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Effect of day of prostaglandin F2α treatment on selection and development of the ovulatory follicle in heifers
Animal Reproduction Science, 23 (1990) 169-180
169
Elsevier Science Publishers B.V., Amsterdam
Effect of day of prostaglandin F2~ treatment on selection and development of the ovulatory follicle in heifers J.P. K a s t e l i c , L. K n o p f a n d
O.J. Ginther
Department of Veterinary Science, University of Wisconsin-Madison, Madison, WI 53 706, U.S.A. (Accepted 4 May 1990)
ABSTRACT Kastelic, J.P., Knopf, L. and Ginther, O.J., 1990. Effect of day of prostaglandin F2,~ treatment on selection and development of the ovulatory follicle in heifers. Anita. Reprod. Sci., 23:169-180. In previous studies of heifers with two follicular waves during an estrous cycle, the dominant follicle of Wave 1 was first detected ultrasonically on approximately the day of ovulation (Day 0) when its diameter was 4-5 ram. On average, it grew linearly for 6 days (growing phase), remained the same size for 6 days (static phase), and then regressed (regressing phase). The dominant follicle of Wave 2 was first detected on approximately Day 9 and became the ovulatory follicle. In the present experiment, nonbred and bred heifers were treated with a luteolytic dose of prostaglandin F2,~ (25 mg) on Days 5, 8, or 12, when the dominant follicle of Wave 1 was expected to be in the growing, static, and regressing phase, respectively. There were no significant effects of breeding status on any end point. The hypothesis that growth of the dominant follicle during Wave 1 and response to prostaglandin F2,~ treatment is different between bred and nonbred heifers was not supported. Ovulation occurred from the dominant follicle of Wave 1 in 5 of 5, 6 of 6 and 0 of 4 heifers treated on Days 5, 8, and 12, respectively (P<0.005). Wave 2 was not detected in the Day-8 heifers, but was the origin of the ovulatory follicle in the Day-12 heifers. The results supported the hypothesis that the dominant follicle of Wave 1 is viable (capable of ovulation ) before detection of Wave 2. For heifers treated on Days 5, 8, and 12, the ovulatory follicle had a mean diameter of 13.8, 17.3, and 11.8 mm, respectively, on the day of treatment and a mean diameter of 16.0, 19.5, and 16.4 ram, respectively, on the day prior to ovulation (significant increase between treatment and day prior to ovulation for each group). The results supported the hypothesis that the static-phase dominant follicle of Wave 1 is capable of further growth after luteolysis, even though it has apparently reached maximum diameter. The interval from treatment to ovulation was significantly shorter in Day-5 heifers (mean, 3.0 days) than in Day-12 heifers (mean, 4.5 days). In summary, the viable dominant follicle present at the time of luteolysis increased in diameter and became the ovulatory follicle.
INTRODUCTION A wave of ovarian follicular growth in cattle involves the synchronous development of a group of follicles with each wave culminating in the development of a dominant follicle. In studies utilizing daily ultrasonic monitoring
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of individual follicles in heifers, some laboratories found that most ( --- 80%) estrous cycles had three follicular waves (Savio et al., 1988; Sirois and Fortune, 1988). However, most ( - 80%) of the cycles monitored in our laboratory had two waves (Ginther et al., 1989b). For two-wave estrous cycles, on average, the dominant follicle of Wave 1 was first detected retrospectively on Day 0 (day of ovulation ) when its diameter was 4-5 mm. It grew linearly for 6 days (growing phase), remained approximately the same size for 6 days (static phase; mean diameter, 15.8 m m ) , and then began to regress (regressing phase; Ginther et al., 1989a). Wave 2 was first detected on approximately Day 9; the dominant follicle of Wave 2 reached a diameter not significantly different from the diameter of the static-phase follicle of Wave 1 and became the ovulatory follicle (Ginther et al., 1989a). The dominant follicle of Wave 1 was in the static phase for a mean of 3 days prior to first detection of Wave 2; a reduction in diameter of the dominant follicle of Wave 1 was not detected until approximately 3 days after the first detection of Wave 2 (Ginther et al., 1989a). Therefore, the dominant follicle of Wave 1 may still be viable during much of its static phase. The size and viability of a dominant follicle at the time of induced luteolysis with prostaglandin F2, (PGF) may affect the length of the interval from treatment to ovulation. Cattle treated with PGF during mid-diestrus had a longer interval from treatment to estrus than cattle treated during early or late diestrus (King, et al., 1982; Macmillan and Henderson, 1984; Stevenson et al., 1984). In a comparison of interovulatory intervals with two vs. three waves (Ginther et al., 1989b), the dominant follicle that was in its growing phase at the time ofluteolysis became the ovulatory follicle. In a comparison of nonbred and pregnant heifers (Ginther et al., 1989c), the diameter of the dominant follicle of Wave 2 was similar between nonbred and pregnant groups on the day of luteolysis in the nonbred group. Within a few days, thereafter, this follicle began to regress in pregnant heifers, but maintained or increased in diameter in nonbred heifers. As a result, the largest mean diameter attained by the dominant follicle of Wave 2 in the nonbred group was greater than for the pregnant group. Many experiments have been conducted to study selection and development of the ovulatory follicle in cattle. Dufour et al. (1972) marked the largest and second largest follicle with India ink on various days of the estrous cycle. In that study, the largest follicle became the ovulatory follicle only when it was marked __<3 days prior to estrus. Between 4 and 12 days after estrus, Staigmiller and England ( 1982 ) removed the ovary with the largest follicle or the ovary contralateral to the largest follicle and assessed the remaining ovary 4 days later. Removal of the ovary with the largest follicle resulted in an increase in follicular size and follicular fluid weight on the remaining ovary, but removal of the contralateral ovary did not. Parfet et al. (1989) marked the largest follicle and administered PGF 5 days after estrus; the marked follicle
