Effects of a dominant follicle on ovarian follicular dynamics during the oestrous cycle in heifers J. C. H. Ko, J. P. Kastelic, M. R. Del Campo and O. J. Ginther Department of Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
Summary. Two hypotheses were tested: (1) a dominant follicle causes regression of its subordinate follicles, and (2) a dominant follicle during its growing phase suppresses the emergence of the next wave. Cyclic heifers were randomly assigned to one of four groups (6 heifers/group): cauterization of the dominant follicle of Wave 1 or sham surgery (control) on Day 3 or Day 5 (day of ovulation Day 0). Ultrasonic monitoring of individually identified follicles was done once daily throughout the interovulatory interval. The onset of regression (decreasing diameter) of the largest subordinate follicle of Wave 1 was delayed (P < 0\m=.\01)by cauterization of the dominant follicle of Wave 1 on Day 3 compared to controls (mean onset of regression, Days 10\m=.\8 \m=+-\2\m=.\1vs 4\m=.\3 \m=+-\0\m=.\4).Cauterization of the dominant follicle of Wave 1 on Days 3 or 5 caused early emergence (P < 0\m=.\01)of Wave 2 when compared to controls (Day-3 groups: Days 5\m=.\5\m=+-\0\m=.\4 vs 9\m=.\6\m=+-\0\m=.\7; Day-5 groups: Days 7\m=.\0\m=+-\0\m=.\3 vs 9\m=.\1 \m=+-\0\m=.\4). The results supported the two hypotheses. In addition, cauterization of the dominant follicle of Wave 1 on Days 3 or 5 increased the incidence of 3-wave interovulatory =
intervals.
Keywords: ovary; follicles; follicular waves; cattle; cauterization
Introduction A wave of ovarian follicular growth involves the synchronous development of a group of follicles with a predominance of 2 (Ginther et ai, 1989a) or 3 (Savio et ai, 1988; Sirois & Fortune, 1988) follicular waves per oestrous cycle. Wave 1 was first identified on Day 0 (day of ovulation) when the follicles were 4-5 mm in diameter (Ginther et ai, 1989a). A retrospectively identified dominant follicle grew linearly for approximately 6 days (growing phase), remained approximately the same size for 6 days (static phase), and then began to regress (regressing phase as indicated by decreasing diameter). Wave 2 was first identified on an average of Day 9; for 2-wave interovulatory intervals, the dominant follicle became the ovulatory follicle. Comparisons of temporal associations between heifers with 2-wave and 3-wave interovulatory intervals (Ginther et ai, 1989b) and between unmated and pregnant heifers (Ginther et ai, 1989c) showed that (1) subordinate follicles of a wave ceased to grow after a few days, and (2) a new wave did not appear while a dominant follicle was in its growing phase or in the initial portion of its static phase. These temporal relationships suggested that a growing-phase dominant follicle exerted a profound inhibitory effect on its subordinates and suppressed development of the next wave. In previous reports (Matton et ai, 1981; Staigmiller & England, 1982), it was suggested that a single large follicle inhibited the development of smaller follicles. Matton et ai (1981) utilized marking or cauterizing selected follicles; replacement of the largest follicle was more rapid at the end of the oestrous cycle than at the beginning. In another study (Staigmiller & England, 1982), when the ovary containing the largest follicle was removed, follicular size and follicular fluid weight
of the remaining ovary increased by 4 days after surgery. However, there was no change in follicular development when the ovary contralateral to the largest follicle was removed. These studies utilizing follicle monitoring, follicle cauterization or ovary removal suggested that a dominant follicle produced an inhibitory factor which played a key role in interfollicular associations. Bovine follicular fluid is known to contain a non-steroidal factor that has follicleinhibiting activity in heifers (Miller et ai, 1979). It has been concluded that a dominant follicle suppressed other follicles through systemic channels since no intraovarian relationships were detected (Ginther et ai, 1989d). Individual monitoring of ovarian follicles throughout an interovulatory interval following removal of the dominant follicle apparently has not been done. In the present experiment, the dominant follicle of Wave 1 was destroyed and individually identified follicles were monitored by transrectal ultrasound. The following hypotheses were tested: (1) a dominant follicle causes regression of its subordinate follicles, and (2) a dominant follicle during its growing phase suppresses the emergence of the next wave.
