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Information in follicular development in Bos indicus cattle following hormonal intervention is lacking. Therefore a study was conducted to determine the follicular wave development in terms of size and number of dominant follicles and number of follicular waves in local zebu cattle following CIDR® and prostaglandin treatments. Thirty cows of three breeds (Kedah-Kelantan, n=10; Brakmas, n=10; and Charoke, n=10) ranging from first to third parity were used in the study. The cows were inserted with controlled intra-vaginal progesterone releasing devices (CIDR®) containing 1.9 g progesterone for 7 days and given intramuscular injection of 25 mg Prostaglandin-F2Î± (Estrumate®: PGF2Î±) two days prior to CIDR® removal. The mean diameter of dominant ovulatory follicles after CIDR removal was 11.73 ± 0.07 mm, and no significant difference (P = 0.26) was found among the breeds of cattle studied. The newly developed follicles emerged on 1.37 ± 0.16 days at a mean size of 5.1 ± 0.02 mm after ovulation with no significant difference detected among breeds (P= 0.23). The number of follicles at emergence was significantly higher in Kedah-Kelantan (4.4±0.63, P=0.036) compared to Brakmas (3.2±0.36) and Charoke (2.6±0.37). In conclusion, the results of the present study concurred with the hypothesis of follicular growth followed a wave like pattern, and there were differences in the development of the first, second and third follicular wave in Brakmas, Charoke and Kedah-Kelantan cattle.
Keywords: beef cows, follicular development, oestrus synchronisation, CIDR®
Research in cattle reproduction in Malaysia had focused on the development and growth
of follicles in the ovaries (Azizah et al., 2005; 2007). Most studies on follicular dynamics
were conducted in Bos taurus European breeds of cattle (Savio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989), and a report on Zebu cattle (Bos indicus) was limited to studies reported by Figueiredo et al. (1997), Rhodes et al. (1995), and Pinheiro et al. (1998). These studies used different protocols for oestrous synchronisation. The same synchronization protocol had been proved to react differently, depending on the type of cows, age, breed and type of hormones used for ovarian superstimulation. Kawate et al. (2004) had reported that the addition of an intravaginal controlled internal drug release (CIDR®) device to the ovsynch protocol which combines gonadotropin releasing hormone (GnRH) and PGF2Î± had improved the rate of conception following timed-artificial insemination (timed-AI) in postpartum Japanese Black beef cows. Fertility to timed-AI using the protocol of estradiol and CIDR® and followed by PGF2Î± and GnRH, had been reported only for beef heifers (Colazo et al., 2003) and Bos indicus cattle. These studies were conducted to improve the protocols of synchronization of oestrus and ovarian superstimulation in order to adapt with artificial insemination at specific time or timed-AI (Fricke et al., 1998).
Synchronising the oestrous cycle involves the manipulation of ovarian activity so that the
time of ovulation can be predicted. Using this method, over 90% of cattle can be induced
to enter oestrus within 24 hours (Cavalieri et al., 2003) and a greater proportion of anoestrus of prepubertal animals can be induced to ovulate following treatment (Fike et al., 1997). The administration of GnRH during the oestrous cycle resulted in LH release (Chenault, 1990), causing ovulation or luteinization of the dominant follicles in the ovary, synchronized recruitment of a new follicular wave (Thatcher, 1989) and follicular development (Wolfenson, 1994).
In cattle and sheep, prostaglandin given during luteal phase for oestrous synchronization reduces the progesterone level and length of the oestrous cycle, thus enhancing the formation of follicular growth and occurrence of the ovulation earlier than expected (Pursely, 1997). Therefore, the development of oestrous synchronization method could best be preceded by understanding the physiology of bovine oestrous cycle. The objective of the study is to determine the follicular wave development in terms of dominant size follicles and number of waves and follicles existing in each follicular wave in Zebu cattle following CIDR® and prostaglandin treatments.
