaxation, which is usually based on prostaglandin F2α (PGF), estradiol benzoate (EB), and oxytocin (OT) administrations [4]. These hormones relate to extracervical matrix remodeling, inducing the dispersion of collagen fibers and increasing the cervix flexibility [5]. The major problem, however, is that these are also associated with the luteolytic process, making them undesirable in superovulated ewes [6].

Physiologically, the cervical relaxation protocol may trigger deleterious effects that are similar to the early luteal regression and may be harmful to the embryo. A PGF discharge, induced by OT and/or E2 during the early luteal phase, plays an embryotoxic role in reducing fertility in estrus-induced ewes [7] and impairs embryo quality and recovery rates in superovulated ewes [8]. Endocrinologically, cervical relaxation protocol administered six days after ovulation has been shown to affect luteal function by reducing luteal area and progesterone concentration [6,9], at the same time as increasing estradiol levels by approximately two to five-fold [6]. To date, the embryo-level effects of administering these luteolytic drugs before embryo recovery have not been fully elucidated.

It is well known that, after cervical relaxation protocols with PGF alone in goats [10] or PGF-OT-E2 in sheep [11], fresh or frozen-thawed embryos from donors undergoing NSER achieve acceptable to good pregnancy rates. However, in addition to the recovery rate, Santos et al. [12] reported that NANOG and OCT-4 (important markers of embryonic pluripotency at the blastocyst stage) had affected expression, after the cervical relaxation protocol, although these were normalized after 24 h of in vitro embryonic culture. Despite this observation, the authors did not observe effects on embryonic morphology or apoptotic index. In view of this, we can speculate that the time taken between the induction of luteolysis and embryonic recovery may be short enough to compromise the embryo's morphological viability. However, it is possible that changes – even if transient – at molecular or cellular levels may lead to irreversible damage to the subsequent embryo development.

Considering the endocrine impacts of cervical relaxation protocols with or without EB and its possible embryotoxic effects, this study has focused on evaluating the efficiency of NSER procedure, embryonic morphology, and in vitro viability and survival of cryopreserved ovine embryos recovered after cervical relaxation protocols using 0.0 mg, 0.5 mg, or 1.0 mg of EB associated with PGF and OT. Additionally, the genes involved in maintaining pluripotency (OCT4 and NANOG), cell stress (HSP90 and PRDX1), and apoptosis (BCL2 and BAX) have also been assessed.