Ovarian response to P4‐PGF‐FSH treatment in Suffolk sheep and P4‐PGF‐PMSG synchronization in cross‐bred ewes, for IVD and ET protocol

Abstract Background The success of an embryo transfer protocol in sheep depends on many factors, but the choice of drugs for the desired superovulation as well as the conception rate (CR) are most essential. Reproductive activity in sheep is characterized by a seasonality influenced by several factors such as photoperiod, latitude, temperature, nutrition and breed. Reproductive seasonality and nutritional condition are the main factors that influence embryo production in sheep. In sheep, some anatomical peculiarities limit the application of traditional reproductive biotechnologies used in cattle. Objectives The aim of this study was to conclude on the effectiveness of a wider on farm in vivo embryo transfer development programme in Suffolk sheep by streamlining hormone therapies and optimizing technique. Methods A total number of 60 sheep and three rams were included in this study, divided into two groups (receptors and donors). Donor Suffolk sheep were treated for superovulation using the P4‐PGF‐FSH multiple ovulation embryo transfer (MOET) protocol, while the cross‐bred recipients’ group was synchronized with P4‐PGF‐PMSG. Results On the first day after superovulation, all ovaries had more than five dominant follicles, while corpora lutea were later observed in 83.3% sheep. The recovery rate was 83.3%, while 72.9% embryos were transferable. Embryos were transferred directly into recipients. Fertility after 30 days was 68.57%, lambing rate was 91.6% and CR was 62.85%. This study showed that veterinary drugs (P4, FSH, LH, PMSG, PGF) used for superovulation optimized by us were capable of producing by this improved technique the optimization of the reproduction indices at embryo‐transfer (ET) and to be able to be used successfully. Conclusions The application of an MOET protocol has a positive effect in the production of in vivo embryo production (IVD) embryos in Suffolk sheep and can guarantee the success of embryo transfer activity to ewes with lower genetic merit. Our research aimed at representing a model for sheep farms for a rapid improvement of productive traits.


