Introduction

In recent years, there has been an increasing demand for the use of various breeds in domestic sheep herds to implement breeding programs. The high price of imported rams, the sensitivity and mortality of these rams due to the various diseases in domestic herds, as well as the presence of rump in the most Iranian breeds as a cause of inefficient mating prevent the extensive use of such breeds of rams in the Iranian herds (Vodjgani et al., 2017). Artificial insemination (AI) can be applied as a useful method in breeding programs (Sathe, 2018). Due to the special anatomy of ewes’ cervix, it is difficult to access inside the uterus and thus, insemination of ewes with frozen semen has been encountered with a big challenge (Sathe, 2018; Noakes et al., 2019).

Laparoscopic AI is currently one of the well-known intrauterine methods of insemination with frozen sperm in sheep (Casali et al., 2017). Successful synchronization of estrus and ovulation is one of the major contributing factors affecting conception rate following laparoscopic AI in sheep (Olivera–Muzante et al., 2019).

In this context, various estrus synchronization protocols have been used in sheep including application of 12- or 14-day progestogen-dependent protocols (Abecia et al., 2012; Ávila-Castillo et al., 2019; Martemucci and D’Alessandro, 2010). Yet, synchronization of estrus by protocols merely including progestogens or prostaglandin F2α (PGF2α) during the breeding season may not lead to the desired outcomes (Martemucci and D’Alessandro, 2010) since prolonged exposure with progestogens (≥ 9 days) reduces the quality of oocytes, and in turn, conception rate, and impairs embryonic development (Fierro et al., 2016). It also affects sperm transport and survival in the female reproductive tract (Martemucci and D’Alessandro, 2010). Moreover, devices containing progestogens could escalate the risk of environmental and tissue contamination (Ávila-Castillo et al., 2019). To overcome these issues, some studies have shown short-term exposure to progesterone; short-term synchronization protocols have shown acceptable results in sheep during breeding as well as non-breeding seasons because they not only enhance estrus synchronization but also culminate in oocytes of higher quality (Titi et al., 2010).

Administration of a single dose of PGF2α during the breeding season does not influence synchrony of estrus and ovulation, because it can induce luteolysis but not synchrony in follicle development. (Martemucci and D’Alessandro, 2011). Nevertheless, two injections of PGF₂α at 7-16-day intervals have been reported to improve synchrony of estrous and ovulation (Fierro et al., 2016; Carvalho et al., 2018), but the interval between two administrations of PGF2α could impact fertility (Ataman and Akoz, 2006). In this sense, administration of GnRH was incorporated at the beginning of the protocol to control follicular growth prior to PGF2α injection (Ataman and Akoz, 2006; Titi et al., 2010; Vallejo et al., 2019). As a result, short-term GnRH-PGF2α-based protocol (5-day interval) has been recommended as a practical alternative to common progestogen-based estrous synchronization protocols during the breeding season (Ataman and Akoz, 2006; Titi et al., 2010; Martemucci and D’Alessandro, 2010; Hashem et al., 2015).

Studies on ewes (Bartlewski et al., 2017; Titi et al., 2010) and cows (Simões et al., 2018) have shown that the use of exogenous progesterone between GnRH and PGF2α injections prevents the spontaneous luteolysis resulted from irresponsiveness of the dominant follicle to GnRH injection. Response to GnRH depends on the stage of the estrous cycle in which the injection is made. Furthermore, exogenous progesterone also increases pregnancy rates and boosts follicular growth (Martinez and Gonzalez, 2019).

In cows, the highest efficiency of Ovsynch program is achieved when there is a dominant responsive follicle at the time of GnRH injection and a suitable corpus luteum at the time of PGF2α injection (Youngquist and Threlfall, 2007). Studies have shown that starting Ovsynch at the beginning and at the end of the estrous cycle has not good results due to the presence of new follicles that are not responsive to GnRH, and premature luteolysis and onset of estrus before the second GnRH administration, respectively (Youngquist and Threlfall, 2007). Therefore, pre-synchronization using two PGF2α injections 14 days apart was used 12 days before the start of the Ovsynch program in cows, until the cows were in ideal phase of estrus cycle at the beginning of the Ovsynch program (days 5-12) (Zarkouny et al., 2004).

