Study on comparative efficacy of different protocols in estrus synchronization of sheep

Introduction

Sheep husbandry is an important component of the livestock sector in India, providing livelihood and nutritional security for millions of rural farmers. Estrus synchronization is an important reproductive biotechnology used to control the timing of estrus and ovulation in sheep. It facilitates planned breeding, artificial insemination and embryo transfer programs (Kusina et al., 2000; Jainudeen et al., 2000) [15,12] . Progesterone based intravaginal devices such as Avikesil-S ® sponges are widely used for estrus synchronization because they mimic the luteal phase and suppress ovulation until removal of the sponge (Kumar et al., 2016; Mahendra, 2016; Yadav et al., 2020) [14,18,25] . Recently, non-steroidal drugs such as clomiphene citrate and aromatase inhibitors like letrozole have been explored as alternatives to conventional hormonal protocols due to concerns regarding hormone residues in animal products (Land and Scaramuzzi, 1979; Rateb et al., 2019; Gupta et al., 2021) [16,22,10] . Clomiphene citrate (CC), a selective estrogen receptor modulator (SERM) that acts by blocking hypothalamic estrogen receptors, thereby reducing negative feedback and stimulating GnRH, FSH and LH release, which in turn promotes follicular development and ovulation. Its advantages include low cost and ease of oral administration, making it attractive for low-input production systems. And letrozole inhibits the aromatase enzyme and suppresses estrogen synthesis, thereby increasing gonadotropin secretion and promoting follicular development (Goss and Strasser, 2001; Casper and Mitwally, 2012) [9,3] . Therefore, the present study was conducted to compare the efficacy of progesterone sponge, clomiphene citrate and letrozole based estrus synchronization protocols in ewes.

Materials and Methods

The study was conducted on eighteen healthy (aged one to four years) non pregnant, cycling ewes maintained under semi-intensive management conditions. Animals were randomly divided into three groups with six animals in each group.

Group I: Avikesil-S ® progesterone intravaginal sponge inserted for 7 days followed by intramuscular injection of cloprostenol sodium (PGF₂α) at sponge removal.

Group II: Oral clomiphene citrate (150 mg) administered for five consecutive days followed by intramuscular injection of cloprostenol sodium (PGF₂α) on fifth day.

Group III: Letrozole intravaginal sponge (7.5 mg) inserted for seven days followed by intramuscular injection of cloprostenol sodium (PGF₂α) at sponge removal.

Letrozole sponges were prepared by using polyurethane material 30 mm length × 20 mm width × 20 mm thickness and thread of 15 cm length. Sponges were autoclaved and stored in the air tight polythene bags. The sponges were loaded with 7.5 mg of letrozole (3 tablets of Letroz ® 2.5 mg suspended in one mL distilled water (w/v) and sponge were dipped in suspended and dried before intravaginal insertion for ewes.

Estrus detection and mating

All the study animals were observed for the behavioural estrus twice daily for 30 minutes each time (06:00 -07:00 and 18:00 -19:00 hour) for five days after PGF 2 α injection. Stand to be mounted by the Ram till it refuse to mate. Ewes were isolated and exposed to the sexually active ram to aid estrus detection. Stand to be mounted by the ram was considered to be in estrus.

Determination of pregnancy

Pregnancy diagnosis was performed 50 days of post mating using ultrasonography and (Honda, HS/2000, Japan) with a 3.5-10 MHz linear array rectal transducer. The parameters recorded included sponge retention rate, estrus response rate, interval to onset of estrus, duration of estrus and conception rate.

Results and Discussion

Intravaginal sponge retention rate

In the present study, all ewes in Group I (Avikesil-S ® progesterone sponge) and Group III (letrozole sponge) retained the intravaginal sponges throughout the 7-day treatment period, resulting in a 100 per cent sponge retention rate (Table 1 andFigure 1). None of the animals exhibited signs of estrus while the sponges were in situ and no abnormal behavioural changes were observed except mild turbid vaginal discharge, indicating good tolerance of the devices under field conditions. The high retention rate observed in the present study may be attributed to factors such as sponge material characteristics (polyurethane), duration of insertion, vaginal anatomy, breed differences and parity of the animals. Comparable results were also reported with Avikesil-S ® sponges applied for 7 to 12 days in different ewe breeds (Mahendra, [18,14,7,25,23] . Similarly, Abdel Dayem et al. (2020) [1] reported complete retention of letrozole intravaginal sponges in Farafra ewes. However, slightly lower retention rates (94%) have been reported with longer treatment durations (14 days) (Swelum et al., 2018) 24 . Overall, these findings confirm that intravaginal sponges are reliable and well tolerated for estrus synchronization in ewes.

