The effect of prostaglandin and gonadotrophins (GnRH and hCG) injection combined with the ram effect on progesterone concentrations and reproductive performance of Karakul ewes during the non‐breeding season

Abstract The effect of prostaglandin and gonadotrophins (GnRH and hCG) combined with the ram effect on the progesterone (P4) concentrations and reproductive performance of Karakul ewes was investigated during non‐breeding season. Ewes (n = 93) received a male effect and were divided into two treatment groups including GnRH ‐ hCG (hCG, n = 32), GnRH ‐ GnRH (GnRH, n = 30) and a control (n = 31) group. This study was carried out from April (hormonal injection) to October (lambing). The first doses of GnRH (4.2 μg, Buserelin) were injected at the beginning of the study in treatment groups. These ewes were treated with hCG (250 IU) or the second GnRH dose five days later. All animals received two injections (ten days apart) of 150 μg PGF2α five days after the hCG or the second GnRH injection. Mating was initiated two days after the second prostaglandin injection and lasted for 34 days. Blood samples were collected by jugular venipuncture on days −10, −5, 0 (first PGF2α injection), 17 and 30 during the study. Pregnancy diagnosis was performed through transabdominal ultrasonography on day 40 after the removing of ram. Conception rate was 93.8, 90 and 87.1% in the hCG, GnRH and control groups, respectively. Lambing rate tended to increase in the hCG group compared with the control group (87.1 versus 58.1%; p < .1). There was no significant difference in P4 concentrations among studied groups in identical sampling times (p > .05). In conclusion, the administration of prostaglandin and hCG in combination with the ram effect tended to decrease lambing period. In other words this protocol tended to increase lambing rate at the first cycle. Treatment with hCG or GnRH did not increase serum P4 concentrations of treated Karakul ewes during the non‐breeding season.


| INTRODUC TI ON
Ewes are known as animals with seasonal reproductive activity.
The most commonly used synchronization methods are based on the use of progesterone or prostaglandin protcols. The intravaginal administration of progesterone with eCG injection is a common method of synchronization in sheep (Swelum, Alowaimer, & Abouheif, 2015). Although progesterone-based programmes are preferred, these programmes are doubtful. There is a need to reconsider the protocols based on the use of progestagens for societal reasons such as animal health and welfare, food safety and the environmental impact (Gonzalez-Bulnes, Menchaca, Martin, & Martinez-Ros, 2020). Vinoles, Paganoni, Milton, Driancourt, and Martin (2011) reported that the adding antibiotics before the sponge insertion reduced the amount of mucus and odour compared with the control group. There was a possibility of the intravaginal sponges and antibiotic residues used to prevent vaginal infections (Berruga, Rodriguez, Rubio, Gallego, & Molina, 2008).
Prostaglandins are rapidly metabolized in the lung and have no tissue remnants (Davis, Fleet, Harrison, & Walker, 1980). Therefore, the use of PGF2α or its analogues is a good alternative synchronization method in sheep.
The use of gonadotropin releasing hormone (GnRH) or the male effect in PGF2α-based protocols was reported previously (Mirzaei et al., 2017;Olivera-Muzante, Gil, Fierro, Menchaca, & Rubianes, 2011). Human chorionic gonadotropin (hCG) and GnRH administrations on the day of mating or post-mating were applied for improving the reproductive performance (conception, lambing, twining rate and litter size) of different breed (Akkaraman, fat tailed, Afshari × Booroola-Merino crossbred, Anatolian Merino) ewes (Ahmadi & Mirzaei, 2016;Ataman, Aköz, Sarıbay, Erdem, & Bucak, 2013;Dursun, 2019;Rostami, Hajizadeh, Shahir, & Aliyari, 2017). A higher plasma P 4 concentrations was reported in post-mating hCG-treated groups in Afshari × Booroola-Merino crossbred ewes by Rostami et al. (2017). Injection of GnRH on the day of oestrus or at the time of mating and 7 or 9 days later increased serum P4 concentration. and enhanced pregnancy rate and litter size (Hashem, El-Azrak, Nour El-Din, Taha, & Salem, 2015;Zonturlu et al., 2018). High pregnancy rates with natural mating were reported after treatment including GnRH injection at device insertion of progesterone during the out of breeding season (Martinez et al., 2015). In non-breeding season, there was a tendency to a greater formation of CL and pregnancy rate in GnRH-treated ewes compared with control (ram introduction alone) group (Jordan, Inskeep, & Knights, 2009). One injection of GnRH in a PGF2α-based protocol combined with the ram effect enhanced lambing rate and litter size during non-breeding season in Karakull ewes (Mirzaei et al., 2017).
The hypothesis being that GnRH or hCG treatments combined with the ram effect could induce ovulation in the ewes. The aim of the present study was to evaluate the effect of prostaglandin and GnRH or hCG in combination with the ram effect on progesterone concentrations and reproductive performance of Karakul ewes during the non-breeding season. Plasma progesterone concentrations were used for assessing the response to hormonal injection, with regard to ovulation.