The use of pre-synchronization to increase the efficiency of GnRH-PGF2α based programs in sheep is a new practice and only has been carried out sparingly in one study, with two PGF2α injections nine days apart (Ávila-Castillo et al., 2019).

To increase the efficiency of short-term protocols based on GnRH-PGF2α, Ferdowsi et al. (2018) evaluated eCG performance at different time intervals to PGF2α. The results of the study showed that the concomitant use of eCG with PGF2α can increase the effectiveness of short-term GnRH-PGF2α-based programs.

This study aimed to increase the efficiency of GnRH-PGF2α- eCG short-term protocols to achieve the appropriate program for increasing the fertility rate resulting from laparoscopic insemination. Therefore, the effects of pre-synchronization with two PGF2α injections, nine days apart, three days before the start of GnRH-PGF2α- eCG-based short-term programs, and the effect of using 5-day exogenous progesterone were evaluated in this study.

Materials and Methods

Location of Study

The study was carried out in an ewe flock located in Alborz province of Iran in Eshtehard District with a longitude of 50 degrees and 22 minutes and latitude of 35 degrees and 43 minutes during the breeding season.

Animals

In this study, 120 healthy ewes of Zandi breed were used (age range: 2-6 years). Average weight and body condition score (BCS) were 57.3±1.4 kg and 3.1±0.1, respectively. All ewes received the same diet and had ad labium access to fresh water.

Experimental Design

Ewes were randomly assigned into four experimental groups (n=30 in each group) considering age, weight, and BCS. All ewes received GnRH (25 μg of alarelin acetate, Vetaroline, Aboureihan, Iran), and five days afterwards, PGF₂α (75 μg d-cloprostenol, Vetaglandin, Aboureihan, Iran) plus eCG (400 IU; Gonaser, Hipra, Spain) (Ferdowsi et al., 2018). In controls (Group 1), ewes received no additional treatment. In controlled internal drug releasing (CIDR) group (Group 2), ewes received 5-day CIDR between GnRH and PGF2α of main estrus synchronization protocol. In Pre-synch group (Group 3), ewes received two injections of PGF₂α at 9-day interval three days before GnRH administration of main estrus synchronization protocol. In Pre-synch-CIDR group (Group 4), ewes received both injections of PGF₂α at 9-day interval and 5-day CIDR (Figure 1).

Figure 1. Experimental design including blood sampling (BS), injection and timing in different experimental groups (30 ewes in each group)

Estrus Detection

Estrus detection using teaser rams equipped with aprons and crayons was started six hours after PGF₂α injection and lasted for 48-hour after that (Ferdowsi et al., 2018).

Laparoscopic AI

All the ewes were inseminated based on fixed timelaparoscopic AI at 48 hours after administration of the last PGF₂α (Youngquist and Threlfall, 2007). To this end, the ewes were deprived of food and water for 12 h prior to laparoscopy (Sathe, 2018). Animals were sedated with 0.3 ml acepromazine maleate (Neurotanq; 10 mg/mL, Alfasan, Holland) 10–30 minutes before insemination (Sathe, 2018). Ewes were placed in laparoscopic cradles and kept in dorsal recumbency. Local anesthesia was performed by administration of 2 mL lidocaine hydrochloride (Luracaïne; 20 mg/mL; Vétaquinol S.A., France) in the site of abdominal puncture 5 min prior to insemination (Sathe, 2018). Two trocars were inserted about 3 cm on either side of the midline and about 3 cm cranial to udder to allow for introducing telescope and insemination pipette (Sathe, 2018). Following observation of uterine horns, half of the volume of thawed frozen semen (100 × 10⁶ sperm; Animal Breeding Center, Karaj, Iran) was inseminated in each uterine horn. After insemination, all trocar puncture wounds were sprayed with a combined antiseptic and insect repellant (Vetaque, Animal Health Division, Iran). All laparoscopic operations were performed by an experienced operator.

