Daily transrectal ultrasonographic examinations were conducted in 22 clinically healthy multiparous Boer goats to determine the effects of PGF2α and progesterone methods of oestrous synchronization on ovarian follicular development during the synchronized cycle. The PGF2α group was synchronized with a double intramuscular injection of 125µg cloprostenol 11 days apart while the progesterone group were synchronized with CIDR. Two to six waves were observed, with 3 waves of follicular development most frequently observed (56.1%), while 4 waves (31.7%) were the next most frequently observed pattern in both groups but with no statistical differences (P>0.05) detected between them. The mean ± SEM for number of days to ovulation from cessation of treatment in the PGF2α and CIDR synchronized groups were 3.86±0.26 and 3.63±0.38 respectively and were also not statistically significant. The mean ± SEM for interovulatory intervals were 18.71±0.47 and 19.00±0.76 days for PGF2α synchronized and subsequent natural estrous cycle respectively, and 18.75±1.05, 19.86±1.01 for CIDR synchronized and subsequent natural estrous cycles respectively. There were no significant differences (P>0.05) between groups in the total follicle number, and maximum size of ovulatory follicles. There were however statistically significant differences (P<0.05) in the number of ≥3mm and ≥8mm sized follicles between groups. The mean ± SEM for maximum sizes of ovulatory follicles were 7.11±0.30, 7.33±0.36, 7.43±0.301, and 6.98±0.56, for PGF2α and CIDR synchronized oestrus and natural oestrous cycles respectively. From the results of this study, it could therefore be concluded that oestrus synchronization using PGF2α or intravaginal progesterone inserts does not significantly alter follicular dynamics during the synchronized oestrous cycle in non-seasonally anoestrus goats raised under tropical conditions.
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Key words: Follicular dynamics, Ultrasonography, Oestrus synchronization, Goats
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Goats are highly adaptive and prolific small ruminants that are increasingly becoming a significant socio-economic resource in many countries (Lebbie, 2004; Shrestha and Fahmy, 2007; Lehloenya et al., 2008; Simela and Merkel, 2008). Knowledge of follicular development is becoming increasingly critical in order to develop and improve methods that manipulate the fertility thus increase productivity of goats (Rubianes and Menchaca, 2003). The development and use of serial ultrasonography as a tool to non-invasively monitor follicular development in goats has been described previously (Ginther and Kot, 1994; Gonzalez-Bulnes et al., 2004; Lassala et al., 2004; Simoes et al., 2006; Filho et al., 2007; Fernandez-Moro et al., 2008).
Follicular dynamics in the pre-ovulatory and ovulatory periods have been studied in different goat breeds during natural oestrous cycles (Ginther and Kot, 1994; Simoes et al., 2006; Sarath et al., 2008). Follicular dynamics were also studied in goats synchronized with progesterone or progestagens (Letelier et al., 2009), double injections of PGF2α or its analogue (Medan et al., 2003; Gonzalez-Bulnes et al., 2004; Vazquez et al., 2010) or combinations of the above with or without exogenous gonadotrophins, FSH or eCG (Menchaca and Rubianes, 2002; Motlomelo et al., 2002; Lassala et al., 2004; Menchaca et al., 2007).
Vazquez et al. (2010) observed that synchronization of oestrus and ovulation by administration of prostaglandin analogue, 10-12 days apart caused differences in developmental dynamics and functionality of induced corpora lutea when compared with natural spontaneous oestrus and ovulation. Levya et al. (1998) suggested that oestrus synchronization using progestagens sponge inserts altered the number of follicular waves and modified the size of the largest follicles depending on whether the progestagen treatment was at early, middle or late luteal phase. Maintenance of high concentrations of progestagens and thus low LH concentrations also increased follicular turnover (Levya et al., 1998). Low concentrations of progesterone particularly towards the end of progestogen treatment may lead to inadequate suppression of LH resulting in abnormal follicular development such as large persistent follicles (Kojima et al., 1992; Johnson et al., 1996; Vinoles et al., 1999; Menchaca and Rubianes, 2004)
Previous studies conducted using heifers suggested that exogenous progesterone decreased the number of hypothalamic estradiol receptors, resulting in increased follicular steroidogenesis as a result of increased in gonadotrophin (FSH and LH) release after cessation of treatment with the exogenous progesterone (Evans et al., 1994; Leyva et al.,1998). Adams et al. (1992) also suggested that high dose of progesterone suppressed the dominant follicle in a dose dependent manner during its growing phase.
However, there appears to be very little information on the comparative effects between progesterone and prostaglandin on follicular populations throughout the synchronized oestrous cycle. This study was, therefore conducted to determine the ovarian follicular population dynamics in goats, which had been synchronized with intra-vaginal progesterone insert or with injections of cloprostenol during the interovulatory period.