Estrus response rate

The estrus response rate recorded in the present study was 83.33 per cent (5/6) in Group I, 66.67 per cent (4/6) in Group II, and 66.67 per cent (4/6) in Group III, and no significant difference was observed among the groups (p>0.05) (Table 2 and Figure 2). The estrus response observed in Avikesil-S ® treated ewes was comparable to earlier reports where 90 to 100 per cent estrus response was achieved using progesterone sponge protocols in combination with gonadotropins (Mahendra, [18,14,25,23] . Similarly, De et al. (2015; 2016) [5,6] reported estrus responses of 79 to 83 per cent in Malpura and Kheri ewes. In clomiphene citrate treated ewes, the estrus response rate was also 66.67 per cent, which is lower than reports in goats where response rates above 90% have been documented (Murugan, 2020; Gupta et al., 2021; Rateb et al., 2019) [20,10,22] . Variations in estrus response may be attributed to breed differences, dosage, season, and drug absorption in ruminants. The letrozole treated group showed a moderate estrus response (66.67%), which is slightly lower than reports by Pujara (2022) [21] and Gavimath et al. (2023) [8] who observed responses 83.33 and 87.5 per cent. Similarly, Abdel Dayem et al. (2020) [1] reported 100 per cent estrus induction in Farafra ewes using letrozole sponges.

Mean time to onset of estrus

The mean interval from PGF₂α injection to onset of estrus was 36.00 ± 3.79 hours in Group I, 81.50 ± 5.85 hours in Group II, and 57.00 ± 5.75 hours in Group III. The interval was significantly longer in the clomiphene citrate group (p<0.05) compared to the other treatments (Table 3 and Figure 3). The estrus onset observed with Avikesil-S ® sponges was consistent with previous reports indicating estrus within [20,10,22] . The letrozole group showed an intermediate interval to estrus (57 hours), which is comparable to findings by Abdel Dayem et al. (2020) [1] who reported estrus onset around 54 hours in ewes treated with letrozole sponges. These results indicate that progesterone-based synchronization provides earlier and more predictable estrus onset compared to non-steroidal protocols.

Duration of estrus

The mean duration of estrus recorded in the present study was 26.40 ± 2.40 hours in Group I, 30.00 ± 3.47 hours in Group II, and 30.00 ± 3.47 hours in Group III, with no significant difference among groups (p>0.05) (Table 3 and Figure 4). These values fall within the normal physiological estrus duration in ewes, which ranges between 24 and 36 hours (Hafez and Hafez, 2000) [11] . Similar durations have been reported in progesterone synchronized ewes by Yadav et al. (2020) [25] and Gangadharaiah (2017) [7] . Letrozole treated animals also showed estrus durations comparable to those reported by Abdel Dayem et al. (2020) 1 and Gavimath et al. (2023) [8] . Overall, although synchronization protocols influenced the timing of estrus onset, the duration of estrus remained relatively consistent across treatments.

Conception rate

The conception rate observed in the present study was 60 per cent in Group I, 50 per cent in Group II, and 50 per cent in Group III, with no significant difference among treatments (p>0.05) (Table 4 and Figure 5). The conception rate obtained with Avikesil-S ® sponges was comparable to previous reports using progesterone-based synchronization protocols (Cavalcanti et al., 2012; Lombardo et al., 2020; Martemucci and D'Alessandro, 2011) [4,17,19] . Similarly, the clomiphene citrate group showed a conception rate of 50 per cent, though previous studies have reported variable results depending on species and treatment protocols (Alamdar et al., 2022; Rateb et al., 2019; Gupta et al., 2021) [2, 22, 10] . The letrozole group showed a moderate conception rate (50%), which is slightly lower than reports by Abdel Dayem et al. (2020) [1] and Pujara (2022) [21] where conception rates above 70 per cent were achieved.

Conclusion

In conclusion, intravaginal progesterone sponges produced the most predictable and earliest estrus expression with robust luteal-phase progesterone support and resulted in tight synchrony. Letrozole sponges offered a non-steroidal alternative that delayed but still effectively induced estrus that is intermediate estrus onset and achieved comparable conception under these conditions. Whereas, oral clomiphene citrate yielded the least synchronized response with a markedly prolonged interval to estrus despite similar estrus duration. Given the smaller sample size, these findings support the viability of letrozole as a promising non-steroidal option but indicate that larger studies are needed to optimise dosing, timing and to confirm fertility outcomes and pharmacokinetic behaviour before field adoption.

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