| Animals and flock management
The present study was performed in a Karakul flock during the non-breeding (April to September) season in Saadat Shahr, Fars province, Iran. Saadat Shahr is located at latitude of 30° 3' N and longitude of 53° 7' E. Its altitude is 1892 m above sea level. A total of 93 non-lactating Karakul fat-tailed ewes were used during the study period.This study was carried out from April (hormonal injection) to October (lambing). The ewes were vaccinated and received anti-parasitic drugs before starting the study. Ewes were divided into three groups, with respect to body condition score (BCS) and age, including two treatment groups (hCG and GnRH groups) and an untreated control group. Body condition score (BCS) of each ewe was determined through back vertebral palpation (1-5 points) (Pugh, 2002). The mean (± SD) of BCS of ewes in GnRH -hCG (n = 32), GnRH -GnRH (n = 30) and control (n = 31) groups were 3.20 ± 0.33, 3.28 ± 0.31 and 3.16 ± 0.27, respectively. The teeth formula and farm's records were the criteria for excluding very young and old ewes (Pugh, 2002). The mean (±SD) age of ewes in hCG, GnRH and control groups were 3.22 ± 0.38, 3.20 ± 0.36 and 3.18 ± 0.42 years old, respectively. They are multiparous (parity 2 and 3) and dry ewes. All ewes grazed on the natural pastures and were flushed with balance (alfalfa hay (23%), corn silage (67%) and barley grain (10%) ad libitum) from 3 weeks before breeding to the removing of ram (about 55 days). Mineral salt (containing 19.6% calcium and 9.6% phosphorus) and water were offered ad libitum. Ewes were kept away (3 months) from the rams before starting the study.
At the time of first GnRH injection (Day -10 before the first injection of PGF2α), ewes and rams were kept in close proximity to each other through fences at nights.

| Hormonal treatments
Ten days before the first injection of PGF2α (Day -10 before the first injection of PGF2α; Figure 1), ewes in the GnRH -hCG (hCG) and GnRH -GnRH (GnRH) groups received an intramuscular injection of GnRH (4.2 μg, Buserelin, 4.2 μg/ml, Aburaihan, Iran). This injection was given in order to induce ovulation or luteinize for mature and immature follicles, respectively. Five days later after the GnRH dose (Day -5 before the first injection of PGF2α; Figure 1), ewes were treated with hCG (250 IU, IM, LG Chem, Ltd.; South Korea; hCG group) or the second GnRH dose (4.2 μg, Buserelin; GnRH group) for inducing ovulation. Animals considered as a control group received distilled water as a placebo on the same days.
Mating was initiated two days after the second prostaglandin injection and lasted for 34 days later (from day 12 to 46 after the first injection of PGF2α, two oestrous cycles; Figure 1). Twelve fertile Karakul rams (four males per each treatment) with good body condition scores were released into the ewes. The hormonal treatments of the present study are shown in Figure 1

| Sampling and Ultra-sonographic examination
Blood samples (n = 93) were collected by jugular venipuncture on days −10, −5, 0, 17 and 30 during the study (Figure 1). Serums were separated through centrifugation at 1,500 × g for 10 min and were stored at −20°C. Serum P4 concentrations were determined using a validated commercial ELISA kit (IBL International, Hamburg, Germany). The intra-assay and inter-assay CV were 5.4% and 8.6%, respectively. The sensitivity of the assay was 0.1 ng/ml. The ewes with serum P4 concentrations of more than 1 ng/ml on day 0 (first dose of PGF2α injection) were considered as ovarian responders (ovulation) to hCG or second GnRH administration (Mirzaei et al., 2017). Pregnancy diagnosis was performed by transabdominal ultrasonography on days 40 after the removing of ram in standing position using a real-time ultrasound scanner equipped with a 3.5 MHz convex probe (KX5200, Kaixin, China). The probe was applied at the inguinal region of right side after adding the gel (Aziz & Lazim, 2012). The conception rate (based on the pregnancy diagnosis), lambing rates, fetal mortality rate, litter size (lambs/ lambed ewe) and weights of lambs were recorded for the evaluations.
The reproductive performance of ewes was evaluated by calculating the following variables: Conception rate = (number of pregnant ewes at pregnancy diagnosis/number of exposed ewes) × 100; Lambing rate = (number of ewes lambing/number of exposed ewes) × 100; Fetal mortality rate = [(number of detected embryos at pregnancy diagnosis -number of lambs born)/number of detected embryos at pregnancy diagnosis] ×100. We recorded the data of lambing (lambing period) on two consecutive cycles. First and second cycles were considered from 151 to 159 and 166 to 181 days after ram release, respectively. Pregnant ewes conceived at induced and first spontaneous oestrus, they lambed during the first and second cycle, respectively. Overall lambing rate was defined as the proportion of ewes that lambed in two consecutive cycles.  Data were presented as the percentage or mean (±SE), and values of p ≤ .05 were considered as significant data.