MATERIALS AND METHODS
Experimental animals and design
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A total of 22 clinically healthy multiparous female Boer goat crosses of 3-4 years old were selected for this study from a population of 146 goats. The mean weight and body condition score for the experimental animals were 35±2.7 kg and 3-4 (scale1 to 5) respectively. The goats were raised intensively in raised sheds/barns with slatted floors at a commercial goat farm, (Lat: 3° 15N and Long: 101° 32' 60E), in Selangor, Malaysia. Transrectal ultrasonographic screening was performed on each goat during the selection process to exclude those with ovarian (follicular or luteal cyst) or uterine pathology (pyometra, hydrometra) or pregnancy. The does were fed rations daily, comprising palm leaves, commercial pellets, soya bean waste and palm leaf silage. Water and salt licks were provided ad-libitum.
The goats were initially divided into two main groups: PGF2α synchronized group (n=11) and CIDR synchronized group (n=11). In the PGF2α synchronized group, oestrus was synchronized with a double injection of 125µg (0.5ml) of the PGF2α analogue, cloprostenol (Estrumate™, Schering-Plough) 11 days apart (Ahmed et al., 1998; Kusina et al., 2000., Vazquez et al., 2010). The natural oestrous cycle immediately subsequent to the PGF2α synchronized oestrus cycle of was also studied in the same group.
The CIDR synchronized group (n=11) was synchronized with Controlled Internal Drug Releasing Device (CIDR, EAZI-BREED™) containing 0.3g progesterone inserted into the vagina and left in place for 17days (Wildeus, 2000; Motlomelo, et al., 2002). The natural oestrous cycle immediately subsequent to the CIDR synchronized oestrus cycle of was also studied in the same CIDR synchronized group.
The natural oestrous cycle in does randomly selected from the PGF2α and CIDR synchronized groups was also studied, one month after the end of the synchronized and subsequent natural oestrous cycles studied above.
Ultrasonographic evaluation of follicular development
Daily ultrasonographic scanning of the ovaries to study ovarian follicular development commenced one day after removal of intravaginal CIDR insert in the first group or one day after the second PGF2α injection. Each goat was scanned once daily and the scanning periods were from 0800 to 1200 hours, using a real-time B-mode ultrasound scanner (Aloka, 500 SSD, Japan), with a transrectal 7.5MHz linear probe (UST-660-7.5 model). Ovaries were visualized and the number, size and position of follicles of ≥ 3mm (range of ≥ 3 to ≥8mm) in diameter were measured using the ultrasound scanner's in-built callipers. Images were frozen and sketches of all ovarian structures observed in each ovary were made as they were visualized in real time (Ginther and Kot, 1994; Simoes et al., 2005; Simoes et al., 2006).
Follicles were distinguishable as hypoechoic, roughly circumscribed image within the outline of the ovary. Some of the images were frozen and printed for subsequent comparison to the ovarian charts. Ovulation was considered as the collapse of a large preovulatory follicle (≥ 5mm in diameter), which had been monitored daily by ultrasonography, and the subsequent appearance of a corpus luteum on the same location (Ginther and Kot, 1994; Simoes et al., 2006; Menchaca et al., 2007; Vazquez et al., 2010)
Ultrasonographic data were summarized for each goat. Follicle data were combined for both left and right ovaries. Follicular waves were characterized according to previously described methods (Ginther and Kot, 1994; Gonzalez-Bulnes et al., 1999; Simoes et al., 2005; Simoes et al., 2006). A follicular wave was defined as one in which one or more follicles appeared within 48 hours and grew to at least 5 mm in diameter (Ginther and Kot, 1994; Gonzalez-Bulnes et al., 1999; Simoes et al., 2005; Simoes et al., 2006). Wave onset (emergence) was the day in which follicles were firstly detected at 3 mm, growing to at least 5 mm on the following 3 day period (Simoes et al., 2006). End of wave was considered as when the proportions of growing and regressing 3mm follicles were the same (Simoes et al., 2006).
Descriptive statistics, repeated measures analysis of variance (ANOVA), Kruskal-Wallis' test and Chi-square analysis were conducted where necessary using SPSS statistical software (SPSS Inc. Version 17). Analyses were considered to be statistically significant at P < 0.05.
Data from four and three animals in the PGF2α and CIDR groups respectively were not included in the analysis for the following reasons, either as a result of pregnancy diagnosed within the first week of commencement of the study (1 goat from CIDR group), persistent large follicles resulting to indistinct follicular wave pattern (2 goats from PGF2α group) or due to non-scanning for 2 or more consecutive days (4; 1 goat from PGF2α group and 3 goats from CIDR group). Each group of animals in the study had different number of follicular wave represented in different proportions as shown in Table 1.
