Repeated trans-cervical embryo recoveries in Santa inês ewes subjected to short- or long-term superovulatory treatment regimens
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.
Section snippets
Animals and superovulatory treatments
This study was approved by the Animal Care Committee of Embrapa Dairy Cattle (protocol 15/2014) and was conducted during a period from July to September (period of increasing day lengths characterized by the occurrence of recurrent estrous cycles in 50%–70% of ewes; Oliveira et al., 2016) at the Experimental Campus of Embrapa Dairy Cattle, in the rural area of Coronel Pacheco, MG, Brazil (latitude 21°35′S, longitude 43°15′W and altitude of 435 m.a.s.l.). A total of 16 multiparous, non-lactating
Results
Plasma P4 concentrations were greater (P < 0.05) in ewes of the G-6.5d group compared with those of the G-14.5d group at the time of first pFSH injection, MAP sponge removal and embryo collection (Table 1). The proportion of ewes with plasma P4 concentrations >1 ng/mL (mid-luteal phase concentration) was greater (P < 0.05) in ewes of the G-6.5d compared with G-14.5d group at the time of MAP sponge insertion and immediately before the time of embryo collection; at the time of embryo recovery (
Discussion
Results from quantification of circulating P4 concentrations in the ewes of the present study revealed that 28 % of animals were in the luteal phase of the estrous cycle at the time of MAP sponge insertion. These results are not completely consistent with those of a previous study by Balaro et al. (2014) where it was reported that a small percentage of Santa Inês ewes had recurrent estrous cycles between July and September when located in a subtropical and tropical climate of Brazil. A
Conclusions
The results of the present study indicate that duration of treatment regimens to induce super-ovulation may potentially alter the effects that endogenous and exogenous steroids have on oocyte/embryo quality in ewes treated to induce super-ovulations. The duration of progestin priming and multiple-dose pFSH treatment, however, had a limited effect on embryo yields in Santa Inês ewes treated to induce super-ovulations approximately 60 d apart. Furthermore, non-surgical embryo recovery (NSER) is a
Author contributions
JFF conceived the present study; MEFO, JMGS-F, JDG and JFF finalized the experimental design; FNZ, JMGS-F and JFF collected the data from the animals; MEFO, JMGS-F, JDG, JFF and FZB performed initial analyses of the data; FZB performed hormone assays and hormonal data analyses; MEFO and PMB performed additional statistical analyses; MEFO wrote the first version of the manuscript; PMB revised it critically, and MEFO, FNZ, PMB, JDG, FZB, JMGS-F and JFF all approved of the final version of the
Declaration of Competing Interest
Nothing to declare.
Acknowledgements
This study was funded by the grants from Embrapa (Brazilian Agricultural Research Corporation; Project Superovi # 22.13.06.026.00.03), CNPq (National Council for Scientific and Technological Development; Project # 310166/2012-8), and Fapemig (Foundation for Supporting of the State of Minas Gerais; Project # CVZ-PPM 00042-14) and CAPES (Coordination for the Improvement of Higher Education Personnel). MEFO, FZB, JFF and JMGS-F are CNPq fellows and JMGS-F is a FAPERJ (Research Funding Agency of
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