MATERIALS AND METHODS
Animal Management and Treatment
The study was conducted at MARDI Research Station, Kluang, Johor (1°56'57"N
103°21'56"E). Thirty cows of three breeds of local zebu (Bos indicus) cattle: were Kedah-Kelantan (KK, n=10), Brakmas (BK, n=10), and Charoke (CK, n=10) ranging from first to third parity and 2 to 4 years of age with body weight ranging from 250 to 350 kg and average body condition score of 4 (1=emaciated, 4=moderate, 8=overweight) were used in this study. KK was a local Zebu breed, whereas BK was developed from the crossing of Brahman and KK breeds, and CK from crossing of Charolais and KK breeds. Cows were managed in a semi-intensive system - cows were released for grazing in the morning and maintained indoor for the rest of the day in individual pens and fed pellet feed and had free excess to water. Feed was offered to the cow based on maintenance requirement of beef cows (ARC, 1980). The study was conducted for two consecutive oestrous cycles.
Synchronisation of Oestrus
Cows received a controlled intra-vaginal progesterone releasing device (CIDR®, Pharmacia & Upjohn, Australia) containing 1.9 g progesterone for 7 days. Intramuscular injection of 25 mg Prostaglandin-F2Î± (PGF2Î±; Estrumate® containing 250 Î¼g/mL cloprostenol, Schering - Plough Animal Health, Australia) was given on Day 5 after CIDR® insertion. The synchronisation was conducted twice, 14 days apart, and the cows were observed for oestrus signs after CIDR withdrawal (Day 8) on the second part of synchronisation (Figure 1).
Ovarian Ultrasonography and Follicular Mapping
Prior to the ultrasonogaphy, the rectum of the cow was emptied of faeces and the position
of the ovaries was localized and identified by rectal palpation. The ovaries were located either slightly underneath the uterus or to the side of the uterus at a variable distance.
Both ovaries were scanned using 7.5 MHz linear array transrectal transducer attached to the portable ultrasound device (Aloka®, SSD-500) to visualize the image of the ovaries onto a ultrasound monitor. Scanning was carried out from six hours at the end of oestrus and was repeated every six hours until ovulation occurred.
The diameter of the follicle was obtained by freezing the image, followed by its measurement with electronic callipers at the interface of the follicular wall with ovarian stroma. For the non-spherical shape, the largest and the smallest width were measured, and the average width was recorded. The number of follicles greater or equal than 4 mm (â‰¥4 mm) in diameter was counted, measured and mapped from both ovaries on the follicle map. Ovarian data were combined for the 2 ovaries of each animal. Size and relative dimension of follicles and corpus luteum (CL) were sketched on a follicle map.
The day of first detection of a follicle of diameter â‰¥ 4 mm was identified as a dominant follicle and taken as the first day of a wave following the method of Ginther et al (1989). A dominant follicle (DF) is the largest follicle which occupies the most space of ovarian stroma during its growth phase. Ovulation is confirmed by the disappearance of large antral grafian follicles of size greater than 10 mm in diameter as evident with the formation of a corpus luteum in the same location on the ovary. The day of ovulation at the beginning of an interovulatory interval was designated as Day 0.
Blood Collection and Progesterone Assays
During each ultrasound examination, 10 ml of blood was sampled from all cows through jugular venipunture into plain tubes (Vacutainer®, Becton Dickinson Limited, England) using a hypodermic disposable needle 1.2 x 38 millimetre (mm) for determination of progesterone (P4) hormone concentration. Blood was kept at room temperature for an hour and stored at 4oC for 24 hours. Serum was obtained from all samples by blood centrifugation at 700 G for 20 minutes. Serum was decanted and kept in small bottles before stored frozen at -20oC. Serum was transported on dry ice for radioimmunoassay RIA), P4 assay was performed using the developed kit assembled by the Animal Production Unit (Diagnostic Products Corporation, Los Angeles, CA 90045). The sensitivity of the assay was 0.02 nanogram millilitre-1 (ng/mL).
Statistical analysis for detection of breed differences in follicular dynamics and hormonal concentration was carried out using analysis of variance (ANOVA). The proportion of cows with specific number of waves of follicular development was tested using chi square analysis. Both analyses used a statistical package SPSS version 17.0. For the purpose of the present study, size and growth of the dominant follicles and emergence of new follicular wave were analysed. Progesterone concentration data was analysed as repeated measures. ANOVA to examine the influence days of oestrus cycle on progesterone concentration in the breeds of cattle.
