The aim of the study was to compare the effect of short and long-term progesterone treatment and their combination with or without equine chorionic gonadotropin (eCG) on oestrous response and hormonal profiles during non-breeding season. Seventy-six (76) Merino ewes aged between 2–5 years were allocated into two groups, 36 for short-term and 40 for long-term progesterone treatments. In the short and long-term progesterone treatment groups, controlled internal drug release devices (CIDR) were inserted for 11 and 14 days, respectively. At withdrawal of CIDR, half of each group were injected intramuscularly with 300 IU of eCG. Blood samples were collected to measure the level of progesterone and oestradiol. Ewes treated with both short or long-term progesterone combined with or without eCG resulted in the same oestrus response, onset of oestrus and duration of oestrus. The levels of progesterone hormone were the same at CIDR insertion, and removal and 48 hours post CIDR removal. However, oestradiol hormone was significantly higher at CIDR removal and 48 hours post CIDR removal than at insertion in all treated groups. Therefore, it can be concluded that oestrous synchronisation protocols of both short and long-term progesterone combined with or without eCG during non-breeding season can be used to synchronise oestrus during non-breeding season.
Research Article
Effect of Progesterone Treatment on Synchronising Oestrus during Non-Breeding Season in South African Merino Ewes
https://doi.org/10.21203/rs.3.rs-3910399/v1
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short-term progesterone
long-term progesterone
controlled internal drug release
equine chorionic gonadotropin
oestradiol
Sheep are a significant part of the agribusiness economy of South Africa as they are used for meat and wool production (Hasani et al., 2018). Therefore, sheep play a vital role in supporting the livelihood of the human population as a whole (Hasani et al., 2018). Farmers in many countries favour keeping sheep over other livestock mainly because sheep are easy to manage and dwell in a small area (Lado et al., 2016). Interest in meat and wool keep expanding due to the consistently growing population, and questions emerge concerning how this demand will be met and by who (United Nations, 2013). Sheep are seasonal breeders and influenced by the photoperiod. The two main mating seasons for sheep are breeding season autumn and non-breeding season in spring (Wentzel, 2012). The breeding season is characterised by peak sexual activities which results in higher reproductive performance in sheep due to the short-day photoperiod (Wentzel, 2012). On the other hand, non-breeding season has average sexual activities due to the long-day photoperiod (Wentzel, 2012).
Assisted reproductive technologies such as oestrous synchronisation (Piotroski et al., 2013; López-García et al., 2021), can be used to improve reproductive performance of sheep during non-breeding season (Santos-Jimenez et al., 2020). Oestrous synchronisation is a powerful management tool that has been effectively utilised for artificial insemination and natural mating, particularly in ruminants (Oliveira et al., 2016). Therefore, oestrous synchronisation in ewes is very important in the reproduction efficiency and management processes improvements (Dogan et al., 2005). In sheep, controlled breeding involves artificial regulation of oestrous and ovulation with exogenous hormonal treatments (Martinez et al., 2015a). Intravaginal devices containing progesterone, such as controlled internal drug release (CIDR) are frequently inserted over periods of 6–11 days for short-term progesterone treatment or 12–14 days for long-term progesterone treatment and used in combination with eCG (Martinez et al., 2015a; Santos-Jimenez et al., 2020). The administration of progesterone for between 12–14 days through controlled internal drug release (CIDR) devices, with or without the administration of eCG at device removal, is the commonly used oestrous synchronisation protocol that mimics the luteal phase of the oestrous cycle (Kusina et al., 2000; Martinez et al., 2015b; Santos-Jimenez et al., 2020). Administration of gonadotrophins such as eCG have been proven to induce follicular growth and increases the rate of ovulation and fertility as well as to stimulate an effective synchrony of ovulation in both cycling and anestrous sheep (Martinez-Ros et al., 2019).
This oestrous synchronisation protocol has also proven to be effective in inducing oestrus during non-breeding season in South Africa (Lado et al., 2016). Moreover, although long-term progesterone is a commonly used protocol during non-breeding season but other studies has also proven short-term progesterone treatment (5–11 days) to be more effective in inducing oestrus due to better fertility, resulting from the sufficient suppression of luteinising hormone (LH) pulse frequency and ovulation of the newly recruited follicles (Martinez-Ros et al., 2019; Nakafeeroa et al., 2020). Therefore, this study evaluated the effect and compared the progesterone treatment durations on Merino ewes’ reproductive activity during non-breeding season.