EFFECT OF PROSTAGLANDIN TREATMENT DAY ON OVULATORY FOLLICLE
171
ovulated in 3 of 8 cows. In the above-mentioned studies, growth profiles of dominant follicles were not known because the follicles were evaluated only on two occasions - at the time of treatment and then a few days later. In another study (Quirk et al., 1986), ultrasonography was used to identify individual follicles following spontaneous or PGF-induced luteal regression, but the study was confined to the latter half of the estrous cycle. Ovarian follicular function may be altered in pregnant heifers within several days after ovulation; the concentrations of circulating estradiol-17fl were lower in pregnant than in cyclic heifers 4-7 days after estrus (Schallenberger et al., 1989). In another study (Ginther et al., 1989c), there was a tendency ( P < 0.1 ) for an effect of reproductive status (pregnant vs. nonbred) on the day-to-day diameter profiles of the dominant follicles of Wave 1; the plateau of the dominant follicle appeared to be lower in pregnant heifers. The purpose of the present experiment was to determine the effects of day of PGF treatment and breeding status (nonbred vs. bred) on selection and development of the ovulatory follicle in heifers. The following hypotheses were tested: ( 1 ) the dominant follicle of Wave 1 during the static phase is viable (capable of ovulation) before detection of Wave 2; (2) if luteolysis is induced during the static phase of the dominant follicle of Wave 1 and this follicle becomes the ovulatory follicle, it increases in diameter between luteolysis and ovulation, even though it has apparently reached its natural maximum diameter; and (3) growth of the dominant follicle during Wave 1 and response to PGF treatment is different between nonbred and bred heifers. MATERIALS AND METHODS
Twenty nulliparous Holstein heifers, 1.5-2.5 years of age and weighing 380500 kg, were used during October to December. The design was a 2 X 3 factorial; the factors were breeding status (nonbred or bred) and day of PGF treatment (Days 5, 8, or 12 ). On Days 5 and 8, the dominant follicle of Wave 1 was expected to be in its growing and static phase, respectively. On Day 12, the dominant follicle of Wave 1 was expected to be at the end of its static phase and the dominant follicle of Wave 2 was expected to be early in its growing phase. Heifers were examined daily by transrectal ultrasonic imaging during the last portion of an interovulatory interval and randomized into experimental groups by replicate when an ovarian follicle > 11 mm in diameter, a regressed corpus luteum (Pierson and Ginther, 1984), and a heterogeneous uterine echotexture characteristic of estrus (Pierson and Ginther, 1987) were detected. The ultrasound scanner was a real-time, B-mode instrument with a 5.0-MHz linear-array transducer (Tokyo Keiki LS-200H, Tokyo Keiki U.S.A., Chatsworth, CA, U.S.A. ). Heifers in the bred groups were inseminated approximately 6 h after randomization; most were inseminated a second time,
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J.P. KASTELIC ET AL.
approximately 24 h later. Ovulation was determined ultrasonically as described (Pierson and Ginther, 1984) and was designated the pretreatment ovulation (day of pretreatment ovulation = Day 0). After the pretreatment ovulation, heifers were treated on the designated day with a single intramuscular injection of 25 mg PGF (Lutalyse, The Upjohn Co., Kalamazoo, MI, U.S.A. ) to induce regression of the corpus luteum. The ovulation following PGF treatment was designated the posttreatment ovulation. Ultrasonic examinations were conducted once daily from the day of pretreatment ovulation to the day of posttreatment ovulation or until at least 14 days after treatment if posttreatment ovulation was not detected. Sequential identification of individual follicles was done, as described (Knopf et al., 1989), for most follicles with antral diameters >_4 ram. The operator conducting these examinations did not known which heifers were bred or the day that PGF was administered. Blood samples were collected once daily from the day of pretreatment ovulation to the day of posttreatment ovulation, or until at least 14 days after treatment if posttreatment ovulation was not detected. Plasma progesterone concentrations were determined by a direct enzymeimmunoassay on microtiter plates using the second antibody coating technique and horseradish peroxidase as the enzyme label, as described (Prakash, et al., 1987). Horseradish peroxidase was coupled to progesterone-6fl-hydroxyhemisuccinate by a mixed anhydride method. Microtiter plates were coated with affinity-purified sheep IgG antirabbit IgG. The assay used antiserum raised in rabbits against progesterone-7a-carboxyethylthioether conjugated to bovine serum albumin. The specificity of the antibody has been described previously (Van de Wiel and Koops, 1986 ). The sensitivity of progesterone measurements in terms of the lowest detectable concentrations significantly different from zero was 0.4 pg. A single determination of the progesterone concentration in each sample was done and the interassay coefficient of variation was 17%. Chi square analyses were used to compare, among groups, proportions of heifers that ovulated from the dominant follicle of Wave 1. Two-way analyses of variance were used to determine main effects of day of treatment and breeding status, and the day by status interaction for the following end points: ( 1 ) diameter of ovulatory follicle on day of treatment; (2) diameter of ovulatory follicle on day prior to posttreatment ovulation; (3) increase in diameter of ovulatory follicle from day of treatment to day prior to posttreatment ovulation; (4) length of interval from day of treatment to day of posttreatment ovulation; and (5) diameter of dominant follicle of Wave 1 on Day 8 (Day-8 and Day-12 groups) and on day prior to posttreatment ovulation (Day-8 group). Within each day-group (PGF treatment on Days 5, 8, or 12 ), changes in diameter of the dominant follicle of Wave 1 from the day of pretreatment ovulation to the day before the first heifer in the group ovulated were analyzed by split-plot analyses of variance (Gill and Hafs, 1971; SAS, 1985 ); main effects of day and breeding status, and the day by status inter-
EFFECT OF PROSTAGLANDIN TREATMENT DAY ON OVULATORY FOLLICLE
