Follicular Dynamic and Repeatability of Follicular Wave Development in Peranakan Ongole ( PO ) Cattle

Imron M, Supriatna I, Amrozi, Setiadi MA. 2016. Follicular dynamic and repeatability of follicular wave development in Peranakan Ongole (PO) cattle. JITV 21(1): 26-33. DOI: http://dx.doi.org/10.14334/jitv.v21i1.1349 Superovulation treatment on PO cattle (Bos indicus) was less responsive compared to Bos taurus breed. It might due to the difference of their follicular dynamic. This study was conducted to investigate the follicular dynamics and its repeatability in PO cattle. Follicular dynamics observations conducted on 9 cows trough ultrasound scanning every day. Observations of wave patterns repeatability were performed in 6 cows which its wave pattern already known on the next consecutive IOI. Research result indicated that PO cattle had 3 (66%) and 4-waves (34%) pattern. The first wave of 3 and 4-waves pattern emerged on day 0.4+0.9 and 1.4+1.1 respectively. The second wave of 3 and 4-wave pattern emerged on day 9.8+1.5 and 7.4+1.9 respectively. The pattern of 3 waves has a longer follicle dominant duration (11.6+1.5 day) in the first wave of estrous cycle, compared with 4 waves pattern (10+2.92 and 7+1.00 day respectively). The growth rate of dominant follicle was not different significantly between the 3 and 4-waves pattern (0.87+0.23 and 0.94+0.25 mm/day respectively). Similarly, ovulatory follicle diameter between 3 and 4-waves pattern was also not different significantly (12.24+12.34 and 12.30+12.23 mm respectively). Observation of wave patterns repeatability in 6 PO cows indicated that PO cattle had high repeatability in follicular wave pattern (0.88) and the number of growing follicle was 0.91. This study resulted data for dynamic of follicular development, wave pattern, its repeatability which be expected to design the protocol of superovulation treatment or other reproduction technologies based on follicular dynamic to improve its result in PO cattle.


INTRODUCTION
Follicular wave was identified by growth of a small group follicle with diameter by 3-4 mm (Taylor & Rajamahendran 1991).Wave growth pattern of follicle might be observed in prepubertal (Melvin et al. 1999), during pregnancy (lactating period) (Taylor & Rajamahendran 1991), post-partum (Murphy et al. 1990), and during estrus cycle (Roche et al. 1999).Growth of follicular wave was began with increase of concentration of FSH serum and followed by sudden growth of 8-41 small follicles in 2-3 days (Ginther et al. 1997).One follicle would be selected to continuously grow to be dominant follicle (DF), meanwhile the smaller follicles would be subordinate follicle (SF) and would undergo an atresia.If there was a luteolysis in the growing-phase DF, follicle would undergo final maturation process continued by ovulation.If there was no lutheolysis in the growing DF, the DF would undergo an atresia (Vasenna et al. 2003;Jaiswal et al. 2004;Adam et al. 2008).
Most of cattle breed showed 2 and 3 follicular wave pattern in one estrous cycle (Adam et al. 2008) although sometimes showed 1, 4, and 5 follicular wave pattern (Bleach et al. 2004;Viana et al. 2000).Follicular dynamic was one of research subjects which have been much studied in Europe breed (Bos taurus) but information of zebu cattle (Bos indicus) was very limited.Bó et al. (2003) has reviewed that reproduction characteristic of B. indicus cattle was different from B. taurus such as in length of estrous cycle, estrous time, estrous behavior, growth of dominant follicle and CL.
Superovulation program at Cipelang Livestock Embryo Center (LET) performed to PO cattle (Bos indicus) as donor showed lower ration of transferable embryo per total embryo collected (1.5 embryos) than Angus, Simmental and Limousine cattle (Bos taurus) which had ratio by 4.75, 3.37 and 2.96 embryos respectively (BET 2012).Hormone injection in superovulation was undifferentiated between donor from Bos taurus (Simmental and Limousine cattle) and donor from Bos indicus (PO cattle).Bó & Mapletoft (2014) said that response of superovulation would be optimal if follicle super-stimulation treatment was started in the beginning of follicular growth wave emergence.One day earlier or later gonadotropin treatment from initial follicular wave would decrease superovulation response compared to starting treatment right at the time of the initial follicular wave emergence (Bó et al. 2008).It was assumed that there was a relation of difference of reproduction characteristic between both cattle breeds especially in follicular wave pattern with the obstacles faced in superovulation program in PO cattle.Information of characteristic of ovulary dynamic and its repeatability are needed to optimize superovulation program in PO cattle.
This study was conducted to determine characteristic of ovulary dynamic during estrous cycle and repeatability rate of follicular growth wave pattern in Peranakan Ongole (PO) cattle.