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The mean pooled daily number of follicles from cessation of treatment (second PGF2α dose or CIDR removal) over the 23-day scanning period in synchronized oestrous cycle, the subsequent natural estrous cycle and spontaneous oestrous cycle (control) are presented in Fig. 1 and Fig. 2 respectively. There were between 3 and 8 follicles of ≥3 mm in diameter daily on the ovaries of both PGF2α and CIDR synchronized groups except for the natural cycle subsequent to the PGF2α cycle which ranged from 1 to 9 follicles of ≥3 mm in diameter within the first 6 days after ovulation. The mean daily numbers of follicles for all the groups are shown in Table 2. The mean total follicle number and sizes in natural estrous cycle, PGF2α, and CIDR synchronized goats is represented in Fig. 3. The distribution shows a decrease in the number of follicles with increase in follicle size.
There were no statistically significant differences (P>0.05) in the mean pooled daily follicle number and mean total follicle number between CIDR and PGF2α and in their respective subsequent natural oestrous cycles (Table 2). Repeated measure analysis of variance showed significant main day effects (P<0.05) for mean daily number of follicles. Differences in the mean follicular size were significant (P<0.05) between 3 mm sized follicles in CIDR and PGF2α synchronized cycles. The mean number of 4, 5, 6 and 7 mm sized follicles were however not significantly different between synchronization methods (P>0.05). The mean 8 mm sized follicles were significantly different between CIDR and PGF2α synchronized cycles, the subsequent oestrous cycles and the natural oestrous cycle (control).
The mean pooled daily number of follicles from cessation of treatment in synchronized oestrous cycle, subsequent natural estrous cycle and natural oestrous cycle (control) shows a decrease in the number of follicles but increase in follicle size. Previous studies have shown that synchronous development of a cohort of follicles at the beginning of a wave is followed by senescence of majority of them with only a few follicles growing to become dominant before ovulating or regressing (Ginther and Kot, 1994; Adams et al., 2008).
In this study, there were no statistically significant differences (P>0.05) in the mean pooled daily follicle number and mean total number of follicles between CIDR and PGF2α groups and their respective subsequent oestrous cycles. This finding agrees with Lassala et al. (2004) who similarly found no statistical differences (P>0.05) in follicular dynamics between prostaglandin synchronised and natural oestrous cycles in Serrana goats. Vazquez et al. (2010) also suggested that the mean number of total follicles developing in each cycle did not differ significantly between groups of PGF2α synchronized and natural oestrous cycle in Anglo Nubian goats. Similar studies in both cows and heifers, suggest that prostaglandin treatment did not significantly modify the follicular dynamics (Figueiredo, et al., 1997).
Differences in the mean small (≤3 mm) sized follicles were significant (P<0.05) between CIDR and PGF2α synchronized cycles. Mean follicle size of 4 to 7 mm sized follicles were however not significantly different between CIDR and PGF2α synchronized cycles (P>0.05), while mean large (≥8 mm) follicles were significantly different among synchronized groups, subsequent oestrous cycle and natural oestrous cycle. This finding closely follows that of Vazquez et al. (2010) who reported that an assessment of size distribution of follicles suggested that prostaglandin synchronised goats had a higher number of small and large follicles (P < 0.05) compared with natural oestrous cycle while there were no significant differences between groups in the number of medium follicle. Progesterone has been shown to decrease follicular development in a dose dependent manner (Adams et al., 1992). According to de Castro et al. (1999), high progesterone concentrations has a suppressive effect on oestradiol negative feedback on the hypothalamus, the subsequent reduced number of oestradiol receptors leads to increased release of FSH, LH and follicular steroidogenesis which increases growth and size of the follicles. These positive effects of progesterone on follicle size have been previously suggested (Adams et al., 1992; Vinoles et al., 1999)
Two to six waves of follicular development have been reported to occur during normal oestrous cycles in goats, with 3 and 4 waves being most frequently observed (Ginther and Kot, 1994; de Castro et al., 1999; Schwarz and Wierzchos 2000; Menchaca and Rubianes, 2002; Cueto et al., 2006). In the present study, two to six waves of follicular development were similarly observed, with 3 waves of follicular development, most frequently observed (56.1%), while 4 waves (31.7%) pattern came second.