The study was approved by the University of Zululand’s animal ethics committee (Project no. UZREC 171110-030) and was conducted during non-breeding season in September and November at Cedara Research Station in the uMgungundlovu District Municipality, approximately 16 km from Pietermaritzburg, KwaZulu-Natal, South Africa. A total of 76 Merino ewes aged between 2–5 years were used in this study. All experimental animals were grazing on controlled natural pastures and they had free access to water at all times.
Seventy-six (76) Merino ewes were allocated into four groups and all the groups had a similar average age. Group: 1. short-term progesterone and eCG treated ewes for 11 days, n = 18 (CIDR + eCG); 2. Long-term progesterone and eCG treated ewes for 14 days, n = 20 (CIDR + eCG); 3. Short-term progesterone treated for 11 days, n = 18 (CIDR only); and 4. Long-term progesterone treated for 14 days, n = 20 (CIDR only).
Each ewe received a CIDR device (Pfizer, New Zealand) containing 0.3 g progesterone. The duration of the treatment was for 11 and 14 days for short and long-term groups, respectively. At CIDR withdrawal, 18 of the short-term and 20 of the long-term treated ewes were injected intramuscularly with 300 IU of eCG (Intervet Schering-Plough Animal Health (Pty.) Ltd., SA). The other 18 of the short-term and 20 of the long-term treated ewes were only inserted with a CIDR device and were not injected with 300 IU of eCG at CIDR device removal.
Oestrus was detected by introducing two teaser rams per group for 72 hours following CIDR removal at 12 hour intervals. Tail twitching and standing to be mounted were the main signs targeted for (Ekiz et al., 2006). The parameters recorded were oestrus response (as the number of ewes showing oestrus/number of synchronised ewes × 100), onset of oestrus (as the time from CIDR removal to first acceptance of mounting and tail twitching), and also the duration of oestrus (as the time from the first acceptance of mounting to the first refusal).
For the detection of progesterone and estradiol hormones, blood samples were collected from six ewes per group. The blood samples were collected by means of puncturing the jugular vein using 4 ml sterile BD vacutainer tubes. Blood samples were collected at CIDR insertion, CIDR withdrawal and at 48-hour post CIDR withdrawal. After blood collection, the serum was harvested by means of centrifugation (3000 × g for 20 min at 4°C) and stored at -20°C until analyses (Nogueira et al., 2016).
The serum progesterone and oestradiol-17β concentrations were analysed using enzyme-linked immunosorbent assay (ELISA) kits. Firstly, 50 µL of the sample was added to each well and mixed with 50 µL of Biotinylated Detection Ab was added, followed by incubation for 45 minutes at 37°C. The samples were then aspirated and washed three times, and 100 µL of horseradish peroxidase (HRP) conjugated with avidin was added to each well and incubated for a further 15 minutes at 37°C. The samples were aspirated again and washed five times, after which 90 µL of the kit’s substrate reagent was added, followed by incubation for 15 minutes at 37°C. After incubation, 50 µL of the kit’s stop solution was added. The well plate was inserted into the Multiskan microplate reader and read immediately at 450 nm. The oestrous response, onset of oestrus, duration of oestrus and hormonal profiles was analysed using one-way analysis of variance (ANOVA) of the SPSS® software (IBM SPSS Statistics, Version 23.0, 2015, Armonk, NY., USA). Treatment means were separated using Tukey’s post-hoc test and was considered significantly different (P < 0.05).