Materials
In this study 9 five-seven years old PO cows with body weight of 375-450 kg were used.All cows had Body Condition Value (BCV) ranged in 2.7-3.2 of scale 1-5 with normal estrous cycle.Diet was provided as 30-40 kg of grass per head per day and 2-3 kg of commercial diet per head per day.

Ovulation synchronization
This study was started by ovulation synchronization to synchronize initial observation of ovulary dynamic through installation of intra-vaginal progesterone preparate (Cuemate ® , consisted of 1.56 mg progesterone in 2 pod silicon, Bioniche Animal Health (A/Asia) Pty.Ltd., Australia) followed by injection of 100 µg GnRH (Fertagyl ® , Intervet Schering-Plough Animal Health, German) in the first day.Cuemate ® was entered into vagina for 7 days using special applicator oiled by isotonic gel.PGF2α (Prostavet ® C, 5 mg of etiprostone per 2 ml of solution; Virbac Animal Health, France) was injected intramuscularly along with Cuemate ® releasing in vagina.Two days later, 100 µg GnRH (Fertagyl ® , Intervet Schering-Plough Animal Health, German) was added intramuscularly to induce ovulation synchronization.

Observation of follicular dynamic
Observation of follicular dynamic was performed once a day at the same time and operator using portable ultrasonic (EasyScan Lite, England) equipped with goggle monitor and probe with dynamic frequency by 4-8 Mhz.The main unit of ultrasound was connected to computer and data were recorded in video version.Measurement of diameter of follicle was conducted using Microsoft image tool.Daily observation using ultrasound was started when PO cattle injected by PGF2α.One inter-ovulatory interval (IOI) was defined as time period between 1 ovulation with the next ovulation.First ovulation observed after ovulation synchronization was assumed as day-0 in 1 IOI.
In the initial step, observation was conducted to obtain characteristic of follicular dynamic and for wave pattern in 1 IOI mapping.Nine cows were used in the initial study.To obtain repeatability of follicular wave growth, observation of follicular dynamic was continued in the next1 IOI without a break using 6 cows (3 cows with 3-wave pattern and 3 cows with 4-wave pattern).