Materials and Methods Nulliparous Holstein heifers, 1 -5-2-5 years of age and weighing 330-480 kg were used during January-March. Heifers were examined ultrasonically at 2-day intervals until detection of luteal regression, and then daily until detection of ovulation. This ovulation was designated the pretreatment ovulation (day of pretreatment ovulation Day 0). Ultrasound examinations were done by a single operator under optimized conditions, as previously described (Pierson et al, 1988). The ultrasound scanner was a real-time, B-mode instrument equipped with a 7-5 MHz, linear-array, intrarectal transducer (Aloka SSD-210 DXII; Corometrics Medical Systems, Inc., Wallingford, CT, USA). Heifers were included in the experiment only if the presumptive dominant follicle of Wave 1 was identifiable on Day 3 (exceeded diameter of all other follicles by >2mm). This provision was based on inspection of data from previous studies; in some heifers the dominant follicle was not identifiable by its largest size until after Day 3. Heifers which met this criterion were randomly assigned on Day 3 to the following 4 groups: follicle cautery on Day 3, sham surgery on Day 3, follicle cautery on Day 5, and sham surgery on Day 5 (6 heifers/group). Heifers were randomized in replicates. A replicate consisted of 8 heifers—4 heifers with the dominant follicle ipsilateral to the corpus luteum and 4 heifers with the dominant follicle contralateral to the corpus luteum. A replicate was completely filled before the next replicate was started. Therefore, if the location of the dominant follicle (ipsilateral or contralateral) was opposite to that needed to complete the replicate, the heifer was not used in this or later replicates. The ovulation that occurred at the end of the interovulatory interval was designated the post-treatment ovulation. Ultrasound examinations were done once daily from the day of pretreatment ovulation to the day of post-treatment ovulation. At each examination, follicles >4mm were individually identified by reference to a sketch of the position and diameter of follicles that was made at the previous daily examination as described by Knopf et al (1989). The maximal cross-sectional area of the image of the luteal portion of the corpus luteum (total area minus area of central luteal cavity, if present) was determined as described by Ginther et al (1989b); the area of luteal tissue, as determined by ultrasonography, provides a convenient indication of peripheral progesterone levels (Kastelic el al, 1990). The dominant follicle of a wave was defined retrospectively as the follicle that reached the largest diameter, A subordinate follicle was defined retrospectively as one that appeared to originate from the same follicular pool as the dominant follicle as indicated by: (1) its first detection within 2 days of first detection of the dominant follicle and (2) an increase in diameter for at least 1 day after first detection (Ginther et al, 1989b). In all 24 heifers, a standing flank laparotomy was performed (under local anaesthesia) ipsilateral to the ovary with a dominant follicle. In the follicle-cauterization groups, the dominant follicle of Wave 1 was aspirated and the follicle was cauterized as described by Ginther (1971). In the sham-surgery (control) groups, the ovary that contained the dominant follicle was manipulated for 3 min (approximately the time required for aspiration and cauterization). Pneumoperitoneum following a flank laparotomy can seriously inhibit ultrasonic examination of the reproductive tract (Pierson et al, 1988). Therefore, the amount of air left in the peritoneal cavity was minimized by pushing on the opposite paralumbar fossa before closure of the incision. Cauterization was done on Days 3 or 5 on the basis of previous results (Ginther et al, 1989a). Day 3 was chosen because (1) it was the earliest day that the largest follicle was the dominant follicle in most waves (92%) and (2) the largest subordinate follicle was apparently still viable (increasing in diameter or in early static phase) in most waves. Day 5 was chosen because (1) almost all dominant follicles were still in the growing phase, and (2) in almost all waves, the subordinate follicles were regressing and presumably would be unable to replace a cauterized dominant follicle. For each heifer, the largest subordinate follicle was defined as the subordinate follicle that reached the largest maximum diameter. The second largest subordinate follicle was defined as the subordinate follicle that reached the second largest maximum diameter. The day of onset of regression of the dominant and largest subordinate follicle was defined as the first day of an apparent progressive decrease in follicle diameter that terminated in disappearance of the =
follicle (loss of individual identify). The day of onset of regression of the corpus luteum was determined as described by Ginther et al (1989b). The day of first detection of a 4- or 5-mm follicle that was retrospectively identified as a dominant follicle was taken as the first day of a wave. If the dominant follicle was not detected until it reached 6 or 7 mm, the previous day was taken as the first day of a wave, and 4-5 or 5-5 mm, respectively, was used as diameter of the dominant follicle. This modification was used in a previous study (Ginther et al, 1989b) and was necessary in the previous and present studies in a minority (8%) of waves.
Table 1. Follicular and luteal characteristics (mean ± s.e.m.) of control heifers and heifers that had the dominant follicle of Wave 1 cauterized on Day 3 or 5
Day 3 Control
Cautery (N 6)
Control
0-6 ±0-8 9-6 + 0-3
01+01 90 ± 0-7
01+ 0-3 130 ±0-8
6-3 + 0-7 4-3 + 0-4a 9-6 + 0-7"
5-5 ± 0-5 10-8 + 2-1" 5-5 + 0-4b
(N Dominant follicle, Wave 1 First detection (Day) Diameter on day of treatment (mm) Largest subordinate, Wave 1 Diameter on day of treatment (mm) Onset of regression (Day) No. of days between detection of Waves 1 and 2 Ovulatory follicle First detection (Day) Maximum diameter (mm) Diameter at onset of CL regression (mm) Onset ofCL regression (Day) No. days from: Detection of ovulatory follicle to onset of CL regression Onset of CL regression to ovulation Interovulatory interval
Length (days) Proportion of heifers with 2 waves 3 waves 4 waves
Day 5
11-0 140 12-4 17-0
=
6)
+ IT"
± 0-7" + 0-8
+ 0-5'
5-6 ± 1-1 3-6 + 0-2"
=
± 0-7"
(N
=
6)
6-3 ± 0-8 4-0 + 0-4 91 ± 0-4"
11-8+1-4
Cautery (N 6) =
01 + 0-3 11-7 ± 0-4
61+0-3 5-4 + 11 7-0 ± 0-3"
± 0-6b
140 + 0-3 120 + 0-9 18-1+0-7
13-8 + 1-4 14-3 ± 0-8 110+11 181 ± 0-4
3-8 + 0-3 2-8 ± 01b
60 ± 0-9 2-6 + 0-3
4-1 + 1-0 3-5 + 0-2
15-3 12-2 10-3 191
+ 0-5" + 0-6
20-6 + 0-6
21-8 ± 0-7
200 ± 10
211 ± 0-4
4/6" 2/6* 0/6
0/6" 5/6b
4/6" 2/6"
l/6b 5/6"
1/6
0/6
0/6
abWithin the Day-3 and Day-5 groups, means with different superscripts are different (P < 005 at least) between the control and cautery group; pairs of means without superscripts are not significantly different. Student's / tests were used to determine differences between controls and follicle-cauterization heifers within the Day-3 and Day-5 groups for the end points indicated in Table 1. Fisher's exact tests were used to determine differences between controls and follicle-cauterization heifers within the Day-3 and Day-5 groups for the proportion of heifers with 2-wave and 3-wave interovulatory intervals. For preparation of figures, follicle data were normalized to the mean day of detection of the dominant follicle (follicle diameter, 4-5 mm) as described by Ginther et al (1989b).
