The effects of oestrus synchronization with cloprostenol, flugestone acetate or combinations of both, with or without exogenous eCG or FSH on follicular development, and oestrus response, were studied in 66 nulliparous Boer does that were approximately 1 year old with body condition score of 3 and average body weight of 25.1 kg. The does were randomly assigned into 9 groups and each group was synchronized using different combinations of PGF2α, flugestone acetate or their combinations with 5 mg of FSH or 300IU of eCG. Ultrasound of ovarian follicular development was conducted using a real-time B-mode ultrasound scanner, with a transrectal 7.5 MHz linear probe.
There were statistically significant differences (P<0.05) among the synchronization groups in oestrus response, time of onset of oestrus, duration of oestrus, total number of follicles, and maximum follicle size attained. However there was no significant differences (P>0.05) in the serum cortisol levels (nmol/L) between the synchronization groups. The percentage of does in oestrus was highest when exogenous eCG was used for synchronization (92.7%), compared with FSH (57.1%) and control (52.9%). There were no significant differences (P> 0.05) in the mean number of follicles between eCG, and FSH synchronized groups, but the eCG synchronized groups had significantly larger (P<0.05) mean diameter of the largest follicle compared with the FSH or control group. It was concluded that a single injection of eCG (300IU) to nulliparous Boer does resulted in higher oestrus response and ovulation, and maximum follicle diameter than FSH (5mg)
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Key words: gonadotrophins; PGF2α; flugestone acetate; ultrasonography; oestrus synchronization; Boer does
The increasing world-wide interest in goat production and consequent need to shorten the generation interval, through breeding of young pubertal does as soon as they attain the required body weight, also require the application of efficient methods of oestrus synchronization. Oestrous synchronization is an effective management tool to control reproduction in goats, both for artificial insemination and multiple ovulation and embryo transfer programs (Leboeuf et al., 1998; Wildeus, 2000; Bearden et al., 2004; Whitley and Jackson, 2004; Chao et al., 2008)
Procedures aimed at manipulating the oestrous cycle in different ruminant breeds involve either shortening the luteal phase using luteolytic doses of PGF2α or through extending the follicular phase with exogenous progesterone or progestagens (Kusina et al., 2000; Wildeus, 2000; Lopez-Sebastian et al., 2007). PGF2α-based synchronization protocols are only applicable during the breeding season in cyclic goats with corpus luteum. The most widely practiced methods of oestrous synchronization are progesterone or progestagens-based protocols (Husein et al., 2007; Lopez-Sebastian et al., 2007; Menchaca, 2007; Letelier et al., 2009). Progesterone impregnated intravaginal products used in goats include controlled internal drug-releasing device (CIDR), flugestone acetate (FGA) and methyl acetoxyl progesterone (MAP) (Wildeus, 2000).
Oestrus synchronization methods using either progesterone or prostaglandin were suggested to be more effective when gonadotrophin co-treatments were used (Oliveira et al., 2001; Pierson et al., 2001; Husein et al., 2007). Equine chorionic gonadotrophin (eCG) previously called pregnant mare serum gonadotrophin (PMSG) and follicle stimulating hormone (FSH) were the most often used gonadotrophins in oestrus synchronization protocols (Bearden, et al., 2004). High level of oestrus synchronization has been reported when eCG was incorporated into the synchronization protocol in sheep and goats in and out of the breeding season (Regueiro et al., 1999; Zarkawi et al., 1999; Al-Merestani et al., 2003; Amarantidis et al., 2004; Husein et al., 2007). FSH was similarly reported to be effective in synchronization of oestrus (Gonzalez-Bulnes et al., 2000).
Ozawa et al. (2005) suggested that heat stress during follicular recruitment suppresses subsequent growth to ovulation, accompanied by decreased LH receptor level and oestradiol synthesis activity in the follicles. According to Silanikove (2000a; b), the complexities of the factors associated with thermal heat exchange in ruminants suggest that physical measurements of environmental temperature though useful may be less than satisfactory index of thermal stress, because the impact of environment may be modified by animal behaviour which could differ between specie, breed or individual levels (Silanikove, 2000a; Sejian and Srivastava, 2010). Plasma cortisol levels may thus provide more reliable indications of stress levels associated with production conditions, and heat stress.