The results of this study are in agreement with Cruz et al. (2005) and Vazquez et al. (2010). These 2 groups of researchers reported three wave pattern of follicular development to be most frequent in Saanen and Anglo-Nubian goats raised under tropical environment. However, a four wave pattern of follicular development have been reported by previous researchers to be most prevalent in seasonally anoestrous goats in temperate environments (Ginther and Kot, 1994; Menchaca and Rubianes, 2002; Medan et al., 2003; Gonzalez-Bulnes et al., 2004; Filho et al., 2007). The differences in these reported predominant wave patterns may be related to the tropical conditions of the research area and the non-seasonally poly-oestrous reproductive cycles of the goats studied. Bo, et al. (2003) suggested that some contributing factors that can affect number of follicular waves include low plane of nutrition and heat stress, while Noseir (2003) proposed genetic predisposition and uncontrolled environmental conditions among the contributing factors. Seekallu et al., (2010) however, suggested that there were no apparent endocrine or follicular characteristics that could explain the regulation of the different number of follicular waves (three vs. four) during cycles of similar length. Further studies of the determinants of follicular wave patterns and their significance are therefore necessary.
The mean day of emergence, maximum follicular diameter and duration of the waves among PGF2α and CIDR synchronized cycles, their subsequent natural oestrus cycles, and the natural (unsynchronized) oestrus respectively were not statistically different. These findings are in agreement with Simoes et al. (2006) who similarly found no differences (P>0.05) in the onset, the day of maximum follicular diameter and end of waves in goats with three, four or five waves per cycle synchronized with PGF2α compared with natural oestrous cycle.
There were no statistically significant differences (P>0.05) in time to ovulation from cessation of treatment and the maximum diameter attained by the ovulatory follicles among the PGF2α and CIDR synchronized, subsequent oestrous cycle and the spontaneous cycle. The time of ovulation were also similar to those reported by Simoes et al. (2008) in PGF2α synchronized nulliparous and multiparous Serrana goats during the breeding season. The results of this study concurs with Simoes et al. (2008) who further suggested that no statistical differences were observed in the onset of oestrus to first ovulation interval between nulliparous and multiparous goats or between monovular and polyovulatory oestrus.
The type of oestrus synchronisation method had no significant effect (P>0.05) on the interovulatory interval in the groups studied. The mean interovulatory intervals observed in this study were also not significantly different among the groups studied and were within the range reported in previous studies in synchronized and spontaneous oestrous cycles (De Castro et al., 1999; Gonzalez-Bulnes et al., 1999; Medan et al., 2003; Simoes et al., 2006).
The maximum diameter attained by the largest follicle in the first wave were larger than the maximum diameter of the second and third waves in PGF2α and CIDR synchronized oestrous. These results agree with previous results where the maximum follicular diameters attained by the first and fourth waves were larger than the maximum diameter of the second and third waves (Ginther and Kot, 1994; de Castro et al., 1999; Simoes et al., 2006). Simoes et al. (2006) observed that these observations support the negative effect of different plasma progesterone levels associated with presence and functional status of CL at the onset, middle or end of the oestrous cycle as suggested by Ginther and Kot (1994) in goats or by Leyva et al. (1998) in sheep.
The echographic images of the ovarian structures observed during the daily ultrasonographic scanning were very similar to previous reports (Gonzalez-Bulnes et al., 2004; Lassala et al., 2004; Simoes et al., 2006; Filho et al., 2007; Fernandez-Moro et al., 2008; Simoes et al., 2008). From the results of this study, it could therefore be inferred that changes in follicular dynamics resulting from the use of PGF2α or intravaginal progesterone inserts does not significantly alter post-ovulatory follicular dynamics in non-seasonally anoestrus goats raised under tropical conditions.
Three waves of follicular development were most frequently observed during natural, PGF2α, and CIDR synchronized estrous cycles in the present study. The mean ± SEM day of ovulation after cessation of treatment in the PGF2α and CIDR groups were 3.86±0.26 and 3.63±0.38 respectively with no statistically significant differences between them. The mean ± SEM interovulatory interval were 18.71±0.47, 19.00±0.76 days for PGF2α synchronized and subsequent natural estrous cycle respectively, and 18.75±1.05, 19.86±1.01 days for CIDR synchronized and subsequent natural estrous cycle respectively. There were no significant differences (P<0.05) between groups in the, total follicular number, maximum size of ovulatory follicles, and the maximum follicles size attained per wave. It could therefore be concluded that loestrous synchronization with a luteolytic dose of PGF2α or intravaginal progesterone inserts does not significantly alter the follicular dynamics during the subsequent oestrous cycle in non-seasonally polyoestrus goats raised under tropical conditions.
Aknowledgements. The authors wish to thank the Faculty of Veterinary Medicine, Universiti Putra Malaysia for their support. 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.