Results
Eight and 4 heifers were not used due to an unidentifiable presumptive dominant follicle on Day 3 and inappropriate location of the dominant follicle in relation to the side of the corpus luteum, respectively. For all end points, there were no significant differences between location of the dominant follicle ipsilateral and contralateral to the corpus luteum. Therefore, data for ipsilateral and contralateral relationships within each group were combined. Results, including results of statistical analyses, are summarized in Table 1 for the indicated ovarian end points. Five of 6 and 4 of 6 heifers in the Day-3 and Day-5 control groups, respectively, had 2 follicular waves and the remaining heifers had 3 waves. For illustrative purposes, data for Day-3 and Day-5 controls were combined within 2- and 3-wave interovulatory intervals; mean diameter profiles for
3 waves (
=
3)
8-
Wave 2
Wave 1
Wave 3
a
E
-i-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1
>
2 waves (N
=
9)
64
Wave 1
— — —'— — —r— 10 12 14
8
-1
24
Days after ovulation
Fig.
1. Mean ( + s.e.m.) diameters of the dominant follicles and largest and second largest subordinates follicle from day of pretreatment ovulation to day of post-treatment ovulation ( .) in all control heifers (combined for Days 3 or 5).
the dominant follicle and the largest and second largest subordinate follicles are shown in Fig. 1. In heifers that had the dominant follicle cauterized on Day 3, 5 of 6 had 3 waves (combined data shown in Fig. 2) and the remaining heifer had 4 waves (individual data shown in Fig. 2). In heifers that had the dominant follicle cauterized on Day 5, 5 of 6 had 3 waves (combined data shown in Fig. 3) and the remaining heifer had 2 waves (individual data shown in Fig. 3). The day-to-day diameters of the largest subordinate follicle for each heifer are shown in Fig. 4.
Discussion The
hypothesis that a dominant follicle causes regression of its subordinates was supported. Cauterization of the dominant follicle of Wave 1 on Day 3 significantly delayed the onset of regression of the largest subordinate follicle of Wave 1 compared to controls (means, Day 10-8 and Day 4-3). However, the onset of regression of the largest subordinate follicle appeared to be
3
(
waves
=
5)
14
12
Cautery
10
8'
6'
E E
Wave1
2
Wave 2
Wave 3
——————————— T3 01
ñ
4 waves 14
(N
=
1)
Cautery
10
2-
Wave 1
Wave 2
Wave 3
Wave 4
——
!
10
12
14
16
Days after ovulation 2. Mean ( + s.e.m.) diameters of the dominant follicles and largest and second largest subordinate follicles from day of pretreatment ovulation to day of post-treatment ovulation ( .) in heifers that had the dominant follicle of Wave 1 cauterized on Day 3 (arrow).
Fig.
delayed in only 1 heifer that had the dominant follicle cauterized on Day 5 (Fig. 4); in the remaining 5 heifers, regression of the largest subordinate follicle appeared similar to regression of the largest subordinate follicle in control heifers (Fig. 4). The fate of subordinate follicles therefore became irreversible between Day 3 and Day 5. The results indicated that a cause-and-effect relationship is involved in the previously described temporal relationships between dominant and subordinate follicles (Ginther et ai, 1989b). The temporal associations were confirmed in the controls of the present experiment. In previous studies (Matton et ai, 1981; Staigmiller & England, 1982) it was suggested that a single large follicle inhibited the development of smaller follicles. These studies involved destruction or removal of many follicles. However, in retrospect, the results of these studies are compatible with those of the present study wherein the diameter of individual follicles was determined on a daily basis; the dominant follicle was identified (and selectively destroyed) and the fate of individual follicles was determined.
14
12
108-
6 4 2-
Wave1
Wave 3
Wave 2
— — — — —'— — — — — — — — — — — —>— — —
oE 18-1 E
£
14
Wave 1
Wave 2
16
20
22
—I
24
Days after ovulation 3. Mean ( +s.e.m.) diameters of the dominant follicles and largest and second largest subordinate follicles from day of pretreatment ovulation to day of post-treatment ovulation in heifers that had the dominant follicle of Wave 1 cauterized on Day 5 (arrow).
Fig.
In heifers that had the dominant follicle cauterized on Day 3 the initial regression (decreasing diameter) of the largest subordinate follicle (Heifers A, B, C, E, F; Fig. 4) was usually followed by an apparent increase in diameter. The resurging subordinate follicle appeared to act as a subordinate follicle of Wave 2 (Heifers B, C and D) or as the dominant follicle of Wave 2 (Heifers E and F). These findings indicate that full regression of subordinate follicles requires a continuing suppressive effect of the dominant follicle. When the dominant follicle was destroyed, the sub¬ ordinate follicle was apparently capable of recovery to the extent that it sometimes assumed a dominant position in Wave 2. This profound recovery was unexpected and confirmatory study is indicated. In Heifer A, there was no indication of an effect of cautery on the largest subordinate follicle. In this heifer, the dominant follicle on Day 3 (day of cautery) was larger than the Day-3
Controls, Day 3
Cautery, Day 3
10
12
14
16
18
20
22
Days after ovulation
Fig. 4. Profiles of the diameter of the largest subordinate follicle of Wave 1 for each heifer in the 4 groups. The largest subordinate follicle of Wave 1 became the dominant follicle of Wave 2 in two heifers (E and F) that had the dominant follicle of Wave 1 cauterized on Day 3.