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action were determined. Similar analyses were used for changes in diameter of the dominant follicle of Wave 2 and for changes in plasma progesterone concentrations. Paired t-tests were used to detect increases in diameter of the ovulatory follicle from the day of treatment to the day prior to posttreatment ovulation. For split-plot and two-way analyses of variance, differences were located by Duncan's multiple range test. RESULTS
In 15 of the 20 heifers, luteal regression was rapid and complete and posttreatment ovulation from a single follicle was detected within 5 days following treatment; only data for the 15 responding, single-ovulating heifers were analyzed. Seven of these 15 heifers were bred (2, 3, and 2 bred heifers were treated on Days 5, 8, and 12, respectively). There were no effects of breeding status or interactions involving breeding status for any end point. Therefore, combined data for the two statuses are presented. Ovulation occurred from the dominant follicle of Wave 1 in all heifers treated on Days 5 or 8 and from the dominant follicle of Wave 2 in all heifers treated on Day 12 ( P < 0.005; Table 1 ). Wave 2 was first detected on Days 8, 9, 9, and 9, respectively, in the four Day-12 heifers, but was not detected in any of the six Day-8 heifers. Data have been tabulated and significant differences are shown for diameter of ovulatory follicle on day of treatment, diameter of ovulatory follicle on day TABLE1 Ovarian responses (mean_+ s.e.m.) to PGF2,~ treatment on Days 5, 8, or 12 Item
Day of treatment a 5
No. heifers ovulating from Wave 1 Diameter of ovulatory follicle on day of treatment ( m m ) Diameter of ovulatory follicle on day prior to posttreatment ovulation ( m m ) Increase in diameter of ovulatory follicle from treatment to posttreatment ovulation ( m m ) Length of interval from treatment to posttreatment ovulation (days)
Difference among days
8 5/5
12 6/6
0/4
P<0.005
13.8b+ 1.0
17.3c+_0.6
11.8b_+0.8
P<0.002
16.0b+0.6
19.5¢_+ 1.1
16.4b_+0.5
P<0.04
2.2b+ 0.4
2.2b+ 0.8
4.6c+0.4
P<0.05
4.5d_+ 0.3
P < 0.002
3.0b+ 0.0
3.7¢_+0.2
aDay 0 is the day of pretreatment ovulation. b'c'd'Within each row, means with a different superscript letter are different ( P < 0.05 ).
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J.P. K A S T E L I C E T AL.
A 16
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Days after pretreatment ovulation
Fig. 1. Mean diameters of the dominant follicles of Wave 1 for heifers treated with prostaglandin F2,, on Days 5, 8, or 12 and mean diameters of the dominant follicle of Wave 2 for heifers treated on Day 12. Day before ovulation refers to the day before the first heifer in a group ovulated. Day effect ( P < 0.0001 ) for each profile. There was an increase in the diameter of the ovulatory follicle from the day of treatment to the day prior to posttreatment ovulation ( P < 0.004, P<0.04, and P<0.001 for heifers treated on Days 5, 8, and 12, respectively). 7"
Day of treatment
6"
5
•
No, heifers 5
8
6 ~f
gg 2 .=m Q
3-
O" '0"
2"
4"
'6"
'8 " 1'0 "12 " 1'4 " 1'6
Days after pretreatment ovulation
Fig. 2. Mean plasma progesterone concentrations from day of pretreatment ovulation to 3, 3, and 4 days after treatment for heifers treated with prostaglandin F2,~ (arrow) on Days 5, 8, or 12, respectively. For each group (treatment on Days 5, 8, or 12), there was a day effect ( P < 0.0001 ) and plasma progesterone concentrations decreased significantly between the day of treatment and the following day.
prior to posttreatment ovulation, increase in diameter o f ovulatory follicle from day of treatment to day prior to posttreatment ovulation, and length o f interval from day o f treatment to day o f posttreatment ovulation (Table 1 ). The diameter o f the dominant follicle of Wave l was 17.3 ab mm and 15.2 b mm on Day 8 (Day-8 and Day-12 group, respectively) and was 19.5 a mm on the day prior to posttreatment ovulation (Day-8 group; means without a common superscript are significantly different). Profiles for mean diameters of the dominant follicle o f Waves l and 2 and results o f statistical analyses
EFFECT OF PROSTAGLANDIN
TREATMENT
DAY ON OVULATORY FOLLICLE
l 75
24 20
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Days after pretreatment ovulation
Fig. 3. Dominant follicle diameters and plasma progesterone concentrations in four heifers with a prolonged ( > 10 days) interval from day of prostaglandin F2,~treatment (dashed arrow) to day of posttreatment ovulation (open circle). Heifer A: plasma progesterone concentration decreased precipitously following treatment and remained near baseline. The dominant follicle of Wave 1 reached a diameter of 17 mm, but failed to ovulate. Ovulation from two follicles of Wave 2 was detected on Day 16. Heifer B: two follicles of Wave 1 reached diameters of 20 and 17.5 mm, respectively and developed thick walls (apparent luteinization). Ovulation was not detected by Day 21 (end of observation period). Plasma progesterone concentrations declined following treatment and then increased when the follicles developed thick walls. Heifer C: plasma progesterone concentrations indicated delayed luteal regression. The dominant follicle of Wave 1 regressed and ovulation from the dominant follicle of Wave 2 was detected on Day 18. This heifer developed rectal irritation and follicles were identified but diameters were not determined after Day 12. Heifer D: dominant follicle of Wave 1 regressed. Dominant follicle of Wave 2 reached a diameter of 32 mm with a thick wall (apparent luteinization) and ovulation was not detected by Day 26 (end of observation period). Plasma progesterone concentrations decreased to baseline following treatment and then increased when the follicle developed a thick wall. are g i v e n in Fig. 1. Profiles for m e a n p l a s m a p r o g e s t e r o n e c o n c e n t r a t i o n s a n d results o f statistical analyses are given in Fig. 2. F o u r heifers h a d a p r o l o n g e d ( > 10 d a y s ) interval f r o m day o f t r e a t m e n t to d a y o f p o s t t r e a t m e n t o v u l a t i o n ; d i a m e t e r s o f d o m i n a n t follicles a n d p l a s m a p r o g e s t e r o n e c o n c e n t r a t i o n s are s h o w n (Fig. 3). In a n o t h e r heifer ( n o t s h o w n ) , there were t w o d o m i n a n t W a v e - 1 follicles; this heifer was t r e a t e d o n D a y 5 a n d b o t h follicles o v u l a t e d o n D a y 10.
