Synchronization of Ewe Lambs During Non-Breeding Season: Comparing Clomiphene Citrate-Based Protocols With Progesterone+eCG Protocol

Synchronization can improve reproductive performance of ewe lambs during non-breeding season. Accordingly, the aim of this study was to evaluate reproductive performance of ewe lambs during non-breeding season using different clomiphene citrate (CC) protocols compared with progesterone sponge (PS)+equine chorionic gonadotropin (eCG) protocol. In this study, 40 Ghezel ewe lambs were divided into four experimental groups based on the completely randomized design. Experimental groups were as follows: 1- PS (12 days)+eCG (500 I.U. eCG); 2- PS (12 days)+CC (100 mg CC, 5 days); 3- CC (100 mg CC, 5 days)+eCG group (500 I.U. eCG); 4- CC (100 mg CC, 5 days). Ram introduction was done between 13 and 18 days. Blood samples were taken on 1, 11, 14, and 40 days, and serum levels of estrogen and progesterone were measured. Pregnancy was evaluated at the last trimester of expected pregnancy (by sonography). Based on the results, the effects of treatments and the interaction of treatments × time on estrogen and progesterone concentrations were signicant (P<0.05). Pregnancy and lambing rates were signicantly affected by estrus synchronization treatments and the highest pregnancy was observed in PS+eCG group (P<0.05). Based on the overall results of the present study, the best synchronization method during non-breeding season for ewe lambs was PS+eCG.

It has been shown that synchronization during non-breeding season requires progesterone due to its effect on inducing follicular FSH and LH receptors (Burke and Kissler, 1988;Caraty and Skinner, 1999).
Then, using progesterone compounds with or without equine chorionic gonadotropin (eCG) are conventional protocols for estrus synchronization during non-breeding season and breeding season Clomiphene citrate (CC), a treatment for female infertility, has antiestrogenic effects by blocking estrogen receptors, which increases gonadotropin-releasing hormone (GnRH) secretion (by blocking negative feedback of estrogen on hypothalamus) and in turn, increases FSH and LH secretion (Tiwary,  in ewes increased growing follicle numbers, increased estrogen levels, and induced estrous; however, CC had no effect on ovulating follicles and estrogen levels when administered during superovulation with 2000 I.U. of eCG (EL-sherry et al. 2011). In another study, nine days of feeding 300 mg CC/day/prepubertal heifer along with intravenous injection of 2500 I.U. hCG/heifer induced ovulation in heifers (Bukhari et al. 2016). Treating anestrus cows with single injection of 750 I.U. eCG or oral administration of 300 mg CC/d for ve days reported better results with eCG in comparison with CC for conception rate (Tiwary, 2006). To our information, there is no study regarding the use of CC or related protocols during non-breeding season on ewe lambs. So, the present study was designed to evaluate reproductive performance of ewe lambs during non-breeding season using different CC protocols as compared with PS+eCG protocol. days and orally receiving of 100 mg CC during 7-12 days); 3-CC+eCG group (orally receiving of 100 mg CC during 7-12 days and intramuscular injection of 500 I.U. eCG on day 12 of trial); 4-CC group (orally receiving of 100 mg CC during 7-12 days). Rams were introduced to the ewe lambs between 13 and 18 days of the experiment (one ram per each 10 ewe lambs), in which rams were replaced twice a day to ensure high fertility rate. Estrus signs in PS+eCG group were started 12h after sponge removal and the maximum rate was observed 48h after sponge removal. However, estrus signs in CC, PS+CC, and CC+eCG groups were observed from the beginning of ram introduction and persisted to the last day of ram introduction (from 13 to 18 days of experiment).

Hormonal evaluations
Blood samples of all four groups were collected on days 0 (start of the experiment), 11 (24 hours before progesterone sponge removal), 14 (48 hours after progesterone sponge removal), and 40 of the experiment (28 days after progesterone sponge removal to evaluate pregnancy of ewe lambs). All samples were collected at the same daylight time (11:30 a.m.). Then, blood samples were centrifuged by 3000×g for 15 minutes at room temperature. Afterward, serum samples were stored at -20 °C till the measurement of estrogen and progesterone concentrations. Serum progesterone and estrogen concentrations were measured in one stage and based on enzyme-linked immunosorbent assay method (ELISA) using ELISA reader (Awareness Technology Stat Fax 3200 Microplate Reader, USA). Progesterone kit (Cat. No. 4825-300) was supplied from Monobind company (USA) with 0.1 ng/ml sensitivity and 23.57% intra-assay coe cients of variation of progesterone. Also, estrogen kit (Cat. No. 4925-300) was supplied from Monobind company (USA) with 6.5 pg/ml sensitivity and 19.54% intraassay coe cients of variation of estrogen.

Pregnancy evaluations
In the last trimester of expected pregnancy, ewe lambs were evaluated for pregnancy by ultrasound sonography (UltraScan 900, AMI Co, Canada) using a 5.0 MHz probe to evaluate the presence of placenta.
Pregnancy rate at the last trimester of expected pregnancy was calculated as follows: the number of pregnant ewe lambs in a treatment group/the total number of ewe lambs in that group×100.