dominant follicle in 22 of the remaining 23 heifers, and by the day of cautery the subordinate follicle had already regressed to < 4 mm in diameter. This may account for the failure of recovery of the subordinate follicle in this heifer. The hypothesis that a dominant follicle during its growing phase suppressed the emergence of the next wave was supported. Cauterization of the dominant follicle on Days 3 or 5 decreased the length of the interval between the emergence of the Waves 1 and 2. Removal of the inhibition by the Wave-1 dominant follicle allowed Wave 2 to emerge early. The average interval from cauterization to emergence of Wave 2 was similar for the Day-3 and Day-5 groups (2 day.). Cauterization of the Wave-1 dominant follicle on Days 3 or 5 increased the number of follicular waves within the interovulatory interval compared to the number in control heifers. There was a large proportion of heifers with a 3-wave interovulatory interval in the follicle-cautery groups (10/12 in the Day-3 and Day-5 groups combined versus 3/12 in the control groups; < 0005). In a previous study (Ginther et ai, 1989b), it was concluded that two factors determined whether a third wave emerged during a given interovulatory interval: (1) a shorter interval between the emergence of sequential waves could allow more waves to appear, even when the length of the interovulatory interval was constant, and (2) a longer interovulatory interval (life of corpus luteum) could allow an additional wave to emerge, even when the interval between waves was constant. The higher proportion of 3-wave interovulatory intervals in the heifers that had the dominant follicle cauterized can therefore be accounted for by the facts that (1) the interval between emergence of
Waves 1 and 2 was significantly shorter and (2) the onset of luteal regression was significantly later in heifers that had the dominant follicle cauterized on Day 3. In a previous study (Ginther et ai, 1989b), the ovulatory follicle in 3-wave interovulatory intervals emerged later, was smaller on the day of onset of luteal regression, and had a smaller maximum diameter than the ovulatory follicle in 2-wave interovulatory intervals. In the present study, there was a significant difference between heifers that had the dominant follicle cauterized on Day 3 and control heifers for each of these 3 end points; the differences between heifers that had the dominant follicle cauterized on Day 5 and control heifers were not significant, but were in the same direction as in the Day-3 heifers. For unknown reasons, the corpus luteum began to regress later and ovulation was detected sooner after the onset of luteal regression in heifers that had the dominant follicle cauterized on Day 3 compared to control heifers. In a previous study (Ginther et ai, 1989b), this interval was significantly different between heifers with 2-wave and 3-wave interovulatory intervals. The dominant follicle of Wave 2 was on the same ovary as the dominant follicle of Wave 1 in 8 of 12 heifers that had the dominant follicle cauterized on Days 3 or 5, and in 3 of 12 heifers in the Day-3 and Day-5 control groups, respectively (P < 005). In a previous report (Ginther et ai, 1989d), the location (left or right ovary) of a dominant follicle did not affect the location of the dominant follicle of the next wave. The reason for the apparent local relationship in the present study is not known; perhaps inflammation at the cauterization site caused a unilateral increase in blood flow that favoured development of the dominant follicle of Wave 2. In conclusion, cauterization of the dominant follicle of Wave 1 on Day 3 significantly delayed the onset of final regression of the largest subordinate follicle of Wave 1 and supported the hypothesis that a dominant follicle causes regression of its subordinates. Cauterization of the dominant follicle of Wave 1 on Days 3 or 5 allowed early emergence of Wave 2 and supported the hypothesis that a dominant follicle during its growing phase suppresses the emergence of the next wave. In addition, cauterization of the dominant follicle of Wave 1 on Days 3 or 5 increased the incidence of 3-wave interovulatory intervals.
Supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison. We thank Lisa Kulick for graphic and computer assistance and Maria Westphal for manuscript preparation. J.P.K. is supported by a fellowship from the Medical Research Council of Canada. References Ginther, O.J. (1971) Response of
corpora lutea to cauterization of follicles in sheep. Am. J. vet. Res. 32, 59-62. Ginther, O.J., Kastelic, J.P. & Knopf, L. (1989a) Compo¬ sition and characteristics of follicular waves during the bovine estrous cycle. Anim. Reprod. Sci. 20, 187-200. Ginther, O.J., Knopf, L. & Kastelic, J.P. (1989b) Tem¬ poral associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. J. Reprod. Fert. 87,223-230. Ginther, O.J., Knopf, L. & Kastelic, J.P. (1989c) Ovarian follicular dynamics in heifers during early pregnancy. Biol. Reprod. 41, 247-254. Ginther, O.J., Kastelic, J.P. & Knopf, L. (1989d) Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers. Theriogenology 32, 787-795. Kastelic. J.P., Bergfelt, D.R. & Ginther, O.J. (1990)
Relationship between ultrasonic assessment of the cor¬ pus luteum and plasma progesterone concentrations in heifers. Theriogenology 33, 1269-1278. Knopf, L., Kastelic, J.P., Schallenberger, E. & Ginther, O.J. (1989) Ovarian follicular populations in heifers: test of two wave hypothesis by ultrasonically monitor¬ ing individual follicles. Dom. Anim. Endocr. 6, 111119.
Matton, P., Adelakoun, V., Couture, Y. & Dufour, J.J. Growth and replacement of the bovine ovarian follicles during the oestrous cycle. J. Anim. Sci.
(1981)
52,813-820. Miller, K.F., Critser, J.K., Rowe, R.F. & Ginther, O.J. (1979) Ovarian effects of bovine follicular fluid treatment in sheep and cattle. Biol. Reprod. 21, 537-544.
Pierson, R.A., Kastelic, J.P. & Ginther, O.J. (1988) Basic
principles and techniques for transrectal ultrasonogra¬ phy in cattle and horses. Theriogenology 29,3-20.
Savio, J.D., Keenan, L., Boland, M.P. & Roche, J.F. (1988) Pattern of growth of dominant follicles during the oestrous cycle in heifers. J. Reprod. Fert. 83, 663-671.