Data analysis
One follicular wave was defined as duration of follicular growth started from 4 mm diameter until the dominant follicle back to its original size.Follicle reaching the biggest size from the same wave was stated as dominant follicle (DF), meanwhile smaller follicle was defined as subordinate follicle (SF).Follicular growth phase was a period of follicle detected in 4 mm of diameter until the time the follicle did not grow anymore.Regression phase was started at the time follicle diameter decreased until getting hard to be detected individually.Static phase was defined as a period between the last days of growth phase and first day of regression phase.Data were divided according to pattern of follicular wave amount observed in 1 IOI (3 and 4-wave pattern group) and was tested by Independent-Sample T Test.Repeatability (0-1 value) was defined as proportion of total variance which was able to contribute to individual variance, and was calculated by formula: individual  2 /(individual  2 +  2 error) (Burns et al. 2005).Component of variance for repeatability was estimated using ANOVA with single factor.Percentage of follicular ovulation was amount of ovulation in the left or right ovary divided by total follicular ovulation observed.Percentage of follicular wave occurrence in ovary was amount of follicular wave occurred in the right or left ovary divided by total follicular wave observed.
Wave amount in 1 cycle was not affected by cattle breed (Adam et al. 2008), however Bó et al. (2003) reported that there was 3-wave pattern in poor nutrition and heat-stressed cattle.There was different in wave pattern proportion of heifer and cow in dairy cattle (Wolfenson et al. 2004).In other study, Adam et al. (2008) said that the most heifers (65%) of Nellor cattle (B.indicus) showed 3-wave, while most of cows (83%) showed 2-wave pattern.Satheshkumar et al. (2015) reported that 3-wave pattern emerged more frequently in the winter than the 2-wave pattern, and conversely in the summer.Follicular wave growth was initiated in day -0.4±0.9 and 1.4±1.1 relative against ovulation in cattle with 3 and 4 follicular wave.The next follicular wave started to grow in day 8.1±1.5 and 7.4±1.9for the 3 and 4-wave pattern (Table 1).Three-wave pattern had the longest dominant duration (11.6+1.5 days) on the first wave and shorter on 4-wave pattern (10.0±2.9).This was in line with Adam et al. (2008) who said that there was a highly correlation between dominant duration with its wave amount pattern.Shorter dominant duration would increase the number of wave in 1 IOI.In other study, Jaiswal et al. (2009) said that dominant duration on the first wave in IOI might be used to predict wave amount pattern.Diameter of dominant follicle (DF) of each follicular wave in 3 and 4-wave pattern was not significantly different even diameter of dominant follicle tended to wane by increase of wave amount in 1 IOI (Table 1).Viana et al. (2000) said that decrease of diameter of dominant follicle and its dominant duration was associated with increase of wave amount in 1 estrous cycle.Bó et al. (2003) reported that the number of B. indicus (16%) which had 4 follicular wave pattern was higher than B. taurus (0%) and was allegedly due to a consequence of smaller diameter of dominant follicle and shorter dominant duration of B. indicus.High concentration estradiol suppressed LH impulse inducing synthesis of progesterone of luteal cells (Goodman et al. 1981;William et al. 1978), so that it was expected might affect function of luteal and its lifetime (Jaiswal et al. 2009).
However, on the third wave of 3-wave pattern which was an ovulatory follicle had significantly wider diameter than the third wave of 4-wave pattern (Table 1).As well it had wider diameter of ovulatory follicle than that non-ovulatory follicle in the same wave pattern.Progesterone produced by CL would suppress concentration of estradiol and growth rate of dominant follicle (Ramana et al. 2013;Junior et al. 2010), so that follicle growing during luteal phase had smaller diameter than ovulatory follicle growing during luteolysis.
One IOI cycle in this study tended to longer by increase of 3 and 4 wave pattern by 22±1.6 and 22.8±2.9respectively (Table 2).It possibly related to size of follicular diameter in each wave pattern.Goff et al. (2004) said that estradiol in the first wave of IOI regulated time of oxytocin receptor emergence in endometrium that eventually managing prostaglandin production time to stimulate the luteolisis.There was a suspicion that size of follicular diameter on the first   et al. 2008et al. , Jaiswal et al. 2009).However, Boer et al. (2011) said that mechanism of wave pattern forming was still unclear and was allegedly involving more complex follicular growth regulation.
In this study, growth rate of dominant follicle was not significantly different between 3 and 4-wave (0.87+0.23 and 0.94+0.25 mm/day, respectively) (Table 2).As well DF growth rate was not significantly different from SF until the follicular deviation.This was in line with Adam et al. (2008) who said that a group of follicle had the same growth rate at the beginning of growth until one follicle was selected to continuously grow to become a DF.