The effect of oestrous synchronisation protocols on oestrous response, onset of oestrus and duration of oestrus is indicated in Table 1. Four (4) ewes dropped the CIDR device during the experimental period and were excluded from the experiment. The SA Merino ewes’ overall oestrous response from all treatment groups in the South African non-breeding season was 96%. There was no significant difference (P > 0.05) between short (11 days) and long-term (14 days) progesterone treatments on oestrous response, onset and duration of oestrus. There was no significant difference (P > 0.05) between short and long-term progesterone treatments with eCG on oestrous response, onset and duration of oestrus. There was also no significant difference (P > 0.05) between short and long-term progesterone treatments only on oestrous response, onset and duration of oestrus. There was no significant difference (P > 0.05) between short-term progesterone plus eCG and short-term progesterone only treatments on oestrous response, onset and duration of oestrus. There was no significant difference (P > 0.05) between long-term progesterone plus eCG and long-term progesterone only treatments on oestrous response, onset and duration of oestrus.
Most ewes showed oestrus signs between 48 and 60 hours post the CIDR device’s withdrawal (Table 2).
|
Number of ewes beginning oestrus per hour interval |
|||||
|---|---|---|---|---|---|
|
Treatments |
24h |
36h |
48h |
60h |
72h |
|
Short (11 d) Long (14 d) CIDR + eCG short (11 d) CIDR + eCG long (14 d) CIDR short CIDR long CIDR + eCG CIDR only |
3 0 2 0 1 0 2 1 |
12 4 5 3 7 1 8 8 |
8 19 4 9 4 10 13 14 |
8 13 6 7 2 6 13 8 |
3 3 1 1 2 2 2 4 |
Values with different superscripts within the same row differ significantly (P > 0.05).
The progesterone concentrations analysed for hormonal profiles at different treatment period are indicated in Table 3. There was no significant difference (P > 0.05) between short (11 days) and long-term (14 days) progesterone treatments on the level of progesterone hormone at CIDR insertion, CIDR removal and 48 hours post CIDR removal. There was no significant difference (P > 0.05) between short and long-term progesterone treatments with eCG on the level of progesterone hormone at CIDR insertion, CIDR removal and 48 hours post CIDR removal (P > 0.05). Therefore, there was no significant difference in all treatment groups at insertion, CIDR removal and 48 hours post CIDR removal (P > 0.05).
|
Treatments |
CIDR Insertion |
CIDR Removal |
48 hours post CIDR removal |
|---|---|---|---|
|
CIDR short (11 d) CIDR long (14 d) CIDR + eCG short (11 d) CIDR + eCG long (14) |
10.0 ± 0.1 8.0 ± 0.4 9.0 ± 0.2 10.0 ± 0.3 |
11.0 ± 0,09 11.0 ± 0,01 11.0 ± 0.06 11.0 ± 0,02 |
9.0 ± 0.3 9.0 ± 0.6 10.0 ± 0.2 10.0 ± 0,1 |
Values with same superscripts indicates no significant differences between treatments (P > 0.05).
The oestradiol-17β concentrations analysed for hormonal profiles at different treatment period are indicated in Table 4. Oestradiol concentrations (pg/ml) were higher in all treatment groups following different oestrous synchronisation protocols at CIDR device removal and 48 hours post removal (P < 0.05). Table 4 also shows there was no significant difference (P > 0.05) between short (11 days) and long-term (14 days) progesterone treatments on the oestradiol concentrations at CIDR insertion, CIDR removal and 48 hours post CIDR removal. There was no significant difference (P > 0.05) between short and long-term progesterone treatments with eCG on the oestradiol concentrations at CIDR insertion, CIDR removal and 48 hours post CIDR removal (P > 0.05).. Mean ± SEM values with different superscripts (a or b) on the same row indicate a significant difference (P < 0.05).
|
Treatments |
CIDR Insertion |
CIDR Removal |
48 hours post CIDR removal |
|---|---|---|---|
|
CIDR short (11 d) CIDR long (14) CIDR + eCG short (11 d) CIDR + eCG long (14 d) |
34.4 ± 11.7 a 64.1 ± 47,4 a 30.0 ± 16.9 a 81.8 ± 42.8 a |
176.2 ± 34.3 b 170.3 ± 42.0 b 188.7 ± 49.1 b 210.1 ± 57.6 b |
167.9 ± 44.3 b 178.4 ± 19.9 b 202.5 ± 16.9 b 234.5 ± 17.7 b |
a,b Values with different superscripts within the same row differ significantly (P < 0.05).