The results of follicular wave development characteristic after CIDR withdrawal pre- and post-ovulation of KK, BK and CK cows are presented in Tables 1 to 6. The most common pattern of ovarian follicular development following ovulation was of two or three 4-mm follicles emerging together and developing into 6- or 7-mm diameter follicles, but only one continuing to increase in size with other follicles decreasing in size. A second, third or occasionally fourth wave of follicles would emerge in the same manner during the luteal phase of the oestrous cycle.
Table 1 showed the follicular wave development characteristic following ovulation in the three breeds of cows. Average diameter of dominant ovulatory follicle after CIDR removal was 11.73 ± 0.07 mm, and no significant difference (P = 0.26) of the diameter of ovulatory follicle after CIDR withdrawal was detected in the three breeds of cattle. However, the number of follicles at emergence was significantly higher in KK (4.4±0.6, P=0.04) compared to BK (3.2±0.4) and CK (2.6±0.4). No significant difference in mean diameter of follicle at emergence and mean diameter of preovulatory follicle was observed between BK and the other two breeds of cows, KK and CK.
Table 2 showed the first follicular wave development characteristic after CIDR® withdrawal post-ovulation. The number of follicles at emergence was significantly higher (P = 0.036) in KK (3.8±0.9) compared to BK (3.2±0.5) and CK (2.7± 0.7). The new follicles emerged on average 1.4 ± 0.2 days following CIDR® remove with mean size of 5.1 mm ± 0.02 (4.9±0.02mm; 5.0±0.01mm; 5.4±0.02 mm for KK, BK and CK, respectively) after the ovulation with no difference be detected between breeds (P= 0.227). The first dominant follicle took an average of 8.5±1.6 days to become dominant with no significant difference detected among the breeds (P = 0.75). The first dominant follicle reached an average maximum diameter of 12.5±0.05 mm (11.4±0.08 mm; 13.1±0.08 mm and 13.1±0.07 mm in KK, BK and CK, respectively) with no difference detected among the breeds studied (P=0.19).
The result of the second follicular wave of follicular development is tabulated in Table 3. The average diameter of non-ovulatory and ovulatory dominant follicle of second wave of follicular development was 12.37±0.08 mm, and there was no significant difference (P = 0.062) of the dominant follicle diameter between the three breeds of cattle (9.5 ± 0.2 mm; 12.8 ± 0.08 mm and 12.9 ± 0.08 mm in KK, BK and CK, respectively). The day of follicle emergence was not significantly different between breeds (P = 0.75). It was found that the follicle developed to a dominant status faster in BK (P = 0.03, 13.8 ± 1.1 days) followed by KK (15.0 ± 1.2 days) and CK (17.8 ± 0.5 days).
Mean diameter of ovulatory dominant follicle after CIDR® removal was not significantly different (P = 0.460) between the three breeds of cattle (Table 4). Mean ovulatory dominant follicle diameter was 10.8 ± 0.1 mm, with mean ovulatory dominant follicle diameter in KK was 10.2 ± 0.1 mm, BK 11.6 ± 0.10 mm and CK 10.7 ± 0.1 mm. It was also found that the mean length of oestrous cycle was 20.5 ± 0.6 days with no significant difference found among the three breeds of cows (P = 0.8).
Figures 2a and 2b represent turnover of dominant follicles producing 3-wave and 2-wave pattern of follicular development for the study. Figure 2a shows the first and second dominant follicles developed in similar pattern. However, the first dominant follicles first achieved maximum diameter on day 1 to 3, and the second dominant follicles on day 5 to 9. The third dominant or ovulatory follicles appeared on day 11 and ovulated on day 22. Figure 2b illustrates that the first dominant follicles achieved the maximum diameter on day 8 to 9. Meanwhile the ovulatory follicles emerged on day 7 to 8 and ovulated on day 19 to 20.