INTRODUCTION
Embryo transfer (ET) in sheep is a well-known and insufficiently applied reproductive biotechnology. Therefore, there is a need for an optimization and a continuous improvement of the techniques (Ciornei, 2021).
Sheep size (small ruminants) anatomical features (cervix) and reproductive behaviour (seasonal), which are not common to cattle, present challenges that lead to the need to update the technology to make it easier to apply. (Ciornei, Drugociu, Rosca, & Liliana, 2020;Hafez, 1952).
Reproductive activity in sheep is characterized by a seasonality influenced by several factors such as photoperiod, latitude, temperature, nutrition and breed. Reproductive seasonality and nutritional condition are the main factors that influence embryo production in sheep. Nutrition has a significant effect on several aspects of reproduction including hormone production, fertilization and early embryonic development. The relationship between sheep nutrition and embryo outcomes has not been established conclusively, but undernutrition can compromise follicular waves, luteal secretions and embryo development (Ciornei, Drugociu, Rosca, & Liliana, 2020;Zeleke, 2014). In sheep, some anatomical peculiarities limit the application of traditional reproductive biotechnologies used in cattle. The cervix of small ruminants is a sphincter-like structure at the base of the uterus that is anatomically complicated consisting of a fibromuscular canal with multiple folds of tissues or rings. The size and shape of the cervix vary by breed and can change with the reproductive cycle, history and age of the female (Kershaw et al., 2005;Dayan et al., 2010).
Many combinations of treatments for the purposes of embryo collection and transfer are available. Estrus can be synchronized by the administration of progestogens (P4), prostaglandin F2 alpha (PGF) and pregnant mare serum gonadotrophin (PMSG) (Mircu et al., 2020;Roche et al., 2011).
In vivo embryo production is often referred to as 'multiple ovulation and embryo transfer' (MOET) and involves ovarian superstimulation of the donor female, insemination or mating, uterine flushing for embryo recovery and either cryopreservation or transfer of collected embryos to recipients (Menchaca et al., 2009;Zhu et al., 2018).
MOET has the potential to increase the rates of genetic improvement in sheep. However, better realization of this potential requires a higher yield of transferable embryos (Menchaca et al., 2009). In vivo embryo production activities in small ruminants started as early as the 1930s with the first successful report in 1934. Since then, the great majority of embryo recovery and transfer attempts were performed by surgical procedures.
Laparotomy technique allows exact counting of the number of corpora lutea (CL) and evaluation of total embryo recovery rate (RR). However, disadvantages are the relative high cost of equipment and stress to the animal due to manipulation of the exteriorized reproductive tract (Mircu et al., 2020).
The pregnancy rate following transfer of fresh embryos was satisfactory in small ruminants but not all those that were confirmed pregnant also arrived at parturition, thus reducing the number of lambings in recipient sheep. The pregnancy rate following transfer of vitrifiedthawed embryos was generally low and unsatisfactory (Ciornei et al., 2015).
Suffolk is considered to be a large breed of sheep. Their large frame and muscular bodies make them an ideal breed for meat production, and their wool production is also good. Suffolk rams are commonly used as a terminal sire on cross-bred ewes due to their ability to produce off-spring with excellent growth and carcass traits.
The productivity of Romanian indigenous sheep is low due to poor genetic merit, poor nutrition and weak management (Alam et al., 2001;Zamfirescu et al., 2000). MOET is well accepted and applied worldwide to speed up genetic gain through production of large number of lambs, reducing generation interval and utilization of superior dams (Bari et al., 2003;Menchaca & Hunton, 2020). The MOET programme, followed by direct transfer into recipients, can reduce the cost of ET if the estrus of donors and receptors is concomitant and the time of ovulation is synchronized at the same time (Ghosh et al., 2017). The hormonal methods are intended to shorten the life span of an existing corpus luteum by luteolysis drug or mimicking the corpus luteum function (9-19 days). Alternatively, esrtus synchronization may be achieved by manipulation of both follicular and luteal phase through gonadotropinreleasing hormone (GnRH) in combination with PGF2α. In anoestrous sheep, induction of estrus was accomplished with intravaginal sponges impregnated with progestagens, medroxyprogesterone acetate (MAP) (Evans et al., 2003), inserted from 12 to 14 days. Before this, equine chorionic gonadotropin (eCG) was administered (Zeleke et al., 2014).
The most commonly used gonadotropins for superovulation of the donor are eCG and porcine/ovine (p/oFSH). Of these, eCG was the first drug widely used for superovulation at a dose of 1000-2000 IU.
Because of their short half-life, follicular stimulation hormone (FSH) preparations need to be administered repeatedly, ranging from six to eight applications, in decreasing doses, at 12-h intervals (Brasil et al., 2016;Driancourt, 2001).
The ovarian follicular condition, at the beginning of the overstimulation treatment, is of great importance for the final embryonic production. The ovulatory response and the total number of transferable embryos can be affected by the number of small follicles on the first day of the superstimulatory treatment and by the presence of a dominant follicle. One approach that can be used to increase the control of the follicular growth is the 'day 0 method' , which consists of starting superstimulation with FSH on the day of ovulation, which coincides with the onset of the first follicular wave.
Superovulation protocol by the combination of progesterone and estradiol was frequently used. The mechanism responsible for suppressing the dominant follicle and inducing the emergence of a new follicular wave appears to be a systemic mechanism and involves both FSH and luteinizing hormone (LH) suppression for at least 24 h. In sheep, the administration of 0.5 mg of 17β-estradiol or estradiol benzoate results in the emergence of a new follicular wave between 3.5 and 4 days later (Ramos & Silva, 2018).
The duration of progesterone long time treatment before FSH administration did not affect the ovulation rate, or the embryo yield in cyclic ewes could be enough using a single progesterone insert for 5-7 days, without the need to replace it in 14 days, and follicular waves start every 4-5 days. In addition, from a practical perspective, the possibility to administer the treatment between 5 and 14 days without affecting the results has implications for simplifying implementation of MOET programmes. Progesterone is one of the principal regulators of the endometrium during early pregnancy (Brasil et al., 2016;Shorten et al., 2018;Zamfirescu, 2010;Zeleke, 2014).
In Romania, in the last 15-20 years, the ovulation rate of sheep after eCG treatments was on average 3-12 and 5-16 after superovulation treatments with FSHp (Zamfirescu et al., 2000). The number of viable embryos was between 3 and 8 according to treatment and especially according to the individual variability of the donors. The highest ovulation rate (14) is obtained after treatments with FSH-p and 13.7 after treatments with PMSG and Neutra-PMSG (Zamfirescu, 2010).
There is a genetic component in how each breed (Suffolk) responds to the hormone given to stimulate multiple egg release. Some donors do not respond to a standard dose, while others have a large response. This study sought to evaluate the ovarian response and superovulation protocol (SOP) in Suffolk sheep that were previously acclimatized in Romania, the embryo RR, in vivo embryo production (IVD) and conception rate (CR) after transfer in cross bred recipients. We start from the hypothesis that the ovarian response is similar to sheep subjected to a well-established treatment, and we want to follow to what extent the ovarian reaction, embryo production and gestation rate is applicable to acclimatized meat breeds. Our research aimed at representing a model for sheep farms with low genetic background that wish for a rapid improvement of productive traits.