The overall oestrous response (96%) obtained in this study was revealed by the number of ewes responding to oestrous synchronisation. Comparable to oestrous response of 90% that has been reported during breeding (Vilariño et al., 2013; Swelum et al., 2015; Oliveira et al., 2016) and non-breeding seasons (Cox et al., 2012; Martinez et al., 2015a; Santos-Jimenez et al., 2020). The oestrous response obtained in this study for the short-term treatment without eCG was higher than other studies conducted during breeding season (Martinez-Ros. et al., 2018) and non-breeding season (Ahmed Amer et al., 2009) in short-term progesterone treatment without eCG. In the long-term treatment without eCG, oestrous response of the present study was lower than oestrous response obtained by Martinez-Ros et al (2018) during breeding season but higher than obtained by Ahmed Amer et al (2009) during non-breeding conducted in long-term progesterone treatment without eCG. The oestrous response of the short-term treatment with eCG for this study was higher than other studies during breeding season (Vilariño et al., 2013; Martinez-Ros et al., 2019) and non-breeding season (Ahmed Amer et al., 2009; Martinez et al., 2015a; Texeira et al., 2016) in the short-term progesterone treatment with eCG. For the long-term treatment, oestrous response of the present study was higher than other studies during breeding season (Martemucci et al., 2011; Swelum et al., 2015) and non-breeding season (Texeira et al., 2016; Nakafeeroa et al., 2020) but was lower to the oestrous response obtained by Martinez-Ros et al (2018 & 2019) during breeding and Armed Amer et al (2009) during non-breeding season in the long-term progesterone treatment. The positive oestrous response was due to the progesterone protocol and effective release of progesterone by the inserted CIDR device during the treatment period (Nakafeeroa et al., 2020).
Additionally, this high oestrous response may be credited to the CIDR device’s nature; it is non-absorbent silicone that does not absorb vaginal secretions and holds properly without tightly adhering to the vaginal mucosa (Swelum et al., 2018). The efficient synchrony of oestrus observed at 48 and 60 hours post CIDR removal in this study is supported by other researchers’ results (Vilariño et al., 2013; A. A. Swelum et al., 2015). The CIDR device’s effect on oestrous distribution can be related to the development stage of the follicle when the CIDR device is withdrawn. The efficient synchrony of oestrus shows that the ovulatory follicles are stimulated from a single wave (López-García et al., 2021), which is vital for fixed-time insemination. Recent studies still employ long-term progesterone treatments and have obtained an acceptable oestrous response and onset to oestrus (A. A. Swelum et al., 2015; Oliveira et al., 2016). Furthermore, Nakafeero et al. (2020) reported that both long and short-term progesterone treatments effectively synchronise oestrus in sheep.
In this study, the onset of oestrus was low from previous studies on the use of short-term treatment without eCG during breeding (Martinez-Ros. et al., 2018) but higher than other studies (Ahmed Amer et al., 2009) during non-breeding seasons conducted in short-term treatment without eCG. The onset of oestrus in this study’s long-term treatment without eCG was lower than onset of oestrus obtained by Martinez-Ros et al (2018) during breeding season but similar to Ahmed Armer et al (2009) during non-breeding season in long-term progesterone without eCG. In the short-term treatment with eCG, the onset of oestrus in this study was higher than other studies during breeding (Vilariño et al., 2013; Martinez-Ros. et al., 2018; Nakafeeroa et al., 2020) and non-breeding season (Ahmed Amer et al., 2009; Texeira et al., 2016) in the short-term progesterone treatment with eCG. The onset of oestrus in the long-term treatment with eCG in the present study was higher than other studies also during breeding (Martemucci et al., 2011; Martinez-Ros. et al., 2018; Nakafeeroa et al., 2020) and non-breeding season (Ahmed Amer et al., 2009; Texeira et al., 2016) conducted in the long-term progesterone treatment with eCG. Additionally, variations may be explained by differences in breed, nutrition and season. However, the lack of significance between the treatment groups was possibly due to the small number of ewes per treatment group. However, there was a tendency of earlier onset of oestrus in ewes inserted with the CIDR device for 11 days compared to those treated with CIDR for 14 days; this perhaps shows a faster follicular growth (Hasani et al., 2018). The similar oestrus response to the synchronisation and onset of oestrus between this study’s treatment groups, particularly regarding progesterone duration, suggests that long and short-term periods can effectively synchronise oestrus in a progesterone-based protocol during the non-breeding season.