Sirois, J. & Fortune, J.E. (1988) Ovarian follicular
dynamics during the
estrous
cycle
in heifers moni-
tored by real-time ultrasonography. Biol. Reprod. 39, 308-317. Staigmiller, R.B. & England, B.G. (1982) Folliculogenesis in the bovine. Theriogenology 17,42-52. Received 5 June 1990
Summary. Two hypotheses were tested: (1) a dominant follicle causes regression of its subordinate follicles, and (2) a dominant follicle during its growing phase suppresses the emergence of the next wave. Cyclic heifers were randomly assigned to one of four groups (6 heifers/group): cauterization of the dominant follicle of Wave 1 or sham surgery (control) on Day 3 or Day 5 (day of ovulation Day 0). Ultrasonic monitoring of individually identified follicles was done once daily throughout the interovulatory interval. The onset of regression (decreasing diameter) of the largest subordinate follicle of Wave 1 was delayed (P < 0\m=.\01)by cauterization of the dominant follicle of Wave 1 on Day 3 compared to controls (mean onset of regression, Days 10\m=.\8 \m=+-\2\m=.\1vs 4\m=.\3 \m=+-\0\m=.\4).Cauterization of the dominant follicle of Wave 1 on Days 3 or 5 caused early emergence (P < 0\m=.\01)of Wave 2 when compared to controls (Day-3 groups: Days 5\m=.\5\m=+-\0\m=.\4 vs 9\m=.\6\m=+-\0\m=.\7; Day-5 groups: Days 7\m=.\0\m=+-\0\m=.\3 vs 9\m=.\1 \m=+-\0\m=.\4). The results supported the two hypotheses. In addition, cauterization of the dominant follicle of Wave 1 on Days 3 or 5 increased the incidence of 3-wave interovulatory =
intervals.
Keywords: ovary; follicles; follicular waves; cattle; cauterization
Introduction A wave of ovarian follicular growth involves the synchronous development of a group of follicles with a predominance of 2 (Ginther et ai, 1989a) or 3 (Savio et ai, 1988; Sirois & Fortune, 1988) follicular waves per oestrous cycle. Wave 1 was first identified on Day 0 (day of ovulation) when the follicles were 4-5 mm in diameter (Ginther et ai, 1989a). A retrospectively identified dominant follicle grew linearly for approximately 6 days (growing phase), remained approximately the same size for 6 days (static phase), and then began to regress (regressing phase as indicated by decreasing diameter). Wave 2 was first identified on an average of Day 9; for 2-wave interovulatory intervals, the dominant follicle became the ovulatory follicle. Comparisons of temporal associations between heifers with 2-wave and 3-wave interovulatory intervals (Ginther et ai, 1989b) and between unmated and pregnant heifers (Ginther et ai, 1989c) showed that (1) subordinate follicles of a wave ceased to grow after a few days, and (2) a new wave did not appear while a dominant follicle was in its growing phase or in the initial portion of its static phase. These temporal relationships suggested that a growing-phase dominant follicle exerted a profound inhibitory effect on its subordinates and suppressed development of the next wave. In previous reports (Matton et ai, 1981; Staigmiller & England, 1982), it was suggested that a single large follicle inhibited the development of smaller follicles. Matton et ai (1981) utilized marking or cauterizing selected follicles; replacement of the largest follicle was more rapid at the end of the oestrous cycle than at the beginning. In another study (Staigmiller & England, 1982), when the ovary containing the largest follicle was removed, follicular size and follicular fluid weight
of the remaining ovary increased by 4 days after surgery. However, there was no change in follicular development when the ovary contralateral to the largest follicle was removed. These studies utilizing follicle monitoring, follicle cauterization or ovary removal suggested that a dominant follicle produced an inhibitory factor which played a key role in interfollicular associations. Bovine follicular fluid is known to contain a non-steroidal factor that has follicleinhibiting activity in heifers (Miller et ai, 1979). It has been concluded that a dominant follicle suppressed other follicles through systemic channels since no intraovarian relationships were detected (Ginther et ai, 1989d). Individual monitoring of ovarian follicles throughout an interovulatory interval following removal of the dominant follicle apparently has not been done. In the present experiment, the dominant follicle of Wave 1 was destroyed and individually identified follicles were monitored by transrectal ultrasound. The following hypotheses were tested: (1) a dominant follicle causes regression of its subordinate follicles, and (2) a dominant follicle during its growing phase suppresses the emergence of the next wave.
