Repeated trans-cervical embryo recoveries in Santa inês ewes subjected to short- or long-term superovulatory treatment regimens

Highlights

• Steroids concentration may be altered by progestin priming duration and pFSH treatment.

• The duration of superovulatory regimen may alter the ovine oocyte/embryo quality.

• Transcervical embryo collection is a safe and repeatable method in superovulated ewes.

• The protocol with EB, PGF2α and OX had no adverse effects on ovine embryo viability.

Abstract

Outcomes of short- (6.5 days) and long-term (14.5 days) estrous synchronization for 6.5 d (G-6.5d) or 14.5 d (G-14.5d) and followed by the 4-day or 3-day declining-dose follicle-stimulating hormone superovulatory regimen, respectively, were compared using 16 estrous-cycling Santa Inês ewes. Non-surgical embryo recovery (NSER) procedures were performed 60 d apart starting 6 or 7 d after the onset of estrus; an i.m. injection of estradiol benzoate and of d-cloprostenol at 16 h was followed by an i.v. oxytocin injection administered 20 min before NSER. There was a longer (P < 0.05) period before estrous onset in ewes during the second (September) compared with the first study replicate (July) by approximately 14 h. The NSER could be performed in 11 of 15 ewes that were in estrus, with an average of three viable-embryos/donor and the mean duration of the procedure being 29 min. There were no differences in superovulatory responses between the two groups of ewes, but there were only degenerated embryos in ewes of the G-6.5d group. In summary: i. the duration of progestin-priming and of multiple-dose pFSH treatment had a limited effect on superovulatory responses in estrous-cycling Santa Inês ewes; ii. NSER is a safe and repeatable method of embryo collection in ewes subsequent to superovulation; and iii. duration of the superovulatory treatment regimen may alter the effects of endogenous steroids on oocyte/embryo quality in ewes.

Introduction

The multiple ovulations and embryo transfer (MOET) program is a widely used technology to increase genetics that are considered elite from individual donor females. Presently available hormonal superovulatory procedures, however, are arduous. The superovulatory treatment of sheep and goats usually entails imposing estrous synchronization treatment regimens using vaginal delivery of progesterone/synthetic progestin for 12–14 d and administration of multiple doses of exogenous follicle-stimulating hormone (Fonseca et al., 2007). It has also been suggested that prolonged treatments with progesterone-releasing devices, typically associated with less than mid-luteal phase concentrations of biological actions induced by endogenous progesterone concentrations after approximately 10 d of treatment (Letelier et al., 2009), may negatively affect subsequent fertilization and early embryonic development processes (Gonzalez-Bulnes et al., 2005). In small ruminants, long-term progesterone pre-treatments result in a shorter interval to estrus when compared with when there is imposing of short-term treatment regimens (Harl, 2014), and the timing of estrus and of the preovulatory LH peak can affect superovulatory response (Veiga-Lopez et al., 2008). Even though both the short-term (5–7 d in duration) and long-term (12–14 d) progestin-based estrous synchronization regimens can be used to effectively while the follicular super-stimulation protocols are being imposed, there has only been one study directly comparing the effectiveness of two approaches in the ewes located in a subtropical habitat (Oliveira et al., 2014).

Follicle-stimulating hormone (FSH), a primary choice for hormonal ovarian superstimulation, is typically injected twice a day at 8-h or 12-h intervals over 3–4 d, starting 2 or 3 d before the removal of progestin-containing vaginal devices, respectively (Bartlewski et al., 2016). Decreasing doses of FSH are generally preferred because the imposing of this treatment regimen appears to result in an increase ovulatory responses and embryo viability rates in ewes where there has been super-stimulation of ovarian follicular development (Gonzalez-Bulnes et al., 2000). With the varying durations of progesterone priming-treatments described previously in this manuscript, the outcomes, as a result of ovarian follicular super-stimulation using the 3-d compared with 4-d multiple-dose FSH regimens in ewes, have not been compared in a single study.

There is considerable evidence indicating trans-cervical embryo recovery can be an efficacious alternative to or even completely replace the use of the surgical embryo flushing in ewes having normal estrous cyclic patterns when there are treatments to induce superovulations (Fonseca et al., 2019a). Recently, there has been use of a combination of treatment with estradiol benzoate, d‐cloprostenol and oxytocin that induces cervical dilation, which allows for non-surgical embryo recovery and transfer in ewes (Fonseca et al., 2019b). The use of this technique still requires additional evaluations in various breeds of sheep as well as in animals undergoing different treatments to induce superovulation responses. There have not been evaluations of cervical penetration rates for embryo recoveries and the ease with which the uterine cervix can be traversed where there have been treatments to induce superovulation in ewes that are subjected to short- compared with long-term estrous synchronization treatment regimens. In addition, the effects of recurrent cervical dilation treatment regimens using estradiol benzoate, d-cloprostenol and oxytocin on the efficiency of embryo recovery have not been assessed.

The present study, therefore, was designed to compare the ovarian responses and embryo production in Santa Inês ewes subjected to either short-term (6.5 d) or long-term (14.5 d) progestin-based estrous synchronization treatment regimens and 4-d or 3-d decreasing-dose pFSH superovulatory treatments, respectively. The imposing of the short-term treatment regimens is associated with a delayed onset of estrus (Harl, 2014), indicating the occurrence of a delayed and/or longer period of antral follicular maturation. Embryos were recovered using trans-cervical uterine flushing procedures combined with SOV treatments and embryo recovery was performed at the 60-d intervals, so effects of concurrent ovarian stimulations were also assessed in the present study.