The growth rate of dominant follicle of the present study was similar in the three breeds (1.4 ± 0.18 mm day-1; P=0.48) as shown in Table 5. The regression rate of dominant follicles of BK 1.52 mm day-1 faster compared to the other breeds of the study. The regression rate of BK was 2.3 ± 0.04 mm day-1 and significantly different for those of KK and CK (P=0.05). The pattern of the progesterone concentration is presented in Figure 3. The results showed the progesterone concentration in one oestrous cycle was significantly higher in KK (2.7±0.6 ng ml-1; P = 0.02) compared to the other two breeds of cows
The study determined the wave pattern of follicular characteristic in three Malaysian breeds of beef cattle namely BK, CK and KK. The oestrous cycle of three breeds of cattle evaluated indicated BK had a higher proportion (45%) of 3- follicular waves, followed by CK (35%) and KK (20%). For 2-follicular waves it was highest in proportion for KK (60%), followed by CK (35%) and BK (10%). There was no evidence of these breeds having four or more waves of follicular development. Similarly in Rathi cattle there was no four or more follicular waves found (Gaur and Purohit, 2007).
Evidence of a higher proportion of 2-waves (78.6%) compared to 3-waves (21.4%) of follicular development was also reported by Gaur and Purohit (2007). However, in Gir cows (Vianna et al., 2000) a higher incidence of 3-waves of follicular development and a small proportion of cows having 4- or more follicular waves during the oestrus cycles was found (Vianna et al., 2000). The present study found that BK had a higher proportion of 3-waves follicular development. However, KK had a higher incidence of two-wave follicular development. Similarly, many studies conducted in Europe had reported a higher prevalence of cycles with three waves (Savio et al., 1988; Sirois and Fortune, 1988; and Viana et al., 2000) and two waves (Pierson and Ginther, 1988; Taylor and Rajamahendran, 1991) of follicular development.
To date, this is the first study in which the wave patterns of follicular growth in Malaysian cattle were determined. Majority of Brakmas cattle in the study tended to have 3-follicular waves (45%). Various studies conducted on Brahman cattle have shown that 2-follicular waves pattern was more often recorded (Alvarez et al., 2000; Figueiredo et al., 1997 and Zeitoun et al., 1996). Probably the difference seen could be due to the seasonal effects. Season could be one of the factors that influenced the variation in follicular wave offered in these studies. The discrepancies in the occurrence of 2-wave follicular development in Brahman cows that had been observed were 56, 38 and 84% conducted in summer (July and August) in Florida (Alvarez et al., 2000), spring (May) and Fall (October) in Texas (Zeitoun et al., 1996); and winter (July and August) in Brazil (Figueiredo et al., 1997), respectively. The occurrence of differences in number and follicle size due to the seasonal effects had also been reported by Lammoglia et al. (1996).
It has been documented that the number of follicular waves during the oestrous cycle was regulated by the length of the luteal phase (Ginther et al., 1989; Fortune let al., 1991; Lucy et al., 1992), and cows with 3-follicular waves had been observed to have a longer luteal phase, thus it would lead to longer duration of oestrous cycle (Ginther et al., 1989; Fortune et al., 1991; Lucy et al., 1992). Although BK showed a higher frequency of three waves of follicular development, it did not affect the length of oestrous cycle. Similarly Savio et al.
(1988) found the length of oestrous cycle was not influenced by the number of follicular waves which occurred during the oestrous cycle. The growth rate of the dominant follicle of BK was similar to the other two breeds of KK and CK. The results from the present study postulated a possibility that the regression rate of dominant follicle in the BK occurred faster compared to the other breeds in the study. Thus it led to the duration of each wave to occur almost at a similar time and did not influence the length of oestrous cycle. Other factors such as temperature and rainfall could also be the predisposing factors that influenced the incidence in the occurrence of wave of follicular development affecting the length of the oestrous cycle.
After post-CIDR ovulation, the follicle was capable to develop by itself until 4 mm in size (Garverick et al, 2002). However, it was dependent on FSH when it reached greater than 4 mm in size and composed the cohort that participated in the follicular wave (Garverick, et al., 2002). The FSH-dependent follicle capable of promoting oestradiol led the follicle to maximise its size to a larger diameter (Lucy et al., 2007). FSH was postulated to cause the emergence of the follicular wave. The increased concentration of FSH during and after LH triggered the follicular recruitment. In the study, it could be postulated that the FSH surge occur during postovulatory and midcycle of the first wave leading to the formation of atretic DF. Thus, it then will initiate the first and second follicular waves. Similarly in the 3-follicular wave pattern, the third FSH arises when the second DF becomes atretic.