Materials and Methods Nulliparous Holstein heifers, 1 -5-2-5 years of age and weighing 330-480 kg were used during January-March. Heifers were examined ultrasonically at 2-day intervals until detection of luteal regression, and then daily until detection of ovulation. This ovulation was designated the pretreatment ovulation (day of pretreatment ovulation Day 0). Ultrasound examinations were done by a single operator under optimized conditions, as previously described (Pierson et al, 1988). The ultrasound scanner was a real-time, B-mode instrument equipped with a 7-5 MHz, linear-array, intrarectal transducer (Aloka SSD-210 DXII; Corometrics Medical Systems, Inc., Wallingford, CT, USA). Heifers were included in the experiment only if the presumptive dominant follicle of Wave 1 was identifiable on Day 3 (exceeded diameter of all other follicles by >2mm). This provision was based on inspection of data from previous studies; in some heifers the dominant follicle was not identifiable by its largest size until after Day 3. Heifers which met this criterion were randomly assigned on Day 3 to the following 4 groups: follicle cautery on Day 3, sham surgery on Day 3, follicle cautery on Day 5, and sham surgery on Day 5 (6 heifers/group). Heifers were randomized in replicates. A replicate consisted of 8 heifers—4 heifers with the dominant follicle ipsilateral to the corpus luteum and 4 heifers with the dominant follicle contralateral to the corpus luteum. A replicate was completely filled before the next replicate was started. Therefore, if the location of the dominant follicle (ipsilateral or contralateral) was opposite to that needed to complete the replicate, the heifer was not used in this or later replicates. The ovulation that occurred at the end of the interovulatory interval was designated the post-treatment ovulation. Ultrasound examinations were done once daily from the day of pretreatment ovulation to the day of post-treatment ovulation. At each examination, follicles >4mm were individually identified by reference to a sketch of the position and diameter of follicles that was made at the previous daily examination as described by Knopf et al (1989). The maximal cross-sectional area of the image of the luteal portion of the corpus luteum (total area minus area of central luteal cavity, if present) was determined as described by Ginther et al (1989b); the area of luteal tissue, as determined by ultrasonography, provides a convenient indication of peripheral progesterone levels (Kastelic el al, 1990). The dominant follicle of a wave was defined retrospectively as the follicle that reached the largest diameter, A subordinate follicle was defined retrospectively as one that appeared to originate from the same follicular pool as the dominant follicle as indicated by: (1) its first detection within 2 days of first detection of the dominant follicle and (2) an increase in diameter for at least 1 day after first detection (Ginther et al, 1989b). In all 24 heifers, a standing flank laparotomy was performed (under local anaesthesia) ipsilateral to the ovary with a dominant follicle. In the follicle-cauterization groups, the dominant follicle of Wave 1 was aspirated and the follicle was cauterized as described by Ginther (1971). In the sham-surgery (control) groups, the ovary that contained the dominant follicle was manipulated for 3 min (approximately the time required for aspiration and cauterization). Pneumoperitoneum following a flank laparotomy can seriously inhibit ultrasonic examination of the reproductive tract (Pierson et al, 1988). Therefore, the amount of air left in the peritoneal cavity was minimized by pushing on the opposite paralumbar fossa before closure of the incision. Cauterization was done on Days 3 or 5 on the basis of previous results (Ginther et al, 1989a). Day 3 was chosen because (1) it was the earliest day that the largest follicle was the dominant follicle in most waves (92%) and (2) the largest subordinate follicle was apparently still viable (increasing in diameter or in early static phase) in most waves. Day 5 was chosen because (1) almost all dominant follicles were still in the growing phase, and (2) in almost all waves, the subordinate follicles were regressing and presumably would be unable to replace a cauterized dominant follicle. For each heifer, the largest subordinate follicle was defined as the subordinate follicle that reached the largest maximum diameter. The second largest subordinate follicle was defined as the subordinate follicle that reached the second largest maximum diameter. The day of onset of regression of the dominant and largest subordinate follicle was defined as the first day of an apparent progressive decrease in follicle diameter that terminated in disappearance of the =
follicle (loss of individual identify). The day of onset of regression of the corpus luteum was determined as described by Ginther et al (1989b). The day of first detection of a 4- or 5-mm follicle that was retrospectively identified as a dominant follicle was taken as the first day of a wave. If the dominant follicle was not detected until it reached 6 or 7 mm, the previous day was taken as the first day of a wave, and 4-5 or 5-5 mm, respectively, was used as diameter of the dominant follicle. This modification was used in a previous study (Ginther et al, 1989b) and was necessary in the previous and present studies in a minority (8%) of waves.
Table 1. Follicular and luteal characteristics (mean ± s.e.m.) of control heifers and heifers that had the dominant follicle of Wave 1 cauterized on Day 3 or 5
Day 3 Control
Cautery (N 6)
Control
0-6 ±0-8 9-6 + 0-3
01+01 90 ± 0-7
01+ 0-3 130 ±0-8
6-3 + 0-7 4-3 + 0-4a 9-6 + 0-7"
5-5 ± 0-5 10-8 + 2-1" 5-5 + 0-4b
(N Dominant follicle, Wave 1 First detection (Day) Diameter on day of treatment (mm) Largest subordinate, Wave 1 Diameter on day of treatment (mm) Onset of regression (Day) No. of days between detection of Waves 1 and 2 Ovulatory follicle First detection (Day) Maximum diameter (mm) Diameter at onset of CL regression (mm) Onset ofCL regression (Day) No. days from: Detection of ovulatory follicle to onset of CL regression Onset of CL regression to ovulation Interovulatory interval
Length (days) Proportion of heifers with 2 waves 3 waves 4 waves
Day 5
11-0 140 12-4 17-0
=
6)
+ IT"
± 0-7" + 0-8
+ 0-5'
5-6 ± 1-1 3-6 + 0-2"
=
± 0-7"
(N
=
6)
6-3 ± 0-8 4-0 + 0-4 91 ± 0-4"
11-8+1-4
Cautery (N 6) =
01 + 0-3 11-7 ± 0-4
61+0-3 5-4 + 11 7-0 ± 0-3"
± 0-6b
140 + 0-3 120 + 0-9 18-1+0-7
13-8 + 1-4 14-3 ± 0-8 110+11 181 ± 0-4
3-8 + 0-3 2-8 ± 01b
60 ± 0-9 2-6 + 0-3
4-1 + 1-0 3-5 + 0-2
15-3 12-2 10-3 191
+ 0-5" + 0-6
20-6 + 0-6
21-8 ± 0-7
200 ± 10
211 ± 0-4
4/6" 2/6* 0/6
0/6" 5/6b
4/6" 2/6"
l/6b 5/6"
1/6
0/6
0/6
abWithin the Day-3 and Day-5 groups, means with different superscripts are different (P < 005 at least) between the control and cautery group; pairs of means without superscripts are not significantly different. Student's / tests were used to determine differences between controls and follicle-cauterization heifers within the Day-3 and Day-5 groups for the end points indicated in Table 1. Fisher's exact tests were used to determine differences between controls and follicle-cauterization heifers within the Day-3 and Day-5 groups for the proportion of heifers with 2-wave and 3-wave interovulatory intervals. For preparation of figures, follicle data were normalized to the mean day of detection of the dominant follicle (follicle diameter, 4-5 mm) as described by Ginther et al (1989b).