Evaluation of Blood Serum Progesterone Concentration

Blood serum progesterone concentrations were measured in all ewes prior to injection of PGF₂α (day 5). Blood samples (10 mL) were collected from jugular vein, poured into vacuum tubes without anticoagulants (Sunphoria, Mediplus, China; Figure 1), and centrifuged at 3000 rpm for 15 min. Serum was isolated and transferred to 2-mL tubes and maintained at -20°C until measurement of progesterone (Ferdowsi et al., 2018). Progesterone concentration was measured by ELISA kit (DRG Instruments GmbH, Germany) with detection limit of 0.045 ng to 40 ng progesterone per mL. Intra-assay and inter-assay coefficients of variation were calculated as 5.40% and 9.96%, respectively.

Pregnancy Diagnosis

Diagnosis of pregnancy was performed by transabdominal ultrasonographic examination using a B-mode ultrasound machine (V9, Emperor, China) equipped with a 3.5 MHz convex probe, 50 days after laparoscopic AI (Youngquist and Threlfall, 2007).

Evaluation of Reproductive Parameters

The following reproductive parameters were assessed: time of estrus onset, estrus detection rate (number of estrous ewes divided by total number of ewes × 100), pregnancy rate (number of pregnant ewes divided by number of inseminated ewes × 100), lambing rate (number of lambed ewes divided by number of inseminated ewes × 100), fecundity rate (number of lambs divided by number of inseminated ewes), and prolificacy rate (number of lambs divided by number of lambed ewes × 100). Weight and gender of lambs were also recorded (Vodjgani et al., 2017).

Statistical Analysis

Continuous data (i.e., progesterone concentration, time of estrus onset, and birth weight) were analyzed by General Linear Model (GLM) procedure. Binary data (i.e., estrus detection rate, pregnancy rate, lambing rate, fecundity, prolificacy, and sex ratio) were analyzed using GENMOD procedure including function link logit in the model. Multiple comparisons were performed using LSMEANS statement. All analyses were conducted in SAS version 9.4 (Statistical Analysis Systems, Cary, NC, USA). Data were presented as mean ± SEM or percentage. Differences were considered significant at P-value<0.05.

Results

Pre-synchronization using two administrations of PGF2α nine days apart had no influence on the concentration of progesterone at the time of PGF2α administration in the main synchronization protocol (P>0.05) (Table 1). However, application of CIDR for five days between GnRH and PGF2α injections in the main protocol culminated in higher concentration of circulating progesterone at the time of PGF2α in the main synchronization protocol (P<0.0001) (Table 1), and in turn, progesterone concentration at this time point was greater in CIDR and Pre-synch-CIDR groups as compared with the control and Pre-synch groups (P<0.0001) (Table 1).

Estrus detection rate was not affected by pre-synchronization using two administrations of PGF2α nine days apart, five-day usage of CIDR, and their interaction (P>0.05) (Table 1).

Time to commencement of behavioral estrus expression was advanced by 3.65 hours by the main effect of pre-synchronization using two administrations of PGF2α nine days apart (30.44±1.19 hours in non-pre-synchronized ewes versus 26.79±1.02 hours in pre-synchronized ewes; P=0.022; Table 1). Yet, five-day CIDR and interaction of pre-synchronization using two PGF2α injections nine days apart and five-day CIDR did not impact the estrus onset time (P>0.05) (Table 1).

Pregnancy rate, lambing rate, fecundity, prolificacy, birth weight of lambs, and sex ratio of offspring were not influenced by two administrations of PGF2α nine days apart, five-day usage of CIDR, and their interaction (P>0.05) (Table 1).

