The study was conducted to monitor follicular dynamics and steroid profiles in normal cyclic Sahiwal cows. Based on the diameter, the follicles were categorized into <4 mm, 4-6 mm, >6-8 mm and >8 mm groups. The follicles of <4 mm and 4-6 mm were observed during wave emergence and selection, respectively, whereas follicles of >6-8 mm and >8 mm were observed during dominance.
Follicular wave pattern
The characteristics of the follicular waves observed in Sahiwal cows are presented in Table 1. The ovarian follicles developed in a wave like pattern involving recruitment of small follicle (<4 mm), followed by selection, development and atresia or ovulation of the dominant follicle (DF). Similar wave like pattern of ovarian follicular development had been reported by various workers in cattle (
Adams, 1998 and
Fortune et al., 2001). In the present study, four cows revealed three wave follicular pattern (4/7, 57.1%) whereas, three showed two wave pattern. Similar predominance of three wave estrous cycles had been reported in
Bos indicus breeds like in Brahman and Gir cows
(Zeitoun et al., 1996; Viana et al., 2000), Nelore heifers
(Mollo et al., 2007) and European cattle breeds
(Savio et al., 1988; Sirois and Fortune, 1988). The studies had reported that bovine estrous cycles were composed of either two or three follicular waves (Adams, 1998) with predominance of two wave estrous cycles in
Bos taurus cattle
(Townson et al., 2002; Sartori et al., 2004). Noseir (2003) opined that the number of follicular waves in a single cycle is determined mainly by the size of follicle (>10 mm) and concentration of estradiol (>5.0 pg/ml), when both were achieved after emergence of the 2
nd wave, the estrous cycles were of 2-wave but when not attained, the cycle continued to be 3-wave cycle.
Interovulatory length
The mean interovulatory length recorded in the present study was 20.33±0.33 and 22.50±0.28 days in 2-wave and 3-wave estrous cycle, respectively (Table 1). A similar correlation between number of follicular waves and estrous cycle length had been reported in Angus, Brahman, Senepol
(Alvarez et al., 2000) and in Holstein dairy cows
(Sartori et al., 2004) with average interovulatory interval of 19 and 23 days in 2-wave and 3-wave estrous cycle, respectively.
The length of estrous cycles was shorter by 2.17 days in 2-wave compared to 3-wave estrous cycle in our study which was in agreement with
Ginther et al., (1989) who reported that the interovulatory interval was shorter by 2.4 days in 2-wave compared to 3-wave estrous cycles in Holstein Friesian heifers. Noseir (2003) also reported shorter mean length of estrous cycle in 2-wave compared to 3-wave estrous cycle in cattle (19.8±0.6 vs. 22.5±0.8 days P<0.05).
Baruselli et al., (2003) observed that the wave patterns were affected by parity in Nelore cattle. It was observed that low plane of nutrition and heat stress could also lead to increased proportion of 3-wave estrous cycles in
Bos taurus cattle (
Adams, 1998;
Baruselli et al., 2003). The nutritional supplementation also influence post partum fertility in crossbred cows
(Dhami et al., 2017).
Day of wave emergence
The results of the present study showed that the first follicular wave emerged on the day of ovulation (day 0) in 2-wave as well as 3-wave estrous cycles (Table 1). Similar results with respect to emergence of first follicular wave had been reported in dairy cows by
Noseir (2003). However,
Viana et al., (2000) observed emergence of first follicular wave on day 0.78±0.44 in Gir cows and
Muraya et al., (2015) on day 0.61±0.06 and 0.42±0.16 in the Kenyan Boran cows in 2-wave and 3-wave estrous cycle, respectively. In contrast,
Gaur and Purohit (2007) reported emergence of first follicular wave on day 2.10±0.36 and 0.7±0.5 after ovulation in the Rathi cows in 2-wave and 3-wave estrous cycle, respectively. In the present study, the emergence of the second wave in Sahiwal cows was earlier showing 3-wave compared to 2-wave estrous cycle (on day 7±0.28 vs. 7.5±0.57, respectively).