Results
Eight and 4 heifers were not used due to an unidentifiable presumptive dominant follicle on Day 3 and inappropriate location of the dominant follicle in relation to the side of the corpus luteum, respectively. For all end points, there were no significant differences between location of the dominant follicle ipsilateral and contralateral to the corpus luteum. Therefore, data for ipsilateral and contralateral relationships within each group were combined. Results, including results of statistical analyses, are summarized in Table 1 for the indicated ovarian end points. Five of 6 and 4 of 6 heifers in the Day-3 and Day-5 control groups, respectively, had 2 follicular waves and the remaining heifers had 3 waves. For illustrative purposes, data for Day-3 and Day-5 controls were combined within 2- and 3-wave interovulatory intervals; mean diameter profiles for
3 waves (
=
3)
8-
Wave 2
Wave 1
Wave 3
a
E
-i-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1
>
2 waves (N
=
9)
64
Wave 1
— — —'— — —r— 10 12 14
8
-1
24
Days after ovulation
Fig.
1. Mean ( + s.e.m.) diameters of the dominant follicles and largest and second largest subordinates follicle from day of pretreatment ovulation to day of post-treatment ovulation ( .) in all control heifers (combined for Days 3 or 5).
the dominant follicle and the largest and second largest subordinate follicles are shown in Fig. 1. In heifers that had the dominant follicle cauterized on Day 3, 5 of 6 had 3 waves (combined data shown in Fig. 2) and the remaining heifer had 4 waves (individual data shown in Fig. 2). In heifers that had the dominant follicle cauterized on Day 5, 5 of 6 had 3 waves (combined data shown in Fig. 3) and the remaining heifer had 2 waves (individual data shown in Fig. 3). The day-to-day diameters of the largest subordinate follicle for each heifer are shown in Fig. 4.
Discussion The
hypothesis that a dominant follicle causes regression of its subordinates was supported. Cauterization of the dominant follicle of Wave 1 on Day 3 significantly delayed the onset of regression of the largest subordinate follicle of Wave 1 compared to controls (means, Day 10-8 and Day 4-3). However, the onset of regression of the largest subordinate follicle appeared to be
3
(
waves
=
5)
14
12
Cautery
10
8'
6'
E E
Wave1
2
Wave 2
Wave 3
——————————— T3 01
ñ
4 waves 14
(N
=
1)
Cautery
10
2-
Wave 1
Wave 2
Wave 3
Wave 4
——
!
10
12
14
16
Days after ovulation 2. Mean ( + s.e.m.) diameters of the dominant follicles and largest and second largest subordinate follicles from day of pretreatment ovulation to day of post-treatment ovulation ( .) in heifers that had the dominant follicle of Wave 1 cauterized on Day 3 (arrow).
Fig.
delayed in only 1 heifer that had the dominant follicle cauterized on Day 5 (Fig. 4); in the remaining 5 heifers, regression of the largest subordinate follicle appeared similar to regression of the largest subordinate follicle in control heifers (Fig. 4). The fate of subordinate follicles therefore became irreversible between Day 3 and Day 5. The results indicated that a cause-and-effect relationship is involved in the previously described temporal relationships between dominant and subordinate follicles (Ginther et ai, 1989b). The temporal associations were confirmed in the controls of the present experiment. In previous studies (Matton et ai, 1981; Staigmiller & England, 1982) it was suggested that a single large follicle inhibited the development of smaller follicles. These studies involved destruction or removal of many follicles. However, in retrospect, the results of these studies are compatible with those of the present study wherein the diameter of individual follicles was determined on a daily basis; the dominant follicle was identified (and selectively destroyed) and the fate of individual follicles was determined.
14
12
108-
6 4 2-
Wave1
Wave 3
Wave 2
— — — — —'— — — — — — — — — — — —>— — —
oE 18-1 E
£
14
Wave 1
Wave 2
16
20
22
—I
24
Days after ovulation 3. Mean ( +s.e.m.) diameters of the dominant follicles and largest and second largest subordinate follicles from day of pretreatment ovulation to day of post-treatment ovulation in heifers that had the dominant follicle of Wave 1 cauterized on Day 5 (arrow).
Fig.
In heifers that had the dominant follicle cauterized on Day 3 the initial regression (decreasing diameter) of the largest subordinate follicle (Heifers A, B, C, E, F; Fig. 4) was usually followed by an apparent increase in diameter. The resurging subordinate follicle appeared to act as a subordinate follicle of Wave 2 (Heifers B, C and D) or as the dominant follicle of Wave 2 (Heifers E and F). These findings indicate that full regression of subordinate follicles requires a continuing suppressive effect of the dominant follicle. When the dominant follicle was destroyed, the sub¬ ordinate follicle was apparently capable of recovery to the extent that it sometimes assumed a dominant position in Wave 2. This profound recovery was unexpected and confirmatory study is indicated. In Heifer A, there was no indication of an effect of cautery on the largest subordinate follicle. In this heifer, the dominant follicle on Day 3 (day of cautery) was larger than the Day-3
Controls, Day 3
Cautery, Day 3
10
12
14
16
18
20
22
Days after ovulation
Fig. 4. Profiles of the diameter of the largest subordinate follicle of Wave 1 for each heifer in the 4 groups. The largest subordinate follicle of Wave 1 became the dominant follicle of Wave 2 in two heifers (E and F) that had the dominant follicle of Wave 1 cauterized on Day 3.