Table 1.  Reproductive indices of ewes prepared for timed breeding using GnRH- prostaglandin F₂α₅ days apart, in association with pre-synchronization (two prostaglandin F₂α, ₉  days apart) and CIDR during breeding season

Discussion

Introduction

In recent years, there has been an increasing demand for the use of various breeds in domestic sheep herds to implement breeding programs. The high price of imported rams, the sensitivity and mortality of these rams due to the various diseases in domestic herds, as well as the presence of rump in the most Iranian breeds as a cause of inefficient mating prevent the extensive use of such breeds of rams in the Iranian herds (Vodjgani et al., 2017). Artificial insemination (AI) can be applied as a useful method in breeding programs (Sathe, 2018). Due to the special anatomy of ewes’ cervix, it is difficult to access inside the uterus and thus, insemination of ewes with frozen semen has been encountered with a big challenge (Sathe, 2018; Noakes et al., 2019).

Laparoscopic AI is currently one of the well-known intrauterine methods of insemination with frozen sperm in sheep (Casali et al., 2017). Successful synchronization of estrus and ovulation is one of the major contributing factors affecting conception rate following laparoscopic AI in sheep (Olivera–Muzante et al., 2019).

In this context, various estrus synchronization protocols have been used in sheep including application of 12- or 14-day progestogen-dependent protocols (Abecia et al., 2012; Ávila-Castillo et al., 2019; Martemucci and D’Alessandro, 2010). Yet, synchronization of estrus by protocols merely including progestogens or prostaglandin F2α (PGF2α) during the breeding season may not lead to the desired outcomes (Martemucci and D’Alessandro, 2010) since prolonged exposure with progestogens (≥ 9 days) reduces the quality of oocytes, and in turn, conception rate, and impairs embryonic development (Fierro et al., 2016). It also affects sperm transport and survival in the female reproductive tract (Martemucci and D’Alessandro, 2010). Moreover, devices containing progestogens could escalate the risk of environmental and tissue contamination (Ávila-Castillo et al., 2019). To overcome these issues, some studies have shown short-term exposure to progesterone; short-term synchronization protocols have shown acceptable results in sheep during breeding as well as non-breeding seasons because they not only enhance estrus synchronization but also culminate in oocytes of higher quality (Titi et al., 2010).

Administration of a single dose of PGF2α during the breeding season does not influence synchrony of estrus and ovulation, because it can induce luteolysis but not synchrony in follicle development. (Martemucci and D’Alessandro, 2011). Nevertheless, two injections of PGF₂α at 7-16-day intervals have been reported to improve synchrony of estrous and ovulation (Fierro et al., 2016; Carvalho et al., 2018), but the interval between two administrations of PGF2α could impact fertility (Ataman and Akoz, 2006). In this sense, administration of GnRH was incorporated at the beginning of the protocol to control follicular growth prior to PGF2α injection (Ataman and Akoz, 2006; Titi et al., 2010; Vallejo et al., 2019). As a result, short-term GnRH-PGF2α-based protocol (5-day interval) has been recommended as a practical alternative to common progestogen-based estrous synchronization protocols during the breeding season (Ataman and Akoz, 2006; Titi et al., 2010; Martemucci and D’Alessandro, 2010; Hashem et al., 2015).

Studies on ewes (Bartlewski et al., 2017; Titi et al., 2010) and cows (Simões et al., 2018) have shown that the use of exogenous progesterone between GnRH and PGF2α injections prevents the spontaneous luteolysis resulted from irresponsiveness of the dominant follicle to GnRH injection. Response to GnRH depends on the stage of the estrous cycle in which the injection is made. Furthermore, exogenous progesterone also increases pregnancy rates and boosts follicular growth (Martinez and Gonzalez, 2019).

In cows, the highest efficiency of Ovsynch program is achieved when there is a dominant responsive follicle at the time of GnRH injection and a suitable corpus luteum at the time of PGF2α injection (Youngquist and Threlfall, 2007). Studies have shown that starting Ovsynch at the beginning and at the end of the estrous cycle has not good results due to the presence of new follicles that are not responsive to GnRH, and premature luteolysis and onset of estrus before the second GnRH administration, respectively (Youngquist and Threlfall, 2007). Therefore, pre-synchronization using two PGF2α injections 14 days apart was used 12 days before the start of the Ovsynch program in cows, until the cows were in ideal phase of estrus cycle at the beginning of the Ovsynch program (days 5-12) (Zarkouny et al., 2004).