Lambing evaluations
After parturition, the number of lambs, which were born by each ewe lamb was recorded. Lambing rate in each group was calculated based on the total lambs' number/ewe lambs' number ×100. Twining rate in each group was calculated as the ratio of twine and triplicate lambs/total number of lambs ×100. It should be mentioned that one of the ewe lambs in PS+eCG group delivered triplicate and two ewe lambs in this group delivered twines. Also, proli cacy was calculated in each group based on the total number of lambs/the numbers of delivered ewe lambs ×100.

Data analysis
Proc FREQ and Proc LOGISTIC of SAS software (SAS 9.2) were used to analyze reproductive performance data. In addition, the concentrations of progesterone and estrogen were analyzed based on Proc MIXED (SAS 9.2). A signi cance level of 0.05 was used for comparing treatments.

Results
All ewe lambs showed signs of estrous 24 h after sponge withdrawal, though mating in clomiphene citrate groups lasted more till day 18 of experiment (individual data was not recorded). Ultrasound based pregnancy rate at the last trimester of expected pregnancy was signi cantly affected by synchronization methods (P<0.05). The highest pregnancy rate was observed in PS+eCG ewe lambs (90%), while PS+CC group indicated 10% pregnancy rate and CC+eCG and CC groups showed no pregnancy (0%), (Table 1). 2 Embedding progestogen sponge for 12 days+oral receiving of 100 mg CC during 7-12 days (n=10). 3 Oral receiving of 100 mg CC during 7-12 days+eCG injection (500 IU) at 12 days of experiment (n=10). 4 Oral receiving of 100 mg CC during 7-12 days (n=10). 5 Pregnancy of ewe lamb's based on blood progesterone concentration at 40 days of experiment. 6 Ewe lamb's pregnancy based on ultrasound at the last expected trimester of pregnancy.
Lambing rate was signi cantly affected by estrus synchronization methods (P<0.05) and the highest lambing rate was observed in PS+eCG group with 130% lambing rate (Table 1). On the other hand, 10% lambing rate was observed in PS+CC group and 0% lambing rate was observed in CC+eCG and CC groups ( Table 1).
The impact of estrus synchronization methods on twining rate was not signi cant (P>0.05); however, twining rate was higher in PS+eCG group (53.85%) compared with PS+CC group (0%), (Table 1).
Based on the results, estrus synchronization treatments had a signi cant effect on blood progesterone concentration (P<0.05), in which progesterone concentration was 1.68±0.32 ng/ml in PS+eCG group, 1.56±0.36 ng/ml in CC+eCG group, 1.03±0.32 ng/ml in PS+CC group, and 0.153±0.36 ng/ml in CC group. Although blood progesterone concentration was not affected by blood sampling times (P>0.05), the interaction effects of estrus synchronization treatments by sampling times on serum progesterone concentration was signi cant (P<0.01), (Fig. 1). Different treatments had a signi cantly different progesterone concentration on 0, 11, 14, and 40 days of experiment (P<0.05), (Fig. 1). On day 11 of the experiment, progesterone concentration was the highest in CC+eCG group as compared with other groups (P<0.05), (Fig. 1). Also, PS+eCG group had the highest progesterone concentration on day 40 of the experiment (P<0.05), (Fig. 1).

Discussion
Pregnancy rate of ewe lambs was 90% in PS+eCG group and 10% in PS+CC group. Also, results indicated 130% lambing rate, 53.85% twining rate, and 144.44% proli cacy in PS+eCG group. Furthermore, lambing rate, twining rate, and proli cacy in PS+CC group were 10%, 0%, and 100%, respectively. However, CC+eCG and CC groups indicated no pregnancy nor lambing rate. Accordingly, all CC protocols were unsuccessful in improving reproductive performance of ewe lambs during non-breeding season. Regardless of observing estrous signs in clomiphene citrate groups, because of low estradiol concentration on day 14 of experiment in clomiphene citrate groups, it can be concluded that either the level of clomiphene citrate was not su cient or it requires simultaneous administration with copper sulfate to be effective and cause a successful pregnancy in ewe lambs during non-breeding season.

Conclusion
The overall results of the present experiment showed higher pregnancy and lambing rates in PS+eCG group as compared with all clomiphene citrate groups; therefore, the advisable method for synchronization of ewe lambs during non-breeding season is progesterone sponge and eCG. Also, administering clomiphene citrate with the present dosage or protocols can not cause successful pregnancy or lambing in ewe lams during non-breeding season.

Figure 1
The impact of different estrus synchronizations included: PS+eCG (embedding progestogen sponge for 12 days+eCG injection (500 IU) at 12 days of experiment), PS+CC (embedding progestogen sponge for 12 days+oral receiving of 100 mg CC during 7-12 days), CC+eCG (oral receiving of 100 mg CC during 7-12 days+eCG injection (500 IU) at 12 days of experiment), and CC (oral receiving of 100 mg CC during 7-12 days) on blood progesterone concentrations of ewe lambs at day 0 (start of the experiment), 11 (24 hours before progesterone sponge removal), 14 (48 hours after progesterone sponge removal), and 40 of the experiment (28 days after progesterone sponge removal to evaluate pregnancy of ewe lambs).

Figure 2
The impact of time of blood sampling on serum estrogen concentrations of ewe lambs. Figure 3