dominant follicle in 22 of the remaining 23 heifers, and by the day of cautery the subordinate follicle had already regressed to < 4 mm in diameter. This may account for the failure of recovery of the subordinate follicle in this heifer. The hypothesis that a dominant follicle during its growing phase suppressed the emergence of the next wave was supported. Cauterization of the dominant follicle on Days 3 or 5 decreased the length of the interval between the emergence of the Waves 1 and 2. Removal of the inhibition by the Wave-1 dominant follicle allowed Wave 2 to emerge early. The average interval from cauterization to emergence of Wave 2 was similar for the Day-3 and Day-5 groups (2 day.). Cauterization of the Wave-1 dominant follicle on Days 3 or 5 increased the number of follicular waves within the interovulatory interval compared to the number in control heifers. There was a large proportion of heifers with a 3-wave interovulatory interval in the follicle-cautery groups (10/12 in the Day-3 and Day-5 groups combined versus 3/12 in the control groups; < 0005). In a previous study (Ginther et ai, 1989b), it was concluded that two factors determined whether a third wave emerged during a given interovulatory interval: (1) a shorter interval between the emergence of sequential waves could allow more waves to appear, even when the length of the interovulatory interval was constant, and (2) a longer interovulatory interval (life of corpus luteum) could allow an additional wave to emerge, even when the interval between waves was constant. The higher proportion of 3-wave interovulatory intervals in the heifers that had the dominant follicle cauterized can therefore be accounted for by the facts that (1) the interval between emergence of
Waves 1 and 2 was significantly shorter and (2) the onset of luteal regression was significantly later in heifers that had the dominant follicle cauterized on Day 3. In a previous study (Ginther et ai, 1989b), the ovulatory follicle in 3-wave interovulatory intervals emerged later, was smaller on the day of onset of luteal regression, and had a smaller maximum diameter than the ovulatory follicle in 2-wave interovulatory intervals. In the present study, there was a significant difference between heifers that had the dominant follicle cauterized on Day 3 and control heifers for each of these 3 end points; the differences between heifers that had the dominant follicle cauterized on Day 5 and control heifers were not significant, but were in the same direction as in the Day-3 heifers. For unknown reasons, the corpus luteum began to regress later and ovulation was detected sooner after the onset of luteal regression in heifers that had the dominant follicle cauterized on Day 3 compared to control heifers. In a previous study (Ginther et ai, 1989b), this interval was significantly different between heifers with 2-wave and 3-wave interovulatory intervals. The dominant follicle of Wave 2 was on the same ovary as the dominant follicle of Wave 1 in 8 of 12 heifers that had the dominant follicle cauterized on Days 3 or 5, and in 3 of 12 heifers in the Day-3 and Day-5 control groups, respectively (P < 005). In a previous report (Ginther et ai, 1989d), the location (left or right ovary) of a dominant follicle did not affect the location of the dominant follicle of the next wave. The reason for the apparent local relationship in the present study is not known; perhaps inflammation at the cauterization site caused a unilateral increase in blood flow that favoured development of the dominant follicle of Wave 2. In conclusion, cauterization of the dominant follicle of Wave 1 on Day 3 significantly delayed the onset of final regression of the largest subordinate follicle of Wave 1 and supported the hypothesis that a dominant follicle causes regression of its subordinates. Cauterization of the dominant follicle of Wave 1 on Days 3 or 5 allowed early emergence of Wave 2 and supported the hypothesis that a dominant follicle during its growing phase suppresses the emergence of the next wave. In addition, cauterization of the dominant follicle of Wave 1 on Days 3 or 5 increased the incidence of 3-wave interovulatory intervals.
Supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison. We thank Lisa Kulick for graphic and computer assistance and Maria Westphal for manuscript preparation. J.P.K. is supported by a fellowship from the Medical Research Council of Canada. References Ginther, O.J. (1971) Response of
corpora lutea to cauterization of follicles in sheep. Am. J. vet. Res. 32, 59-62. Ginther, O.J., Kastelic, J.P. & Knopf, L. (1989a) Compo¬ sition and characteristics of follicular waves during the bovine estrous cycle. Anim. Reprod. Sci. 20, 187-200. Ginther, O.J., Knopf, L. & Kastelic, J.P. (1989b) Tem¬ poral associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. J. Reprod. Fert. 87,223-230. Ginther, O.J., Knopf, L. & Kastelic, J.P. (1989c) Ovarian follicular dynamics in heifers during early pregnancy. Biol. Reprod. 41, 247-254. Ginther, O.J., Kastelic, J.P. & Knopf, L. (1989d) Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers. Theriogenology 32, 787-795. Kastelic. J.P., Bergfelt, D.R. & Ginther, O.J. (1990)
Relationship between ultrasonic assessment of the cor¬ pus luteum and plasma progesterone concentrations in heifers. Theriogenology 33, 1269-1278. Knopf, L., Kastelic, J.P., Schallenberger, E. & Ginther, O.J. (1989) Ovarian follicular populations in heifers: test of two wave hypothesis by ultrasonically monitor¬ ing individual follicles. Dom. Anim. Endocr. 6, 111119.
Matton, P., Adelakoun, V., Couture, Y. & Dufour, J.J. Growth and replacement of the bovine ovarian follicles during the oestrous cycle. J. Anim. Sci.
(1981)
52,813-820. Miller, K.F., Critser, J.K., Rowe, R.F. & Ginther, O.J. (1979) Ovarian effects of bovine follicular fluid treatment in sheep and cattle. Biol. Reprod. 21, 537-544.
Pierson, R.A., Kastelic, J.P. & Ginther, O.J. (1988) Basic
principles and techniques for transrectal ultrasonogra¬ phy in cattle and horses. Theriogenology 29,3-20.
Savio, J.D., Keenan, L., Boland, M.P. & Roche, J.F. (1988) Pattern of growth of dominant follicles during the oestrous cycle in heifers. J. Reprod. Fert. 83, 663-671.
Sirois, J. & Fortune, J.E. (1988) Ovarian follicular
dynamics during the
estrous
cycle
in heifers moni-
tored by real-time ultrasonography. Biol. Reprod. 39, 308-317. Staigmiller, R.B. & England, B.G. (1982) Folliculogenesis in the bovine. Theriogenology 17,42-52. Received 5 June 1990