This study was therefore conducted to depict the follicular development, oestrus response, time of onset and duration of oestrus behavior following oestrous synchronization using PGF2α, FGA or their combinations with exogenous eCG or FSH in non-seasonally polyoestrous, peri-pubertal Boer goats intensively raised under tropical farm conditions.
MATERIALS AND METHODS
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A total of 66 nulliparous Boer does that were approximately 1 year old with body condition score of 3 and mean ± SD body weight of 25.11±4.01 (20-35 kg) were selected for this study. The goats were kept in raised sheds with slatted floors at a commercial goat farm located at Lat: 3° 15N and Long: 101° 32' 60E in Selangor, Malaysia. The minimum and maximum temperatures and relative humidity during the period of study were 20-35oC and 67-83% respectively. The does were fed mixed rations based on 3% of their body weight daily, comprising palm leaves, commercial pellets, soya bean waste and palm leaf silage. Water and salt licks were provided ad libitum.
The goats were randomly assigned into 9 groups and oestrus was synchronized using different combinations of PGF2α (Estrumate™, Schering-Plough), flugestone acetate (FGA) impregnated vaginal sponges (Ovakron™ Johannesburg). Two PGF2α injections were given 11 days apart in the PGF2α only protocols (Control group) while the FGA sponges were inserted and left in place for 14 days. Five mg of FSH (Ovagen™ ICP, Auckland, New Zealand) and 300IU of eCG (Sergon™ Bioveta-Ivanovice) were administered according to manufacturers' instructions in the respective treatment groups. The nine treatment groups are shown in Table 1.
Observations to detect behavioural oestrus was conducted twice daily at 0800 hrs and 1500 hrs with the aid of 2 vasectomised bucks using methods described by Amarantidis et al. (2004). Goats were considered to be in oestrus when they accepted mounting by the buck, and/or when vocalization, tail wagging and oedematous vulva were observed. Time to onset of induced oestrus was considered to be the period from the cessation of treatment to observed oestrus behaviour, while the duration was considered to be the period from commencement of oestrus to disappearance of overt oestrus signs.
Ultrasonographic monitoring of ovarian follicular development following cessation of treatment was conducted using a real-time B-mode ultrasound scanner (Aloka, 500 SSD, Japan), attached to a transrectal 7.5 MHz linear probe (UST-660-7.5 model). The ultrasound scanning was performed once daily for the first 3 days after cessation of treatment, then once every two days.
Standing restraint was used for ultrasonographic procedure (Ginther and Kot, 1994; Riesenberg et al., 2001; Gonzalez-Bulnes et al., 2003; Simoes et al., 2005). The linear probe lubricated with carboxymethocellulose gel was introduced into the rectum and the transducer directed dorsally. Faeces were not evacuated (Simoes et al., 2005). The transducer was then rotated 180o to be directed downwards while maintaining contact with the rectal wall. The bladder was located and followed cranially to visualize the uterine horns. The transducer was then slowly rotated 45-90o clockwise and counter clockwise to locate and observe the right and then the left ovaries and their associated structures (Ginther and Kot, 1994; Simoes et al., 2005).
Ovaries were visualized in real time and the number, size and position of follicles ≥3mm in diameter were measured and sketched on ovarian charts (Ginther and Kot, 1994). Ovulation was considered as the collapse of a large ovulatory follicle (≥5mm in diameter), which had been monitored by ultrasonography and the subsequent appearance of a corpus luteum on the same location (Ginther and Kot, 1994; Simoes et al., 2005; Simoes et al., 2006; Menchaca, 2007; De Santiago-Miramontes et al., 2008; De Santiago-Miramontes et al., 2009; Vazquez et al., 2010). A minimum of 5 scanning sessions were performed for each doe.