This study’s results contradict with Harl (2014), who observed that short-term protocols resulted in a longer interval to oestrus compared to long-term protocols. Harl (2014) observed that ewes receiving a CIDR treatment for 14 days showed a shorter interval to oestrus. The shorter interval to oestrus observed in this study for all treatment groups could be because the more prolonged CIDR treatment period increased the possibility that the ewe’s existing corpus luteum regressed during treatment. However, short-term progesterone treatments decrease hormone usage quantities, offer the benefit of the CIDR device’s re-use (Vilariño et al., 2013) and also lead to the ovulation of high-quality follicles (Lado et al., 2016; Martinez-Ros. et al., 2018; Farahavar et al., 2020).
The similar interval to the onset of oestrus between the treatments with progesterone CIDR devices, with and without eCG, implies that both treatments stimulated a similar follicular growth pattern in ewes. Few studies have compared CIDR devices with eCG and without eCG at the end of treatment on progesterone treated ewes on the same flock or under the same conditions. An injection of eCG after CIDR removal not 48 hours before CIDR removal in this study, may explain the similarity in the interval to onset of oestrus between the groups treated with CIDR plus and without eCG. In studies where a dosage of 300 IU has been used, an increment in ovulation rate was observed (Cox et al., 2012; El-Mokadem et al., 2019; López-García et al., 2021), which resulted in an increase in the oestrous response and onset to oestrus (Swelum et al., 2018).
The addition of eCG to a progesterone protocol, which is known to increase the recruitment of new follicles, could perhaps stimulate an earlier onset to oestrus in both long and short-term progesterone treatments (Oliveira et al., 2016; Martinez-Ros et al., 2018; Biehl et al., 2019). This study’s results shows that using CIDR with or without eCG at the end of the progesterone treatment has similar effects on oestrous response and the onset of oestrus between treatment groups. Therefore, the results suggest that using CIDR alone at the end of progesterone treatment can be effective in progesterone-based oestrous synchronisation protocols during non-breeding season.
The duration of oestrus in the short-term treatment without eCG in this study, was lower than study conducted by Martinez-Ros et al (2018) during breeding season in the short-term progesterone treatment without eCG. For the long-term treatment without eCG, the duration of oestrus was also lower in this study compared to other studies (Martinez-Ros. et al., 2018) during breeding season conducted in the long-term progesterone treatment without eCG. The duration of oestrus obtained by Texeira et al (2016) during non-breeding and martinez-Ros et al (2018) during breeding season on the short-term treatment with eCG was higher than the duration of oestrus obtained in the present study in the short-term treatment with eCG. The duration of oestrus was lower for the long-term treatment with eCG in the present study than in other studies during breeding and non-breeding season (Texeira et al., 2016; Martinez-Ros. et al., 2018) performed in the long-term progesterone treatment with eCG. The progesterone treatments’ duration had no significant effect on the duration of oestrus between the treated groups in this study. The lack of a significant effect between the oestrous synchronisation protocols on the duration of oestrus may be due to similar effects of CIDR with and without eCG to induce LH surge. The mean duration of oestrus (26.5 ± 0.5 hours) found in this study is slightly comparable to that (30.07 ± 1.00 hours) obtained by Gore (2016). However, when comparing this study’s results with other studies, the duration of oestrus was shorter (Oliveira et al., 2016).
It is typical that the level of progesterone in blood increase rapidly after insertion of the CIDR device and decrease rapidly after the CIDR device’s withdrawal. In this study, the duration of progesterone or CIDR treatment did not significantly alter the serum progesterone concentration of the ewes at CIDR withdrawal and 48 hours post withdrawal. The progesterone level was similar in all the treatment groups at CIDR device insertion. Similar results have been observed in a study conducted by Vilariño et al. (2013) in sheep, who found that the progesterone levels were similar in all the treated groups at the CIDR device’s insertion. Furthermore, the progesterone level within a group was not different at the CIDR device’s removal in all groups.