The use of pre-synchronization to increase the efficiency of GnRH-PGF2α based programs in sheep is a new practice and only has been carried out sparingly in one study, with two PGF2α injections nine days apart (Ávila-Castillo et al., 2019).

To increase the efficiency of short-term protocols based on GnRH-PGF2α, Ferdowsi et al. (2018) evaluated eCG performance at different time intervals to PGF2α. The results of the study showed that the concomitant use of eCG with PGF2α can increase the effectiveness of short-term GnRH-PGF2α-based programs.

This study aimed to increase the efficiency of GnRH-PGF2α- eCG short-term protocols to achieve the appropriate program for increasing the fertility rate resulting from laparoscopic insemination. Therefore, the effects of pre-synchronization with two PGF2α injections, nine days apart, three days before the start of GnRH-PGF2α- eCG-based short-term programs, and the effect of using 5-day exogenous progesterone were evaluated in this study.

Materials and Methods

Location of Study

The study was carried out in an ewe flock located in Alborz province of Iran in Eshtehard District with a longitude of 50 degrees and 22 minutes and latitude of 35 degrees and 43 minutes during the breeding season.

Animals

In this study, 120 healthy ewes of Zandi breed were used (age range: 2-6 years). Average weight and body condition score (BCS) were 57.3±1.4 kg and 3.1±0.1, respectively. All ewes received the same diet and had ad labium access to fresh water.

Experimental Design

Ewes were randomly assigned into four experimental groups (n=30 in each group) considering age, weight, and BCS. All ewes received GnRH (25 μg of alarelin acetate, Vetaroline, Aboureihan, Iran), and five days afterwards, PGF₂α (75 μg d-cloprostenol, Vetaglandin, Aboureihan, Iran) plus eCG (400 IU; Gonaser, Hipra, Spain) (Ferdowsi et al., 2018). In controls (Group 1), ewes received no additional treatment. In controlled internal drug releasing (CIDR) group (Group 2), ewes received 5-day CIDR between GnRH and PGF2α of main estrus synchronization protocol. In Pre-synch group (Group 3), ewes received two injections of PGF₂α at 9-day interval three days before GnRH administration of main estrus synchronization protocol. In Pre-synch-CIDR group (Group 4), ewes received both injections of PGF₂α at 9-day interval and 5-day CIDR (Figure 1).

Figure 1. Experimental design including blood sampling (BS), injection and timing in different experimental groups (30 ewes in each group)

Estrus Detection

Estrus detection using teaser rams equipped with aprons and crayons was started six hours after PGF₂α injection and lasted for 48-hour after that (Ferdowsi et al., 2018).

Laparoscopic AI

All the ewes were inseminated based on fixed timelaparoscopic AI at 48 hours after administration of the last PGF₂α (Youngquist and Threlfall, 2007). To this end, the ewes were deprived of food and water for 12 h prior to laparoscopy (Sathe, 2018). Animals were sedated with 0.3 ml acepromazine maleate (Neurotanq; 10 mg/mL, Alfasan, Holland) 10–30 minutes before insemination (Sathe, 2018). Ewes were placed in laparoscopic cradles and kept in dorsal recumbency. Local anesthesia was performed by administration of 2 mL lidocaine hydrochloride (Luracaïne; 20 mg/mL; Vétaquinol S.A., France) in the site of abdominal puncture 5 min prior to insemination (Sathe, 2018). Two trocars were inserted about 3 cm on either side of the midline and about 3 cm cranial to udder to allow for introducing telescope and insemination pipette (Sathe, 2018). Following observation of uterine horns, half of the volume of thawed frozen semen (100 × 10⁶ sperm; Animal Breeding Center, Karaj, Iran) was inseminated in each uterine horn. After insemination, all trocar puncture wounds were sprayed with a combined antiseptic and insect repellant (Vetaque, Animal Health Division, Iran). All laparoscopic operations were performed by an experienced operator.