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J.P. KASTELIC ET AL.
DISCUSSION
There were no significant effects of breeding status on any end point. Therefore, the hypothesis that the dominant follicle of Wave 1 is altered when a conceptus is present was not supported. The diameter of the dominant follicle of Wave 1 was not different between bred and nonbred heifers on Day 8, and the previously reported tendency for a lower plateau in the follicle profile of pregnant heifers was not confirmed. It cannot be stated unequivocally that the bred heifers in the present experiment were pregnant on the day of PGF treatment because luteolysis was induced before reliable ultrasonic pregnancy diagnosis could be made (Kastelic et al., 1989 ). However, in contemporary studies, pregnancy was confirmed on Day 24 in 5 of 6 (83%) heifers from the same herd and bred with the same semen by the same technician. In all heifers treated with PGF on Day 5, the dominant follicle of Wave 1 had not yet reached its static phase, and in all heifers treated on Day 12, Wave 2 was detected before PGF was given. For both days of treatment, therefore, a dominant follicle was in its growing phase on the day of treatment. It is concluded that, for both waves, the dominant follicle became the ovulatory follicle when luteolysis was induced during the growing phase of the dominant follicle. This result is consistent with the report (G.inther et al., 1989b) that the viable dominant follicle present at the onset of spontaneous luteolysis became the ovulatory follicle for both two-wave and three-wave estrous cycles. The rhythmic emergence of follicular waves during early pregnancy (Ginther et al., 1989c) is consistent, therefore, with the absence of luteolysis. After induced luteolysis, ovulation occurred from only the dominant follicle of a wave, indicating that subordinate follicles lost their capability for growing and ovulating. In previous studies (Sirois and Fortune, 1988; Ginther et al., 1989a; Knopf et al., 1989 ), subordinate follicles ceased growing in an average of 3 days after detection of a wave (follicle diameter at wave detection, 4-5 mm). Wave-2 follicles did not reach a detectable size (diameter, 4-5 m m ) in any of the Day-8 heifers with rapid and complete luteal regression. In a previous study, the average day of detection of Wave 2 was Day 9.8 (range, days 8-11; Ginther et al., 1989a). In the Day-8 group, the dominant follicle of Wave 1 was its static phase on the day of treatment, but was viable as indicated by its ovulatory capability. The diameter of the dominant follicle of Wave 1 increased an average of 2.2 m m after treatment, even though it was in its static phase and had apparently reached m a x i m u m diameter. These results indicate that the Day-8 static-phase dominant follicle of Wave 1 in these heifers was not only capable of ovulating, but was also capable of further growth and that a stimulus for growth was present immediately after luteolysis. In this regard, Schallenberger et al. (1984) showed an increased frequency of FSH pulses after luteolysis.