Swelum et al. (2018) and Santos-Jimenez et al. (2020) observed that the CIDR insertion led to an effective increase in the serum progesterone concentration of sheep for 3 or 4 days, followed by a decrease 6 days after treatment. Furthermore, the increase in progesterone level due to insertion of the CIDR device resulted in ovarian activity inhibition via increasing the negative feedback mechanism on the hypothalamus, which further inhibits pituitary LH secretion (Smith et al., 2010; El-Mokadem et al., 2019). Hasani et al. (2018) had similar results and observed that continual exogenous progesterone administration adequately regressed the dominant follicles and suppressed the release of gonadotropin from the anterior pituitary until the time of progesterone withdrawal. Following the removal, the decline in progesterone concentration stimulated the initiation of ovarian activity through declining the negative feedback on LH secretion in the pituitary gland (A. A. Swelum et al., 2015; Manes et al., 2018; Martinez-Ros. et al., 2018). However, this study’s results +++contradict with those of Biehl et al. (2019), who observed that the level of progesterone at the insertion and withdrawal time was significantly higher in long than in short-term CIDR treatments.
The level of oestradiol concentration was significantly higher at the CIDR device’s removal and 48 hours post withdrawal; this is due to the stimulation of ovarian activity through decreasing the negative feedback mechanism on pituitary LH secretion following the decrease in progesterone concentration after the CIDR device’s withdrawal (Swelum et al., 2018). These results agree with those of Swelum et al. (2015; 2018), who observed that serum oestradiol levels were higher at the CIDR device’s removal and 48 hours post withdrawal (P > 0.05), providing an excellent advantage for fixed-time artificial insemination. It is well known that oestradiol is considered an excellent quality follicle marker during the oestrous cycle. This is because the level of oestradiol is directly proportional to the number of persistent or large follicles (Martinez-Ros et al., 2019). The duration of the CIDR treatment did not change the serum oestradiol concentration between the treatment groups at all stages of treatment. However, this may be due to the small number of ewes used for this study’s hormonal profiles. Similar results have been observed in other studies during both the breeding and non-breeding seasons (Vilariño et al., 2013; Swelum et al., 2015; Oliveira et al., 2016; Hasani et al., 2018; Swelum et al., 2018; Farahavar et al., 2020).
The use of short and long-term progesterone treatment resulted in the same oestrous response, onset, and duration. The use of short and long CIDR treatment can be used to synchronise oestrus for sheep during non-breeding season. The study also showed that there is no need to use CIDR in conjunction with eCG during non-breeding season. A high Estradiol concentration at 48 hours post CIDR removal, indicates the effectiveness of both progesterone durations with or without eCG in stimulating follicular growth during non-breeding season. It is recommended that further studies should be conducted using a larger number of ewes, which would strengthen this study’s conclusions. Furthermore, future studies should inject eCG at 48 hours before CIDR removal.
Acknowledgements
The authors would like to thanks the University of Zululand, Agricultural Research Council, and Moses Kotane Institute for assistance with funding/research.
Funding
This study was funded by Agricultural Research Council (ARC)
Authors Contributions
The study's conception and design involved contributions from all authors. Nhlakanipho Sam Zulu conducted material preparation, data collection, and analysis. The initial manuscript draft was crafted by Nhlakanipho Sam Zulu, while Fhulufhelo Vincent Ramukhithi and Khoboso Christine Lehloenya provided feedback on earlier versions of the manuscript. The final manuscript was reviewed and approved by all authors.
Statement of Animal Rights
The ethical treatment of sheep in this research necessitated a commitment to uphold their rights, ensuring freedom from unnecessary suffering, access to appropriate living conditions, and their respect inherent value as sentient beings. This study adhered to established principles of animal welfare, recognizing the importance of minimizing stress and prioritizing the well-being of sheep subjects throughout the research process.
Conflict of Interest Declaration
There is no conflict of interest to declare.
Data availability
The datasets produced and examined in this study can be obtained upon reasonable request from the corresponding author.
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