MATERIALS AND METHODS
The present study was conducted during normal breeding season (autumn) in the North-Eastern region of Romania.
An initial number of 60 ewes and three rams were included in this study. According to ET, in vivo protocol, a donor needs 10 sheep for synchronization for ET direct. The response of the receptors to the stimulation of estrus and to the grouping of ovulation with the donor varies depending on the treatment and the breeding season (40%-90%). In fact, the average transferable embryos per flushing session is 3-5, which must be transferred immediately to the uterus of perfectly synchronized recipient.
The females were divided into two groups: Group 1-donors' group (10 Suffolk sheep with superior genetics) and Group 2-recipients' group (50 local crossbred sheep).

Donors management
For this experiment, 10 Suffolk ewes were initially selected to be included in the ET protocol. Following offspring evaluation and a complete clinical and gynecological examination, performed according to embryo donor's selection criteria (IETS manual 2020), only six ewes (60%, 6/10) aged 2.5-5 years were subjected to SOP.
One month before the beginning of the study, each ewe was subjected again to general physical examination and deworming. The epidemiological health record of the farm was in accordance with the official regulations.
The ewes were kept on a natural pasture from 06:30 to 18:00 h and were housed in outdoor pens overnight. Water and a mineral supplement were available ad libitum. The management of the farm did not change throughout the whole experimental period.

Rams management
Three Suffolk rams were selected for biostimulation, estrus detection and breeding. The ram with the best pedigree and reproductive performance was retained for donor mounting, while the other two were used as teaser rams. About a month before, they were separated and fed with a high-energy ratio as well as vitamin-mineral supplements. In order to stimulate libido and spermatogenesis, a dose of GnRH agonist-10.5 μg busereline acetate (Receptal, MSD, Holland) was administered 30 days before mounting. Seven days before starting the protocol, an andrological examination was performed including sperm collection and qualitative evaluation of the ejaculate.

Estrus management
In order to improve estrous behaviour and ovulation (inducing the male effect), the presence of teaser males was permitted from the moment of intravaginal device removal until 60 h later. This was also required for estrus detection (which occurred 36-60 h after FGA sponge removal). Upon estrus detection, three mounts were allowed at 12 h interval in the donor group using the designated ram. The other two teaser rams detected the ovulatory moment in the recipient group.
In order to ensure accurate donors' and recipients' synchronization, the recipient ewes which accepted rams' mounting in the same time as donors (or within an interval of 6-12 h) were identified.

Embryo collection
Seven days after mounting, the embryos were recovered by the laparoscopic surgical technique (Berg et al., 2010).
Donors were not allowed to eat for 18 h before surgery, while water removal was done 12 h before surgery. Donors were premedicated with a suitable epidural anaesthesia (60 mg procaine hydrochloride-Procarom 2% Romvac, Romania), antibiotic (enrofloxacin 5 mg/kg, MI, 7 days -Enrofloxarom 5%, Romvac, Romania) and non-steroidal anti-inflammatory (flunixin 1.5 mg/kg, IN, 3 days-Megadyne, Virbac, India), given well in advance of any procedure. The ewe was placed in dorsal recumbency on a specially designed laparoscopy cradle. Both ovaries were examined by ultrasound and laparoscopy. If a superovulatory response of ≥3 well-developed CL was recorded, the donor was considered acceptable for embryo collection. Uterine flushing was performed using Vigro complete flush (Vetoquinol, USA) a two-way catheter (Vortech 14Ch) and a filter (EmSafe Filter).

Evaluation of in vivo-derived sheep embryos
Examination of the recovered flushing fluid was performed under a 20-80× magnification using a stereomicroscope. Criteria for embryo evaluation and classification were the same as for bovine embryos and as The excellent and good embryos (code 1) were loaded into the Tomcat catheter in a small volume of holding medium between two air bubbles positioned on both sides of the embryo.

Embryo transfer procedure
ET in the recipients' group was performed by a minimally invasive surgical method using laparoscopy, which allows the placement of embryos directly into the cranial portion of the uterine horn. This is the standard method used worldwide (Menchaca et al., 2020). The embryo was placed in the ipsilateral horn of the good quality CL.