EFFECT OF PROSTAGLANDIN TREATMENT DAY ON OVULATORY FOLLICLE
| 77
At the time of PGF treatment, the dominant follicle of Wave 1 was in its late growing and early static phase in heifers treated on Days 5 and 8, respectively, and the dominant follicle of Wave 2 was in its early growing phase in heifers treated on Day 12. Therefore, at the time of treatment, the diameter of the ovulatory follicle was smallest in Day-12 heifers, intermediate in Day5 heifers, and largest in Day-8 heifers. In all three treatment groups, the diameter of the ovulatory follicle increased significantly from the day of treatment to the day prior to posttreatment ovulation. This increase was significantly greater in the heifers treated in Day 12 than in heifers treated on Day 5 or 8, consistent with the diameter of the ovulatory follicle on the day of treatment. The dominant follicle of Wave 2 (treatment on Day 12 ) was an average of 2.0 mm smaller (not significant) on the day of treatment and had a 2.4-mm greater increase in diameter after treatment (significant) than the dominant follicle of Wave 1 (treatment on Day 5 ). The diameter of the ovulatory follicle on the day prior to posttreatment ovulation, however, was not significantly different between these two groups. Therefore, the interval from treatment to posttreatment ovulation was longer for heifers treated on Day 12 than for heifers treated on Day 5 (difference, 1.5 days). The longer intervals from treatment to ovulation in the Day-12 group than in the Day-5 group were consistent with the results of previous studies (King et al., 1982; Stevenson et al., 1984) and with the proposal (Scaramuzzi et al., 1980; Macmillan and Henderson, 1984) that the size of the largest nonatretic follicle at the time of treatment is a determining factor in the time required for a follicle to reach ovulatory size. Several reports (King, et al., 1982; Macmillan and Henderson, 1984; Stevenson, et al., 1984 ) have concluded that the interval from PGF treatment to luteolysis is similar when PGF is given to cattle with a mature corpus luteum, regardless of day of treatment. Results of the present study are consistent with these reports in regard to the uniformity of luteolysis for the various days of treatment, as indicated by the profiles of plasma progesterone concentration. In Heifer A and another heifer treated on Day 5, two dominant follicles developed and ovulated. The incidence of twin ovulations in this experiment was consistent with the reported incidence of twin ovulations in Holstein cattle ( 5-13%; Kidder et al., 1952; Labhsetwar et al., 1963 ). Our working hypothesis is that an emerging dominant follicle initiates or is an important factor in the regression of its subordinates and the previous dominant follicle (Ginther et al., 1989c). In Heifer A, development and ovulation of the Wave-2 follicles was not accompanied by a decrease in diameter of the dominant follicle of Wave 1. It is not clear why the dominant follicle failed to cause regression of the previous dominant follicle in this heifer. In Heifers A, B, and D, at least one dominant follicle reached preovulatory diameter after luteolysis, but ovulation was not detected. Prolonged intervals
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J.P. KASTELICETAL.
( > 8 days) of low peripheral progesterone concentrations following treatment with a PGF analogue have been reported previously (Jackson et al., 1979; Baishya et al., 1980). However, ovarian structures were not monitored in these studies. Impairment of positive feedback of estradiol on LH release has been suggested as the cause of anovulatory (cystic) ovarian follicles in postpartum cows (Kesler et al., 1979) and may have been the cause of ovulation failure in the present experiments. In Heifer A, the impairment was apparently short-lived, as ovulation occurred from two dominant follicles of Wave 2. In Heifers B and D, ovulation was not detected during the study period; perhaps a preovulatory LH surge was suppressed by the increased plasma progesterone concentrations associated with follicle luteinization. In Heifer D but not in Heifer B, there was emergence of other follicles in the presence of large, anovulatory follicles. In a previous study (Kesler et al., 1980 ) anovulatory follicles regressed and were replaced by other anovulatory follicles in 6 of 8 cystic cows. In Heifer C, plasma progesterone concentrations declined following PGF treatment, but remained above baseline for approximately 1 week. In this heifer, the interval from treatment to posttreatment ovulation was prolonged, but the viable dominant follicle present at the time of luteolysis became the ovulatory follicle. In conclusion, growth of the dominant follicle of Wave 1 and response to PGF treatment was not different between bred and nonbred heifers. In heifers with rapid and complete luteal regression, ovulation occurred from the dominant follicle of Wave 1 in all heifers treated on Days 5 and 8 and from the dominant follicle of Wave 2 in all heifers treated on Day 12. In all groups, the ovulatory follicle increased in diameter from the time of treatment to the day before ovulation. Therefore, the viable dominant follicle present at the time of luteolysis increased in diameter and became the ovulatory follicle. ACKNOWLEDGEMENTS
Supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison and USDA Grant 87-CRSR-2-3158, and by an NIH Biomedical Research Support Grant to the School of Veterinary Medicine and Department of Veterinary Science, University of Wisconsin-Madison. We thank Lisa Kulick for graphic and computer assistance, Inge Redl for technical assistance, Sophie Yap for statistical assistance, Sharon Schade and Maria Westphal for manuscript preparation, and Dr. E. Schallenberger for performing the progesterone assays. Lutalyse and bull semen were provided by The Upjohn Company, Kalamazoo, MI and American Breeders Service, De Forest, WI, respectively. J.K. is supported by a fellowship from the Medical Research Council of Canada. L.K. is Research Fellow of the Deutsche Forschungsgemeinschaft and the
EFFECT OF PROSTAGLANDIN TREATMENT DAY ON OVULATORY FOLLICLE
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H.W. Schaumann Stiftung and is on leave from Department of Physiology, Technical University Munich, Weihenstepan, Federal Republic of Germany.