Blood samples were collected via the jugular venipuncture into plain vacutainer tubes and allowed to clot at room temperature for 30 minutes. The serum was separated by centrifugation at 1006.2 x g for 15 min, and stored at -20oC until assayed. Hormonal analysis of serum cortisol was by previously validated radio immunoassay using 125 I RIA kit (DSL-2000, Diagnostic Systems Laboratories, Inc. Webster, Texas) for the quantitative measurement of cortisol in serum. The assay sensitivity was 0.11µg/dl. The range of standards was 0 to 60 µg/dl. The intra and inter assay coefficients of variation (CV's) were 4.15 and 20.69 respectively
Follicular population data for the left and right ovaries were combined and were summarized for each goat. Differences between means were tested using Duncan's multiple range tests. Data with unequal variances were analyzed using Kruskall Wallis non parametric test. Chi-square analysis was used to test oestrus response. Spearman correlation analysis was conducted to determine the relationships between the oestrus response, onset, duration, total follicle number, maximum diameter of ovulatory follicles and cortisol levels. Data were analyzed using a statistical software program (SPSS Inc. Version 17). Analyses were considered to be statistically significant at P < 0.05.
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Figs. 1 to 5 show the distribution of observed responses to oestrus synchronization methods using PGF2α, FGA or their combinations with exogenous eCG or FSH. On the other hand, Figs. 6 to 9 show the effects of exogenous eCG or FSH on the variables studied irrespective of whether PGF2α, FGA or their combinations were used for oestrus synchronization.
The percentage oestrus response was highest (100%) in goats synchronized using PGF2α+eCG, FGA+PGF2α+eCG, FGA+eCG with the least percentage oestrus response (20%) in the PGF2α+FSH synchronized group (Fig. 1). Onset of oestrus was more advanced from cessation of treatment (76 hours) in PGF2α+eCG synchronized group compared with FGA-based protocol and control (Fig. 2). The oestrus duration was lowest in the PGF2α+FSH group (4.80 hours) as shown in Fig. 3.
The mean total numbers of observed follicles are shown in Figs. 4. The FGA+PGF2α+FSH PGF2α+FGA+FSH groups had the highest mean number of follicles (12.44 and 11.78 respectively). The PGF2α+FSH synchronized groups had the lowest mean follicle number (5.80). The mean maximum size attained by the largest follicle ranged from 5.65 mm in the PGF2α+FSH synchronized group to 8.38 mm in PGF2α+FGA+eCG group (Fig. 5).
Further analysis of the results obtained based on the type of gonadotrophin used show that the percentage of oestrus response were highest when exogenous eCG was used in the synchronization (92.7%) compared with FSH (57.1%) and control (52.9%) respectively (Fig. 6). Time to oestrus onset from cessation of treatment was also more advanced in eCG synchronized compared with FSH synchronized groups (Fig. 7). The groups that were synchronized with eCG had significantly longer oestrus duration (P<0.05) compared with the FSH groups (Fig. 8).
There were no significant differences (P> 0.05) in the mean total number of follicles among eCG, FSH and control groups (Fig. 9). However, eCG synchronized groups had significantly larger (P<0.05) mean maximum diameter of the largest follicle than the FSH or control groups (Fig. 10). The mean serum cortisol levels were lowest in the control group (3.96 nmol/L) while eCG and FSH synchronized had 9.78 nmol/L and 11.43 nmol/L respectively (Fig. 11)
There were statistically significant differences (P<0.05) between the synchronized groups in percentage oestrus behaviour, time to onset of oestrus, duration of oestrus, total follicle number, maximum follicle size attained by largest follicle (Tables 1 and 2). There were however no statistically significant differences (P>0.05) in the serum cortisol levels (nmol/L) between the groups studied (Tables 1 and 2). Time to ovulation from oestrus onset approached significance (P=0.080) as shown in Table 2.