Noseir (2003);
Gaur and Purohit (2007) reported emergence of second follicular wave on day 7.2±1.0 in 3-wave cycle which was comparable to emergence of second follicular wave on day 7.0±0.28 in 3-wave cycle in the present study.
The emergence of second wave observed in the present study was earlier in 2-wave as well as in 3-wave estrous cycle compared to Gir cows and Kenyan Boran cows reported by
Gaur and Purohit (2007);
Muraya et al., (2015), respectively. In the present study, the third wave emerged on day 14.5±0.81 in Sahiwal cows showing 3-wave estrous cycle, whereas,
Muraya et al., (2015) reported emergence of third wave on day 15.00±0.98 in Kenyan Boran cow having 3-wave estrous cycle. However,
Gaur and Purohit (2007) observed emergence of third wave on day 13.2±3.4 in Rathi (
Bos indicus) cows which was earlier compared to the emergence of third wave observed in Sahiwal cows in the present study.
Mean number of antral follicles
The mean±SEM number of antral follicles at first wave emergence was 25.0±5.93 in Sahiwal cows. Similar mean number of antral follicles at wave emergence had been reported in
Bos taurus cattle by
Ginther et al., (1996) while this number was less in Kenyan Boran cow (17.5±2.67;
Muraya et al., 2015) and in Angus (21±4;
Alvarez et al., 2000). In Nelore heifers, the number of antral follicles observed at the time of onset of wave emergence was double (approximately 50;
Buratini Jr.et_al2000) than the present study.
Alvarez et al., (2000) also reported higher number of follicles at wave emergence in Brahman (39±4) and Senepol (33±4) cows.
Selection of first wave dominant follicle
The day of dominant follicle selection was defined as the day on which the difference in diameter of dominant follicle and SOF reached ³2 mm. The mean day of selection of dominant follicle was 2.71±0.28 days and the average diameter of dominant follicle at the time of selection was 7.14±0.24 mm in seven Sahiwal cows under the study (Table 2).
The selection of dominant follicles was earlier and the diameter of selected follicle was higher in Sahiwal cows under the study compared to Kenyan Boran cows
(Muraya et al., 2015). However, in
Bos taurus breeds, follicular selection occurred when the largest developing follicle reached 8.5 to 9.0 mm in diameter
(Ginther et al., 1996, Sartori et al., 2001) whereas, in case of ovulatory wave the diameter at the time of selection was between 8.3 to 9.8 mm in Holstein cattle
(Bastos et al., 2010).
Various studies had shown that the diameter of follicle at selection differed in
Bos indicus and
Bos taurus breeds. However,
Sartori et al., (2016) observed that the time for follicle deviation after ovulation or after wave emergence was similar between
Bos taurus and
Bos indicus (Nelore) which could be due to a slower growth rate of follicle in Nelore (0.8 to 1.2 mm per day;
Sartori and Barros, 2011) compared to Holstein cattle (1.2 to 1.6 mm per day;
Sartori et al., 2001).
The inhibin produced by follicles along with increased circulating estradiol (0.2 to 1 pg/ml) near the time of follicular deviation cause final depression in FSH and was associated with selection of the dominant follicle
(Adams et al., 1992). The inhibition of FSH by estradiol was synergized with inhibin
(Webb et al., 2004).
Growth pattern of Dominant Follicles
The growth pattern of dominant follicle of first wave in Sahiwal cows and having 2-wave and 3-wave estrous cycle was analyzed. The development of dominant follicle was categorized into growth, static and regression phases (Fig 1 and 2).
Growth phase of DF
In the present study, the first wave DF diameter observed at selection was 6.58±0.30 mm and 7.56±0.15 mm and showed a growth rate of 1.04±0.29 and 0.69±0.13 mm in 2-wave and 3-wave estrous cycle, respectively (Table 3, Fig 1 and 2). Whereas, the duration of growth phase were 3.33±0.33 and 3.5±0.50 days in 2-wave and 3-wave estrous cycle, respectively.