REFERENCES Baishya, J., Ball, B.J.H., Leaver, J.D. and Pope, G.S., 1980. Fertility of lactating dairy cows inseminated after treatment with cloprostenol. Br. Vet. J., 136: 227-239. Dufour, J., Whitmore, H.L., Ginther, O.J. and Casida, L.E., 1972. Identification of the ovulating follicle by its size on different days of the estrous cycle in heifers. J. Anim. Sci., 34: 8587. Gill, J.L. and Hafs, H.D., 1971. Analysis of repeated measurements of animals. J. Anim. Sci., 33: 331-336. Ginther, O.J., Kastelic, J.P. and Knopf, L., 1989a. Composition and characteristics of follicular waves during the bovine estrous cycle. Anim. Repro& Sci., 20:187-200. Ginther, O.J., Knopf, L. and Kastelic, J.P., 1989b. Temporal associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. J. Reprod. Fertil., 87: 223-230. Ginther, O.J., Knopf, L. and Kastelic, J.P., 1989c. Ovarian follicular dynamics in heifers during early pregnancy. Biol. Reprod., 41:247-254. Jackson, P.S., Johnson, C.T., Bulman, D.C. and Holdsworth, R.J., 1979. A study of cloprostenol-induced oestrus and spontaneous oestrus by means of the milk progesterone assay. Br. Vet. J., 135: 578-590. Kastelic, J.P., Curran, S. and Ginther, O.J., 1889. Accuracy of ultrasonography for pregnancy diagnosis on Days 10 to 22 in heifers. Theriogenology, 31 : 813-820. Kesler, D.J., Garverick, H.A., Elmore, R.G., Youngquist, R.S. and Bierschwal, C.J., 1979. Reproductive hormones associated with the ovarian cyst response to GnRH. Theriogenology, 12:109-114. Kesler, D.J., Garverick, H.A., Caudle, A.B., Elmore, R.G., Youngquist, R.S. and Bierschwal, C.J., 1980. Reproductive hormone and ovarian changes in cows with ovarian cysts. J. Dairy Sci., 63: 166-170. Kidder, H.E., Barrett, G.R. and Casida, L.E., 1952. A study of ovulations in six families of Holstein-Friesians. J. Dairy Sci., 35: 436-444. King, M.E., Kiracofe, G.H., Stevenson, J.S.. and Schalles, R.R., 1982. Effect of stage of the estrous cycle on interval to estrus after PGF2c~ in beef cattle. Theriogenology, 18:191-200. Knopf, L., Kastelic, J.P., Schallenberger, E. and Ginther, O.J., 1989. Ovarian follicular populations in heifers: test of two wave hypothesis by ultrasonically monitoring individual follicles. Domest. Anim. Endocrinol., 6:111-119. Labhsetwar, A.P., Tyler, W.J. and Casida, L.E., 1963. Analysis of variations in some factors affecting multiple ovulations in Holstein cattle. J. Dairy Sci., 46: 840-842. Macmillan, K.L. and Henderson, H.V., 1984. Analyses of the variation in the interval from an injection of prostaglandin F2,, to oestrus as a method of studying patterns of follicle development during dioestrus in dairy cows. Anim. Reprod. Sci., 6: 245-254. Parfet, J.R., Smith, C.A., Cook, D.L., Skyer, D.M., Youngquist, R.S. and Garverick, H.A., 1989. Secretory patterns of LH and FSH and follicular growth following administration of PGF2, during the early luteal phase in cattle. Theriogenology, 31 : 513-524. Pierson, R.A. and Ginther, O.J., 1984. Ultrasonography of the bovine ovary. Theriogenology 21: 495-504. Pierson, R.A. and Ginther, O.J., 1987. Ultrasonographic appearance of the bovine uterus during the estrous cycle. J. Am. Vet. Med. Assoc., 190: 995-1001.
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Prakash, B.S., Meyer, H.D.D., Schallenberger, E. and Van de Wiet, D.F.M., 1987. Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique. J. Steroid Biochem., 28: 623-627. Quirk, S.M., Hickey, G.J. and Fortune, J.E., 1986. Growth and regression of ovarian follicles during the follicular phase of the oestrus cycle in heifers undergoing spontaneous and PGF2or-induced luteolysis. J. Reprod. Fertil., 77:211-219. SAS, 1985. SAS User's Guide: Statistics, Version 5 Edition. SAS Institute, Cary, N.C, pp. 433506. DaSavio, J.D., Keenan, L., Boland, M.P. and Roche, J.F., 1988. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J. Repro& Fertil., 83:663-671. Scaramuzzi, R.J., Turnbull, K.E. and Nancarrow, C.D., 1980. Growth of Graffian follicles in cows following luteolysis induced by prostaglandin PGF2,, analogue, cloprostenol., Aust. J. Biol. Sci., 33: 63-69. Schallenberger, E., Schams, D., Bullermann, B. and Waiters, D.L., 1984. Pulsatile secretion of gonadotrophins, ovarian steroids and ovarian oxytocin during prostaglandin-induced regression of the corpus luteum in the cow. J. Reprod. Fertil., 7 I: 493-501. Schallenberger, E., Schams, D. and Meyer, H.H.D., 1989. Sequences of pituitary, ovarian and uterine hormone secretion during the first 5 weeks of pregnancy in dairy cattle. J. Reprod. Fertil., Suppl., 37: 277-286. Sirois, J. and Fortune, J.E., 1988. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol. Reprod., 39:308-317. Staigmiller, R.B. and England, B.G., 1982. Folliculogenesis in the bovine. Theriogenology, 17: 43-52. Stevenson, J.S., Schmidt, M.K. and Call, E.P., 1984. Stage of estrous cycle, time of insemination, and seasonal effects on estrus and fertility of Holstein heifers after prostaglandin F2,~. J. Dairy Sci., 67: 1798-1805. Van de Wiel, D.F.M. and Koops, W., 1986. Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma. Anim. Reprod. Sci., 10:201-213.