Initial of growth difference between the two biggest follicles was defined as follicular deviation (Ginther et al. 2003).In this study, deviation was occurred after the selected follicle reached diameter of 6.25+0.37 and 6.44+0.44 in 3 and 4-wave respectively (Table 2).That was lower compared to follicular deviation in B. taurus breed by 8.5-9.0 mm (Ginther & Hoffman et al. 2014, Sartori et al. 2001).This result was in line with Sartorelli et al. (2005) reporting that Nellor cattle (B.indicus) had smaller follicular diameter during deviation and ovulation than B. taurus.B. indicus breed had smaller follicle dominant than B. taurus, so that follicle dominant in B. indicus became smaller during follicular deviation (Bó et al. 2003).
When the DF depended on LH, there were only 2 possibilities: ovulation or regression (Lucy 2007).DF unexposed by LH surge would remind at a certain period (static phase) and then regressed (Valdez et al. 2005).During this period, DF would depend on growth factor supporting transition of G1 into S phase of cellular cycle and prevented apoptosis in granulose cells (Quirk et al. 2004).At the certain time in static phase, DF would lose its dominant function even morphologically this follicle was the biggest size (Ireland et al. 2000).In this study, cows with 3-wave pattern had longer static phase by 3.7+1.9days than 4wave pattern by 1.9+1.4days.
Wave amount pattern in this study had high repeatability value by 0.88 in the same individual (Table 3).It was in line with Jaiswal et al. (2009) who said that wave pattern had high repeatability value in crossed Hereford (B.taurus) cattle.Sichtar et al. (2010) reported almost equal proportion between individual experiencing a change or not in wave amount pattern in the same individual of dairy cattle.
The number of follicle growing in this study had high repeatability value by 0.91 in the same individual.Another studies also reported that the number of follicle recruited into one wave had high repeatability value in the same individual, but was varied in the different individual (Santos et al. 2014;Ireland et al. 2007;David et al. 2005). Singh et al. (2004) reported that the high Repeatability of follicular wave in the right and left ovary 0.14 and 0.10 number of antral follicle had a positive correlation to gonadotropin treatment during superovulation and produced more oocyte and transfer-feasible embryo.So that, characteristic of repeatability of high number of PO cattle might be used as parameter of donor selection to increase effectiveness of superovulation.In this study, ovulation in right ovary was higher (72%) than in the left (Table 3).It was in line with Vasenna et al. (2003) and Nation et al. (1999) who reported that ovulation more often occurred in the right ovary than in the left.However, Purwantara et al. (2006) and Ginther et al. (1989) reported balance ovulation in the right and left ovary.In other hand, percentage of the number of follicular wave formed between the right and left ovary was practically balance (52% and 48% respectively) showing that actually, right and left ovary had the same activity in folliculogenesis.Repeatability of follicular wave emergence in the same ovary was too low both in the right and left ovary (0.14 and 0.10, respectively) showing a process of randomly follicular wave forming in the right and left ovary.This was different from Vasenna et al. (2003) reporting that activity of the right ovary was higher than the left.It was just 1 from a pair of follicle would be selected to be DF both in ipsilateral or contralateral position against the biggest subordinate follicle.
This study provided basic data of follicular dynamic and its repeatability in PO cattle based on USG observation.A better understanding of follicular dynamic was very useful in its utilization for purposes with more specific reproduction management such as in superovulation program.Some field studies had been conducted to implement superovulation program to other native cattle (Bali, Madura, and Aceh cattle) with standard superovulation protocol as applied in Bos taurus breed.However, until now response of superovulation produced was not as expectation.This indicated the urgent of understanding of follicular dynamic characteristics of each native cattle breed to be used as a basic data in implementation reference of reproduction technology, especially that superovulation program.

CONCLUSION
This study showed that characteristic of follicular dynamic of PO cattle was dominated by 3-waves pattern in 1 estrous cycle.Repeatability of wave pattern and follicle number had high value in the same individual.Those characteristics might be used for reproduction management of donor cattle to increase superovulation effectiveness based on individual information.

Figure 1 .
Figure 1.Growth pattern of follicular wave and CL in PO cattle with 3-follicular wave (A) and 4-follicular wave (B).Description: dotted line is CL growth pattern, solid line is follicular growth pattern.

Table 1 .
Characteristic per dominant follicular wave of PO cattle with 3 and 4 follicular wave in 1 IOI Different superscript in the same column and characteristic shows significant different in P<0.05.*: Diameter of ovulatory follicle

Table 2 .
Characteristic of dynamic of dominant follicle, subordinate follicle and corpus lutheum (CL) of PO cattle with 3 and 4

Table 3 .
Value of characteristics of follicular dynamic observed in PO cattle