Gaur and Purohit (2007) reported higher growth rate of 1.81±0.32 and 1.03±0.24 mm/day for first wave DF in 2-wave and 3-wave estrous cycle in Rathi cows compared to the present study, respectively. Whereas, the duration of growth phase of 3.10±0.50 days observed in Rathi cows by
Gaur and Purohit (2007) was comparable to the duration of growth phase recorded in the present study. In contrast to present study,
Viana et al., (2000) reported longer duration of growth phase of 5.67±0.71 days in 3-wave estrous cycle in Gir cows.
Static phase of DF
The duration of static phase was 3±0.57 and 1.75±0.47 in 2-wave and 3-wave estrous cycle, respectively (Table 3, Fig 1 and 2). The static phase of first wave DF was non- significantly longer in 2-wave than 3-wave estrous cycle. This could also be one of the reasons for longer duration of first follicular wave in 2-wave compared to 3-wave estrous cycle in cattle.
Regression phase of DF
The duration of regression phase and regression rate of dominant follicle was 7.66±0.66 and 0.66±0.19 in 2-wave; 7.5±2.21 and 0.95±0.44 in 3-wave estrous cycle, respectively (Table 3, Fig 1 and 2). The difference in regression rate of first wave dominant follicle in 2-wave and 3-wave estrous cycle was non-significant.
Gaur and Purohit (2007) reported that regression rate of first wave DF in 2-wave estrous cycle was 0.55±0.20 mm/day in Rathi cows which was comparable to our results. However,
Muraya et al., (2015) reported higher regression rate of 1.01±0.29 and 0.91±0.10 mm/day compared to the regression rate recorded in the present study in 2-wave and 3-wave estrous cycle, respectively.
Size variations of dominant follicles during 2-wave and 3-wave estrous cycle
The maximum diameter of first wave and the second wave DF was 10.3±1.09 and 11.66±0.60 mm in 2-wave estrous cycle, respectively. The difference between the first and second wave dominant follicle diameter was non-significant (Table 4). The diameters of first, second and third (ovulatory) wave DFs recorded in the present study were 9.90±0.31, 8.82±0.52 and 11.42±0.20 mm in 3-wave estrous cycle, respectively. The diameters of first wave dominant follicle recorded in 2-wave and 3-wave estrous cycle did not differ significantly (10.3±1.09 vs. 9.90±0.31; P>0.05).
However, the diameter of the ovulatory (second dominant) follicle of 2-wave estrous cycle was significantly larger than the diameter of second wave DF of 3-wave estrous cycle (11.66±0.60 vs. 8.82±0.52 mm; P<0.05). In 3-wave estrous cycle the diameter of third DF
i.e. ovulatory follicle was also significantly larger compared to first and second wave dominant follicles (11.42±0.20 vs. 9.90±0.31 and 8.82±0.52 mm, respectively; P<0.05) shown in Table 4. The results of the present study were in agreement with Gaur and Purohit (2007), who also reported significantly larger second wave dominant follicle in 2-wave cycle compared to the second DF in 3-wave estrous cycle in Rathi cows (14.65±1.24 vs. 10.44±2.13 mm, respectively; P<0.05). The maximum diameter of second DF recorded in 3-wave was smaller (P<0.05) than the diameter of first and third DFs in 3-wave estrous cycle in the present study.
Similar results had been reported by
Viana et al., (2000), who reported that the maximum diameter of second DF was smaller than the diameter of first and third DFs in 3-wave estrous cycle in Gir cows (10.44±2.13 vs. 11.78±1.20 and 12.44±1.59 mm, respectively; P<0.05). This difference could be due to the fact that the second wave emerged during the period of higher progesterone production by the corpus luteum, whereas the first and third waves emerged during the luteogenic and luteolytic periods, respectively
(Viana et al., 2000). The maximum diameters of both the ovulatory and non-ovulatory follicles recorded in Sahiwal cow in the present study seemed to be smaller compared to
taurine cows
(Ginther et al., 1989) and Zebu cows
(Figueiredo et al., 1997 and
Sartorelli et al., 2005).