Evaluation of Blood Serum Progesterone Concentration

Blood serum progesterone concentrations were measured in all ewes prior to injection of PGF₂α (day 5). Blood samples (10 mL) were collected from jugular vein, poured into vacuum tubes without anticoagulants (Sunphoria, Mediplus, China; Figure 1), and centrifuged at 3000 rpm for 15 min. Serum was isolated and transferred to 2-mL tubes and maintained at -20°C until measurement of progesterone (Ferdowsi et al., 2018). Progesterone concentration was measured by ELISA kit (DRG Instruments GmbH, Germany) with detection limit of 0.045 ng to 40 ng progesterone per mL. Intra-assay and inter-assay coefficients of variation were calculated as 5.40% and 9.96%, respectively.

Pregnancy Diagnosis

Diagnosis of pregnancy was performed by transabdominal ultrasonographic examination using a B-mode ultrasound machine (V9, Emperor, China) equipped with a 3.5 MHz convex probe, 50 days after laparoscopic AI (Youngquist and Threlfall, 2007).

Evaluation of Reproductive Parameters

The following reproductive parameters were assessed: time of estrus onset, estrus detection rate (number of estrous ewes divided by total number of ewes × 100), pregnancy rate (number of pregnant ewes divided by number of inseminated ewes × 100), lambing rate (number of lambed ewes divided by number of inseminated ewes × 100), fecundity rate (number of lambs divided by number of inseminated ewes), and prolificacy rate (number of lambs divided by number of lambed ewes × 100). Weight and gender of lambs were also recorded (Vodjgani et al., 2017).

Statistical Analysis

Continuous data (i.e., progesterone concentration, time of estrus onset, and birth weight) were analyzed by General Linear Model (GLM) procedure. Binary data (i.e., estrus detection rate, pregnancy rate, lambing rate, fecundity, prolificacy, and sex ratio) were analyzed using GENMOD procedure including function link logit in the model. Multiple comparisons were performed using LSMEANS statement. All analyses were conducted in SAS version 9.4 (Statistical Analysis Systems, Cary, NC, USA). Data were presented as mean ± SEM or percentage. Differences were considered significant at P-value<0.05.

Results

Pre-synchronization using two administrations of PGF2α nine days apart had no influence on the concentration of progesterone at the time of PGF2α administration in the main synchronization protocol (P>0.05) (Table 1). However, application of CIDR for five days between GnRH and PGF2α injections in the main protocol culminated in higher concentration of circulating progesterone at the time of PGF2α in the main synchronization protocol (P<0.0001) (Table 1), and in turn, progesterone concentration at this time point was greater in CIDR and Pre-synch-CIDR groups as compared with the control and Pre-synch groups (P<0.0001) (Table 1).

Estrus detection rate was not affected by pre-synchronization using two administrations of PGF2α nine days apart, five-day usage of CIDR, and their interaction (P>0.05) (Table 1).

Time to commencement of behavioral estrus expression was advanced by 3.65 hours by the main effect of pre-synchronization using two administrations of PGF2α nine days apart (30.44±1.19 hours in non-pre-synchronized ewes versus 26.79±1.02 hours in pre-synchronized ewes; P=0.022; Table 1). Yet, five-day CIDR and interaction of pre-synchronization using two PGF2α injections nine days apart and five-day CIDR did not impact the estrus onset time (P>0.05) (Table 1).

Pregnancy rate, lambing rate, fecundity, prolificacy, birth weight of lambs, and sex ratio of offspring were not influenced by two administrations of PGF2α nine days apart, five-day usage of CIDR, and their interaction (P>0.05) (Table 1).

Table 1.  Reproductive indices of ewes prepared for timed breeding using GnRH- prostaglandin F₂α₅ days apart, in association with pre-synchronization (two prostaglandin F₂α, ₉  days apart) and CIDR during breeding season

Discussion