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Elsevier Science Publishers B.V., Amsterdam
Effect of day of prostaglandin F2~ treatment on selection and development of the ovulatory follicle in heifers J.P. K a s t e l i c , L. K n o p f a n d
O.J. Ginther
Department of Veterinary Science, University of Wisconsin-Madison, Madison, WI 53 706, U.S.A. (Accepted 4 May 1990)
ABSTRACT Kastelic, J.P., Knopf, L. and Ginther, O.J., 1990. Effect of day of prostaglandin F2,~ treatment on selection and development of the ovulatory follicle in heifers. Anita. Reprod. Sci., 23:169-180. In previous studies of heifers with two follicular waves during an estrous cycle, the dominant follicle of Wave 1 was first detected ultrasonically on approximately the day of ovulation (Day 0) when its diameter was 4-5 ram. On average, it grew linearly for 6 days (growing phase), remained the same size for 6 days (static phase), and then regressed (regressing phase). The dominant follicle of Wave 2 was first detected on approximately Day 9 and became the ovulatory follicle. In the present experiment, nonbred and bred heifers were treated with a luteolytic dose of prostaglandin F2,~ (25 mg) on Days 5, 8, or 12, when the dominant follicle of Wave 1 was expected to be in the growing, static, and regressing phase, respectively. There were no significant effects of breeding status on any end point. The hypothesis that growth of the dominant follicle during Wave 1 and response to prostaglandin F2,~ treatment is different between bred and nonbred heifers was not supported. Ovulation occurred from the dominant follicle of Wave 1 in 5 of 5, 6 of 6 and 0 of 4 heifers treated on Days 5, 8, and 12, respectively (P<0.005). Wave 2 was not detected in the Day-8 heifers, but was the origin of the ovulatory follicle in the Day-12 heifers. The results supported the hypothesis that the dominant follicle of Wave 1 is viable (capable of ovulation ) before detection of Wave 2. For heifers treated on Days 5, 8, and 12, the ovulatory follicle had a mean diameter of 13.8, 17.3, and 11.8 mm, respectively, on the day of treatment and a mean diameter of 16.0, 19.5, and 16.4 ram, respectively, on the day prior to ovulation (significant increase between treatment and day prior to ovulation for each group). The results supported the hypothesis that the static-phase dominant follicle of Wave 1 is capable of further growth after luteolysis, even though it has apparently reached maximum diameter. The interval from treatment to ovulation was significantly shorter in Day-5 heifers (mean, 3.0 days) than in Day-12 heifers (mean, 4.5 days). In summary, the viable dominant follicle present at the time of luteolysis increased in diameter and became the ovulatory follicle.
INTRODUCTION A wave of ovarian follicular growth in cattle involves the synchronous development of a group of follicles with each wave culminating in the development of a dominant follicle. In studies utilizing daily ultrasonic monitoring
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of individual follicles in heifers, some laboratories found that most ( --- 80%) estrous cycles had three follicular waves (Savio et al., 1988; Sirois and Fortune, 1988). However, most ( - 80%) of the cycles monitored in our laboratory had two waves (Ginther et al., 1989b). For two-wave estrous cycles, on average, the dominant follicle of Wave 1 was first detected retrospectively on Day 0 (day of ovulation ) when its diameter was 4-5 mm. It grew linearly for 6 days (growing phase), remained approximately the same size for 6 days (static phase; mean diameter, 15.8 m m ) , and then began to regress (regressing phase; Ginther et al., 1989a). Wave 2 was first detected on approximately Day 9; the dominant follicle of Wave 2 reached a diameter not significantly different from the diameter of the static-phase follicle of Wave 1 and became the ovulatory follicle (Ginther et al., 1989a). The dominant follicle of Wave 1 was in the static phase for a mean of 3 days prior to first detection of Wave 2; a reduction in diameter of the dominant follicle of Wave 1 was not detected until approximately 3 days after the first detection of Wave 2 (Ginther et al., 1989a). Therefore, the dominant follicle of Wave 1 may still be viable during much of its static phase. The size and viability of a dominant follicle at the time of induced luteolysis with prostaglandin F2, (PGF) may affect the length of the interval from treatment to ovulation. Cattle treated with PGF during mid-diestrus had a longer interval from treatment to estrus than cattle treated during early or late diestrus (King, et al., 1982; Macmillan and Henderson, 1984; Stevenson et al., 1984). In a comparison of interovulatory intervals with two vs. three waves (Ginther et al., 1989b), the dominant follicle that was in its growing phase at the time ofluteolysis became the ovulatory follicle. In a comparison of nonbred and pregnant heifers (Ginther et al., 1989c), the diameter of the dominant follicle of Wave 2 was similar between nonbred and pregnant groups on the day of luteolysis in the nonbred group. Within a few days, thereafter, this follicle began to regress in pregnant heifers, but maintained or increased in diameter in nonbred heifers. As a result, the largest mean diameter attained by the dominant follicle of Wave 2 in the nonbred group was greater than for the pregnant group. Many experiments have been conducted to study selection and development of the ovulatory follicle in cattle. Dufour et al. (1972) marked the largest and second largest follicle with India ink on various days of the estrous cycle. In that study, the largest follicle became the ovulatory follicle only when it was marked __<3 days prior to estrus. Between 4 and 12 days after estrus, Staigmiller and England ( 1982 ) removed the ovary with the largest follicle or the ovary contralateral to the largest follicle and assessed the remaining ovary 4 days later. Removal of the ovary with the largest follicle resulted in an increase in follicular size and follicular fluid weight on the remaining ovary, but removal of the contralateral ovary did not. Parfet et al. (1989) marked the largest follicle and administered PGF 5 days after estrus; the marked follicle
EFFECT OF PROSTAGLANDIN TREATMENT DAY ON OVULATORY FOLLICLE
171