There were significant positive correlations between maximum size attained by the largest follicle, and the oestrus onset (r=0.45, P=0.000), duration of oestrus (r=0.41, P=0.000), total number of follicles, but a significant negative correlation with oestrus response (r=-0.43, P=0.000). The onset of oestrus showed significantly high negative correlation with oestrus behaviour (r=-0.77, P=0.000) but was positively correlated with duration of oestrus (r=0.59, P=0.000). Duration of oestrus was significantly negatively correlated with oestrus response (r=-0.78, P=0.000)
Characteristic echographic appearance of superovulated ovaries was observed in two of the goats synchronized with PGF2α+FGA+eCG (Fig. 12). The ovaries had multiple follicles greater than 6mm in size that gave the ovaries a spongy appearance. No such response were observed in FSH synchronized group or the rest of the experimental animals though the FSH groups were observed to have higher total number of follicles compared with the eCG synchronized groups.
The percentage of oestrus response observed in this study (100% and 73%) in the PGF2α+eCG and PGF2α+FSH synchronized groups respectively agree with the results of previous studies which suggested that oestrus synchronization methods using either progesterone or prostaglandin were more effective when gonadotrophin co-treatments were used (Oliveira et al., 2001; Pierson et al., 2001; Gonzalez-Bulnes et al., 2005; Husein et al., 2007; Menchaca, 2007). Other studies had however reported very high oestrus response rates without use of gonadotropins. Amarantidis et al. (2004) reported 100% oestrus response in FGA or FGA+PGF2α synchronized indigenous Greek goats with and without PMSG (eCG). Regueiro et al. (1999) similarly obtained 100% oestrus response in Saanen, Nubian goats and their crosses synchronized with MAP, with or without 500IU eCG. Zarkawi et al. (1999) also induced 100% oestrus response outside the breeding season in Damascus goats synchronized with MAP plus injection of eCG at the time of sponge removal. Montlomelo et al. (2002) also reported a slightly lower (96.7%) oestrus response in FGA+PMSG synchronized goats. Dogan et al. (2005) observed 85.7 and 94.4% in FGA+PMSG+PGF2α and FGA+PMSG synchronized Anatolian black does respectively.
Oestrus response in FGA synchronized goats alone or in combination with PGF2α was reported to frequently approach 100% (Freitas et al., 1997; Ahmed et al., 1998; Romano, 2004). The oestrus response observed in this study in PGF2α+FGA+eCG, FGA+PGF2α+eCG and FGA+eCG synchronized goats were significantly higher than response observed in PGF2α+FGA+FSH, FGA+PGF2α+FSH and FGA+FSH synchronized groups suggesting that eCG is more effective in inducing oestrus response than FSH.
Dogan et al. (2005) reported mean time to oestrus onset as 18.0±1.9 and 22.9±1.6 in FGA+PMSG+PGF2α and FGA+PMSG synchronized Anatolian black does respectively. Other reported mean time to oestrus onset includes 25±1.56, (Pierson et al., 2001), 52.3±14.3, (Ahmed et al., 1998) and 49.7±15.7 (Fonseca et al., 2005). Romano (2004) reported that oestrus onset occurred 32.9±9.7 hours in intravaginal FGA pessary synchronized goats given PGF2α at removal of the pessary. Differences in the observed percentage of oestrus response, time to oestrus onset and duration between this study and previous reports could be due to breed, age, parity, body condition or weight differences among the does studied (Simoes et al., 2008; De Santiago-Miramontes et al., 2009).
The percentage of oestrus response, time to onset and duration of the induced oestrus following injection of PGF2α either at the time of sponge insertion or at the time of sponge removal were not found to be significantly different in this study. Dogan et al. (2005) similarly found no significant differences in mean time to onset, and duration of oestrus between FGA+PMSG+PGF2α, FGA+PMSG, MAP+PMSG+PGF2α, MAP+PMSG synchronized groups. Amarantidis et al. (2004) however suggested that priming with FGA before the administration of PGF2α influenced not only the onset, but also the duration of the induced oestrus period. They further suggested that the mean oestrus period was short in the FGA/PGF2α treatment (33.9±5.8h), compared with the double PGF2α injection treatment (50.6±4.7h). In agreement with Amarantidis et al. (2004), the mean time to oestrus onset observed in this study was longer in the PGF2α +eCG synchronized groups (76 hours) compared to the FGA synchronized groups. On the other hand, the duration of induced oestrus was the same among the groups.