Ovarian follicular growth is indicated by the number of follicles in all categories that develop during the oestrous cycle. The study had showed the average size of first, second and third dominant follicle were 12.5 ± 0.05mm, 12.4 ± 0.08 and 12.9 ± 0.06, respectively. The DF is selected from a cohort of follicles and it develops to attain dominance. The DF has the ability to suppress the growth of other smaller subordinate follicles (Mihm, 2002; Hendriksen et al., 2003; Quirk et al., 2004) and has the capability to ovulate with the influence of hormonal conditions (Fortune, 1993), and thus is termed the ovulatory follicle. We noticed in our study that the dominant anovulatory follicle inhibited the subordinate follicle from growing until the next dominant anovulatory or ovulatory follicle irrespective of having 2-wave or 3-wave follicular development as was found by Kulick et al. (2001). The average first dominant follicle in the present study was slightly bigger compared to Angus (11.4mm), and smaller compared to Brahman (15.3mm) and Senepol (13.9mm) (Alvarez, et al., 2000). This study supports the findings of Bo et al., (2003) who concluded that the maximum diameter of dominant follicles in Bos indicus was smaller than Bos taurus cattle. In the present study, the ovarian follicular development and growth as indicated by the diameter of the first dominant and ovulatory follicles after CIDR removal was smaller in Kedah Kelantan (11.8 ± 0.48 mm; P=0.024) than in either Brakmas (14.2± 0.08 mm) or Charoke (12.2 ± 0.07 mm).
In the first-wave follicular development of the study, the follicular wave emergence was at 1.4 ± 0.27 days with the size diameter of 5.1 ± 0.02 mm and the average number of days of follicle emergence of 3.4 ± 0.3 days. However, in Rathi cattle, the two and three waves of follicular development emergence were at 2.1 ± 0.4 and 4.1 ± 1.0 days, respectively. A previous study (Utt et al., 1993) observed that the follicular wave emergence after CIDR withdrawal was 4.8 days when the cows were treated with CIDR for 7 days, and 25 mg prostaglandin given a day prior to CIDR withdrawal. Apparently, in Angus cow that were treated with gonadotrophin releasing hormone (GnRH) or estradiol-17Î² and progesterone at
CIDR insertions, showed the intervals from CIDR withdrawal to follicular wave emergence of 6.6 and 4.7 days (Utt et al., 1993). In the present study, the preovulatory follicular wave development post-CIDR removal was shorter (ranged from 1.2 - 1.5 days). This is probably due to the different protocols and gonadotrophin used in the study, or it could be also related to the different stages or sizes of dominant follicles developing during the synchronisation (Lucy, 2007).
The study observed that the follicle growth wave like-pattern with two or three waves occurred during the oestrous cycle. The formation and turnover of various sizes of dominant follicles and formation of few small sized follicles surrounding the dominant follicles indicated a characteristic of a synchronized group of cohorts to a process of selection, emerging and growing toward ovulation. During ultrasonography, there was a lack of follicles that surrounded the dominant follicles. This could be due DF suppressing the subordinate follicles to become atretic. As in 2-wave follicular development, the second dominant follicle was an ovulatory, whereas the first dominant follicle decreased in size and became an atretic follicle. Similarly in 3-wave follicular development, the first and second dominant follicles became atretic while the third dominant follicle ovulated. This was found at the end of the study, when only 20 out of 30 animals remained for the mapping while the other 10 had ovulated.
The results of the present study concurred with the hypothesis that the follicle growth and development followed a wave like pattern, and there were differences in the development of the first, second and third follicular wave in terms number of follicles at emergence and day at dominance following induction of synchronisation of oestrus in Brakmas, Charoke and Kedah-Kelantan cattle. The follicle growth pattern is dependent on the number of follicular waves developed in certain duration to attain the normal length of oestrous cycle. Therefore, the importance of the study of follicular development is to understand and identify the turnover of follicular development during the oestrous cycles in order to refine the synchronisation protocol for timed-AI or ovum pick-up (OPU) for in vitro fertilisation.