| Progesterone concentrations
The results of P4 concentrations analysis based on repeated measurements are shown in Table 1. The time effect on the increasing of progesterone changes was significant during the study (p < .05).
The mean of P4 concentrations of different studied groups is shown in Figure 2. Analysis of P4 concentrations between days -5 and 0 relative to the first PGF2α dose showed no significant differences among treatment and control groups (p > .05; Figure 2). There was no significant difference among groups in serum P4 concentration (p > .05; Table 2).  Table 2). No significant differences in the other reproductive indices (Ovulation, conception and foetal mortality rate) were found among the studied groups (p > .05; Table 2). The number of lambs born per lambed ewe was similar among groups (p > .05; Table 3). No significant differences in birth weight of lambs were found among groups (p > .05; Table 3).

| D ISCUSS I ON
The results of the present study showed that the lambing rate at the first cycle tended to increase in ewes treated with prostaglandin and hCG in combination with the ram effect when compared with control ewes during the out of breeding season. In this study, GnRH and hCG treatments did not improve ovulation in seasonal anestrus ewes.
Although In this study, in agreement with Kaya, Kaçar, Kaya, and Aslan (2013), the injection of hCG and GnRH had no effect on the lamb weights. It was also observed that the hCG and GnRH injection did not affect the litter size during the anoestrous period. The results of the total lambing rate of treated ewes obtained in this study were similar to those reported by Mirzaei et al. (2017), who examined the together with the ram effect during non-breeding seasons. Similar to previous studies (Kaya et al., 2013;Saharrea et al., 1998), we also found that the GnRH had a lower effect on fertility than hCG in  Pregnancy loss was reduced in synchronized low-prolific Rahmani ewes using a double injection of PGF2α by treatment of GnRH at the time of estrus or 7 days post-mating (Hashem, El-Azrak, et al., 2015).
Tighter synchrony of ovulation was reported in treated ewe with ovsynch-protocol compared with double PGF2α injection; although, conception rate and litter size did not differ between the two regimes (Hashem, El-Zarkouny, Taha, & Abo-Elezz, 2015). In this study, similar to study of Alnimer, Tabbaa, Amasheh, and Alzyoud (2005), there was no difference in conception and the first lambing rates between hormonal treated ewes and the ram effect. Alnimer et al. (2005) reported that the ewes may be synchronized using either GnRH-PGF2α program or two injections of PGF2α 10 days apart. Ovarian activity of postpartum (35 to 60 days after lambing) ewes was resumed by the ram effect (Ferreira-Silva et al., 2017). So, the male effect is efficient to induce ovarian activity and increase progesterone concentration in the control ewes.
In conclusion, the administration of prostaglandin and hCG in combination with the ram effect tended to decrease lambing period. In other words, this protocol tended to increase lambing rate at the first cycle. Treatment with hCG or GnRH did not increase serum P4 concentrations of treated Karakul ewes during the non-breeding season.

This experiment was approved by the Ethical and Research
Committee of the School of the Veterinary Medicine, Shiraz University (96GCU3M83440).

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest and financial, personal or other relationships with other people or organizations.

AUTH O R CO NTR I B UTI O N S
Mohammad Ayaseh is responsible for clinical examiner, data sorting and wrote the manuscript. Abdolah Mirzaei designed and performed the study, analyzed the data and wrote the manuscript and revision. Alidad Boostani is coordinator for all part of this project.
Mansur Mehrvarz helps as assistance for clinical examination and data collection.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.353.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. The first cycle (n) b 1.08 ± 0.05 1.14 ± 0.07 1.16 ± 0.09 .7

O RCI D
The second cycle (n) c 1.33 ± 0.33 1.0 ± 0.0 1.14 ± 0.14 .5 Overall ( TA B L E 3 Litter size in first and second oestrous cycle and weight of lambs (Mean ± SE) of ewes treated with prostaglandin and GnRH or hCG combined with the male effect in seasonal anoestrous ewes