The PGF2α+FSH, PGF2α+FGA+FSH, FGA+PGF2α+FSH and FGA+FSH synchronized groups (groups synchronized with FSH) were observed to have smaller percentage oestrus response, shorter time to onset of oestrus and shorter duration of the induced oestrus compared to eCG synchronized groups suggesting that a single injection of FSH is not as effective as a single injection of eCG in oestrus synchronization. This finding does not agree with Boscos et al. (2002) who suggested that at the beginning of the breeding season in sheep, a single 5 or 10IU FSH treatment at the end of progestagen treatment appeared to be superior in inducing first oestrus and during the mid breeding season, concluding that FSH was equally as effective as eCG.
The total numbers of follicles observed in PGF2α+FSH, PGF2α+FGA+FSH, FGA+PGF2α+FSH and FGA+FSH synchronized groups were higher than the number of follicles observed in the PGF2α+FGA+eCG, FGA+PGF2α+eCG and FGA+eCG synchronized groups. The higher follicle number observed in the FSH groups suggests better stimulation of ovarian follicular development compared with eCG. This finding is supported by the suggested superiority of FSH over eCG given as multiple application for superovulation and embryo recovery in goats (Rosnina et al., 1992; Riesenberg et al., 2001). According to Riesenberg et al. (2001), ultrasonic screening of goats' shows that both eCG (1250IU) and FSH (17mg) given as a single application appears to provide a sufficient stimulus to achieve a satisfactory superovulatory response. The dose levels used were however larger than those used in this study (300IU and 5mg respectively) but superovulatory response was observed in 2 goats both synchronized with PGF2α+FGA+eCG in this study which might suggest differential sensitivity of the ovaries of goats to eCG.
The maximum follicular diameter observed in this study (6.17±0.8mm) in the control group were lower than the maximum diameter of ovulatory follicle in PGF2α synchronized (8.3±0.4mm) and natural (7.2±0.4mm) cycle in Anglo Nubian goats observed by Vazquez et al. (2010). Simoes et al. (2006) suggested that the maximum diameter of the preovulatory follicle in PGF2α synchronized Serrana goats were (7.1±1.0mm), Gonzalez-Bulnes et al. (2004) suggested 7.8±0.4mm for PGF2α synchronized Murciana-Granadina does. Cueto et al. (2006) suggested the maximum diameter of ovulatory follicles to be 6.1mm in short hair and 6.5mm in PGF2α (double injection, 11 days apart) synchronized long hair Neuquen-Criollo goats which closely agrees with the results of our study.
The largest mean diameters of follicles were observed in PGF2α+eCG (9.08±4.18) and PGF2α+FSH (7.17±1.06) groups respectively compared with FGA synchronized groups. This could be attributed to the effect of PGF2α synchronization on follicle size. Gonzalez-Bulnes et al. (2004) suggested that oestrous synchronization with PGF2α would result in dominant follicles of mid-luteal phase with higher maximum diameter and longer permanence that could be related to low progesterone levels found in the cloprostenol-treated goats. Low LH pulse resulting from these low progesterone levels were associated with increase in number, size and permanence of the largest follicles (Rubianes and Menchaca, 2003; Gonzalez-Bulnes et al., 2004).