RESULTS
All superovulated ewes responded to stimulation, and new follicular waves were identified on the ovaries, by transrectal ultrasonography.
On the first day after donors' SOP treatment, all ovaries had more than five follicles (Figure 3a), with no significant differences between ewes and ovaries. Nevertheless, 7 days after estrus, CL were only observed in 5/6 ewes (83.3% had ovulated).
At the time of abdominal laparotomy, an average was 9.1 CL/sheep were identified. The distribution within the six donors was: 0, 11, 12, 15, 9 and 8. The total number of CL observed was 55, 29 on the right ovary and 26 on the left one. The distribution right/left of CL was 0/0, 6/5, 7/5, 7/8, 4/5 and 5/3 (Figure 3b). The management and quality of recipient females are key factors in a successful ET programme. The presence of males (teasers) was from intravaginal device removal to 60 h later and is required for detection of estrus (36-60 h), and for inducing male effect to improve estrous behaviour and ovulation.

DISCUSSION
The development of this reproductive biotechnology in sheep has had a similar development in the past, especially in important breeds of sheep and goats (such as the Suffolk breed). Small ruminant ET is a welldescribed and yet underexploited animal breeding technology. The size of sheep and goats, aspects of their anatomy and seasonal reproductive behaviour present challenges not common to cattle.
The control of ovarian activity in sheep has been and still is of interest to farmers, but the results are variable. Any method of stimulation that increases efficiency is welcome. Hormone therapy is described as a safe method of control, and their association tends to maximize the response.  later use. Obtaining embryos is influenced by the development dynamics of the ovarian follicles, and also by their ovulation (Alam et al., 2001).
Response to superovulation: approximately 25% of programmed donors will not respond to superovulation treatments. Some never respond, some may respond on a subsequent programme. If the donor responds, the donor may produce from 1 to over 30 embryos, with average 8-12 depending on the breed, time of year, and the condition of the animal (Bari et al., 2003;Ghosh et al., 2017;Menchaca et al., 2009).
The superovulatory ovarian response to our donors was 83.3%, a fairly high percentage given that the medication used was long last-ing and used several hormones involved in reproductive function. The result is at the upper limit of those published by other authors in similar experiments (Bari et al., 2003;Hafez, 1952;Shi et al., 2015). Note that 77.8% of females showed ovulation with a mean of 9.6 CL and 3.3 viable embryos (González-Bulnes et al., 2004).
In such an ET direct/in vivo programme (donor flushing and ET on the same day), it is recommended that 8-10 recipients per donor are synchronized to ensure that at least 6-8 recipients will be suitable for direct ET. The number of recipients initially programmed per donor may be adjusted if there is prior information about the expectation of donor response and potential embryo yield (Mircu et al., 2020;Manchera et al 2020, -according to IETS Manual 2020).
An effective estrus synchronization regimen (recipient group) is expected to synchronize the estrus of the treated animals within a 12 to 24-h period, stimulate high rates of estrus and ovulatory response and enable the achievement of a high pregnancy rate. The most common methods used for the hormonal control of the estrus cycle in ewes include the establishment of an artificial corpus luteum function through the administration of progestogens for a certain time period, the stimulation of luteolysis by means of the administration of luteolytic agents and the synchronization of ovulation by means of the combined administration of GnRH.
In this method, the function of the corpus luteum is simulated by application of analogous progesterone compounds. The release of gonadotropins is inhibited by progesterone, and hence the ovulation is also inhibited until progesterone is removed (Figure 2). Applied to a group of receptors, it will synchronize estrus and ovulation.
Progesterone was initially delivered for a period equal to the length of the natural luteal phase. There are various administration time protocols such as long-term progesterone treatments (18-21 days) and short-term progesterone treatments (7-12 days). Therefore, it is crucial to include a luteolytic agent in combination with short-term progesterone treatments in order to get rid of any natural corpus luteum. whereas the viability rate of embryos is higher in September (Shi et al., 2015). In another similar study, CR in Suffolk was 59% (Fair et al., 2006).
Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilization failure as a possible consequence of seasonal shifts in LH secretion and/or associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo, but those produced during in non-breeding season have lower viability following in vitro culture (Mitchell et al., 2002).
This study sought to evaluate the ovarian response to SOP, embryo RR, IVD in Suffolk sheep acclimatized in Romania as well as CR in local recipients. Results proved that the MOET protocol applied had a positive effect on the production of IVD in Suffolk sheep and can guarantee the success of ET activity to ewes with lower genetic merit.

CONCLUSIONS
The These results were even above those published in the literature and can grantee the success of ET activity of this breed in Romania.

ACKNOWLEDGEMENT
We would like to thank the sheep farmer for his cooperation.

CONFLICT OF INTEREST
The authors declare no conflict interest.