Luteal dynamics
The luteal development was divided into three phases; a growth phase characterized by regular increase in the CL diameter, than a lag phase characterized by slight or no increase in the diameter of CL and finally the regression phase where there was decrease in the diameter of CL. In the present study, the maximum diameter of corpus luteum recorded was 19.16±1.30 mm and 17.93±0.85 mm in 2-wave and 3-wave estrous cycle, respectively. The difference was non-significant (Table 5). Similar diameter of corpus luteum ranging from 17 to 21 mm had been reported in
Bos indicus cattle by
Figueiredo et al., (1997).
However, diameter of corpus luteum measuring 20 to 30 mm had been reported in
Bos taurus by Ginther
et al., (1989) and
Sartori et al., (2004) which was higher compared to the present study. It was observed during the study that the onset of CL regression occurred early in 2-wave than 3-wave estrous cycle (12.66±0.88 day vs. 13.75±1.03 day, respectively). Similar findings were observed in Holstein heifers by
Ginther et al., (1989). Jaiswal (2007) observed that the decrease in CL size corresponded with decreasing progesterone and increasing estradiol concentrations resulting in pre-ovulatory LH surge and ovulation.
Mean progesterone concentration in 2-wave and 3-wave estrous cycle
The mean P
4 concentration (ng/ml) varied according to the size of CL in Sahiwal cows under study. The peak progesterone values of 6.00±0.91 and 6.2±1.2 ng/ml were observed in 2-wave and 3-wave estrous cycle, respectively (Fig 3 and 4). It was reported that despite smaller diameter of CL in zebu compared to
taurine breeds of cattle, higher circulating concentrations of estradiol (E
2 ) and progesterone (P4) were recorded in
B. indicus than
B. taurus by Carvalho
et al., (2008) and
Bastos et al., (2010). These physiological differences may due to greater circulating cholesterol, insulin and IGF-1 in
Bos indicus compared to
Bos taurus.
Similar pattern of progesterone concentration had been reported by
Randel (1980) in Brahman cattle and
Hansel (1981) in Holstein heifers.
Liewelyn et al., (1987) observed that plasma progesterone concentration on 2 days before estrus to 3 days after estrus was less than 1.0 ng/ml and reached a maximum 8.0 to 10.0 ng/ml at 11 to 15 days after estrus in Ethiopian highland zebu cattle. The results of the present study were in agreement with
Schams et al., (1977); Coetzer et al., (1978) who observed a drop in the concentration of progesterone in the blood of zebu cows about 13 days after estrus.
Mondal et al., (2004) stated that the mean plasma progesterone concentrations declined from 0.85±0.06 ng/ml (on day-4) to 0.40±0.02 ng/ml on the day of estrus, there after, increased to reach maximum concentration of 3.03±0.91 on day 10.
Mean estradiol levels in 2-wave and 3-wave estrous cycle
The blood plasma levels of estradiol measured during the estrous cycle are represented in Fig 5 and Fig 6. The peak estradiol values of 15.83±0.60 and 14.31±0.44 pg/ml were observed in 2-wave and 3-wave estrous cycle, respectively. The peak values of estradiol in the present study were higher than that of previously reported in Brahman, Angus and Senepol cows
(Alvarez et al., 2000). Rantala and Taponen (2015) reported that estradiol reached to peak value (8.2±1.8pg/ml) on Day 20 in Dairy cows.
Glencross and Pope (1981) cited that plasma estradiol-17β concentration was low at the start of luteal regression (2.2 ±0.5 pg/ml), increased to 3.8±0.6 pg/ml on next day and reached 6.6±0.9 pg/ml when the concentration of progesterone in the blood had fallen to a minimum in the Holstein heifers. The highest concentration of estradiol-17β (10.1 pg/ml) was recorded one or two days after complete luteolysis. Probably the drop in progesterone concentration following luteal regression allowed the pre-ovulatory follicle to increase its secretion of oestradiol-17β
(Karsh et al., 1978).