Time of ovulation from onset of oestrus in PGF2α+PGF2α+eCG, PGF2α +FGA+eCG, FGA+PGF2α+eCG, FGA+FGA+eCG and control were 44.00±32.8, 36.00±9.80, 21.6±13.14, 24.00±12.00, 11.59±12.37 and 24.00±0.00 hours respectively. Time to ovulation from oestrus onset in PGF2α synchronized nulliparous Serrana goats were 30.1±1.1h (Simoes et al., 2008). Riesenberg et al. (2001) suggested that due to the short half-life of FSH (3.4h), a strong exogenous stimulus with FSH might only initiate the superovulatory reaction, while the final follicle development is supported by endogenously produced gonadotrophin. This may explain the lack of ovulations observed in the FSH groups in this study though higher numbers of follicles were observed in these goats. Conversely, eCG which has a longer half life (6 days) resulted in more ovulations.
The significant positive correlations between maximum size attained by the largest follicle, and the oestrus onset, duration of oestrus, total number of follicles, but a significant negative correlation with oestrus response observed in this study suggests that larger maximum diameter of follicles does not result in increased percentage oestrus response .This agrees with Van Eerdenburg, et al. (2002) who suggested that there was no correlation between follicular size and oestrus-detection score in cattle
Serum cortisol levels assayed in this study were found to be within normal range and not different between the groups studied. It was suggested that hyperthermia is deleterious to any form of productivity (Lu 1989; Silanikove 2000a; b; Sejian and Srivastava 2010). Sejian and Srivastava (2010) also reported that plasma cortisol levels in heat stressed goats were significantly higher compared to controls (82.74±2.44 and 18.76±4.33 nmol/L) respectively. Ozawa et al. (2005) suggested that heat stress during follicular recruitment suppresses subsequent growth to ovulation, accompanied by decreased LH receptor level and oestradiol synthesis activity in the follicles. Though the environmental conditions were hot and humid during the period of this study, the goats were kept in well ventilated and shaded pens with unlimited supply of water which ameliorated their stress levels. According to Al-Tamimi (2007) accessibility of goats to shade during summer is a simple and yet an efficient tool to minimize solar radiation-induced heat stress.
There were statistically significant differences (P<0.05) between does synchronized with cloprostenol or flugestone acetate or combinations of both, with or without injection of exogenous eCG or FSH in percentage oestrus response, time to onset of oestrus, duration of oestrus, total follicle number, and maximum follicle size attained. There were no significant differences (P>0.05) in the serum cortisol levels (nmol/L) between the groups studied. The percentages of does in oestrus were highest in eCG compared with FSH and control groups (92.7; 57.1; 52.9%) respectively. There were no significant differences (P> 0.05) in the mean number of follicles between eCG and FSH synchronized groups, but the eCG synchronized groups had significantly larger (P<0.05) mean diameter of the largest follicle compared with the FSH or control groups. There were significant positive correlation between maximum follicle size and the oestrus onset (r=0.45, P=0.000), duration (r=0.41, P=0.000), and total number of follicles, but significant negative correlation with oestrus response (r=-0.43, P=0.000).
The mean total numbers of follicles observed in FSH synchronized groups were higher than mean number of follicles observed in the eCG synchronized groups but the eCG synchronized groups had larger mean diameter of the largest follicle and higher number of ovulations compared to the FSH synchronized groups. It was concluded that there was significant effect of oestrus synchronization method on oestrus response and follicular development and a single 300IU injection of eCG to nulliparous Boer does synchronized with PGF2α, flugestone acetate, or their combinations resulted in higher oestrus response, maximum follicle diameter and ovulations compared with a single injection of FSH (5mg) in intensively raised nulliparous Boer does under tropical conditions. These findings may suggest further studies on an optimum complementary combination of these two gonadotrophins for use in farms as a single cost effective injection, and without adverse effects on future fertility.
The authors wish to thank the Faculty of Veterinary Medicine, Universiti Putra Malaysia for the graduate research fellowship and to Associate Professor Dr. Abdul Rahim Mutalib for providing the RIA kit for cortisol. Special thanks also to Mr. K.C. Yap, P. Ganesamurthi and Mr. M. Fahmi for their technical assistance. We also wish to acknowledge the management and staff of ar-Raudhah Biotech Farm Sdn Bhd, Malaysia for providing the animals and research materials for this study.