ovulated in 3 of 8 cows. In the above-mentioned studies, growth profiles of dominant follicles were not known because the follicles were evaluated only on two occasions - at the time of treatment and then a few days later. In another study (Quirk et al., 1986), ultrasonography was used to identify individual follicles following spontaneous or PGF-induced luteal regression, but the study was confined to the latter half of the estrous cycle. Ovarian follicular function may be altered in pregnant heifers within several days after ovulation; the concentrations of circulating estradiol-17fl were lower in pregnant than in cyclic heifers 4-7 days after estrus (Schallenberger et al., 1989). In another study (Ginther et al., 1989c), there was a tendency ( P < 0.1 ) for an effect of reproductive status (pregnant vs. nonbred) on the day-to-day diameter profiles of the dominant follicles of Wave 1; the plateau of the dominant follicle appeared to be lower in pregnant heifers. The purpose of the present experiment was to determine the effects of day of PGF treatment and breeding status (nonbred vs. bred) on selection and development of the ovulatory follicle in heifers. The following hypotheses were tested: ( 1 ) the dominant follicle of Wave 1 during the static phase is viable (capable of ovulation) before detection of Wave 2; (2) if luteolysis is induced during the static phase of the dominant follicle of Wave 1 and this follicle becomes the ovulatory follicle, it increases in diameter between luteolysis and ovulation, even though it has apparently reached its natural maximum diameter; and (3) growth of the dominant follicle during Wave 1 and response to PGF treatment is different between nonbred and bred heifers. MATERIALS AND METHODS
Twenty nulliparous Holstein heifers, 1.5-2.5 years of age and weighing 380500 kg, were used during October to December. The design was a 2 X 3 factorial; the factors were breeding status (nonbred or bred) and day of PGF treatment (Days 5, 8, or 12 ). On Days 5 and 8, the dominant follicle of Wave 1 was expected to be in its growing and static phase, respectively. On Day 12, the dominant follicle of Wave 1 was expected to be at the end of its static phase and the dominant follicle of Wave 2 was expected to be early in its growing phase. Heifers were examined daily by transrectal ultrasonic imaging during the last portion of an interovulatory interval and randomized into experimental groups by replicate when an ovarian follicle > 11 mm in diameter, a regressed corpus luteum (Pierson and Ginther, 1984), and a heterogeneous uterine echotexture characteristic of estrus (Pierson and Ginther, 1987) were detected. The ultrasound scanner was a real-time, B-mode instrument with a 5.0-MHz linear-array transducer (Tokyo Keiki LS-200H, Tokyo Keiki U.S.A., Chatsworth, CA, U.S.A. ). Heifers in the bred groups were inseminated approximately 6 h after randomization; most were inseminated a second time,
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approximately 24 h later. Ovulation was determined ultrasonically as described (Pierson and Ginther, 1984) and was designated the pretreatment ovulation (day of pretreatment ovulation = Day 0). After the pretreatment ovulation, heifers were treated on the designated day with a single intramuscular injection of 25 mg PGF (Lutalyse, The Upjohn Co., Kalamazoo, MI, U.S.A. ) to induce regression of the corpus luteum. The ovulation following PGF treatment was designated the posttreatment ovulation. Ultrasonic examinations were conducted once daily from the day of pretreatment ovulation to the day of posttreatment ovulation or until at least 14 days after treatment if posttreatment ovulation was not detected. Sequential identification of individual follicles was done, as described (Knopf et al., 1989), for most follicles with antral diameters >_4 ram. The operator conducting these examinations did not known which heifers were bred or the day that PGF was administered. Blood samples were collected once daily from the day of pretreatment ovulation to the day of posttreatment ovulation, or until at least 14 days after treatment if posttreatment ovulation was not detected. Plasma progesterone concentrations were determined by a direct enzymeimmunoassay on microtiter plates using the second antibody coating technique and horseradish peroxidase as the enzyme label, as described (Prakash, et al., 1987). Horseradish peroxidase was coupled to progesterone-6fl-hydroxyhemisuccinate by a mixed anhydride method. Microtiter plates were coated with affinity-purified sheep IgG antirabbit IgG. The assay used antiserum raised in rabbits against progesterone-7a-carboxyethylthioether conjugated to bovine serum albumin. The specificity of the antibody has been described previously (Van de Wiel and Koops, 1986 ). The sensitivity of progesterone measurements in terms of the lowest detectable concentrations significantly different from zero was 0.4 pg. A single determination of the progesterone concentration in each sample was done and the interassay coefficient of variation was 17%. Chi square analyses were used to compare, among groups, proportions of heifers that ovulated from the dominant follicle of Wave 1. Two-way analyses of variance were used to determine main effects of day of treatment and breeding status, and the day by status interaction for the following end points: ( 1 ) diameter of ovulatory follicle on day of treatment; (2) diameter of ovulatory follicle on day prior to posttreatment ovulation; (3) increase in diameter of ovulatory follicle from day of treatment to day prior to posttreatment ovulation; (4) length of interval from day of treatment to day of posttreatment ovulation; and (5) diameter of dominant follicle of Wave 1 on Day 8 (Day-8 and Day-12 groups) and on day prior to posttreatment ovulation (Day-8 group). Within each day-group (PGF treatment on Days 5, 8, or 12 ), changes in diameter of the dominant follicle of Wave 1 from the day of pretreatment ovulation to the day before the first heifer in the group ovulated were analyzed by split-plot analyses of variance (Gill and Hafs, 1971; SAS, 1985 ); main effects of day and breeding status, and the day by status inter-
EFFECT OF PROSTAGLANDIN TREATMENT DAY ON OVULATORY FOLLICLE
173
action were determined. Similar analyses were used for changes in diameter of the dominant follicle of Wave 2 and for changes in plasma progesterone concentrations. Paired t-tests were used to detect increases in diameter of the ovulatory follicle from the day of treatment to the day prior to posttreatment ovulation. For split-plot and two-way analyses of variance, differences were located by Duncan's multiple range test. RESULTS
In 15 of the 20 heifers, luteal regression was rapid and complete and posttreatment ovulation from a single follicle was detected within 5 days following treatment; only data for the 15 responding, single-ovulating heifers were analyzed. Seven of these 15 heifers were bred (2, 3, and 2 bred heifers were treated on Days 5, 8, and 12, respectively). There were no effects of breeding status or interactions involving breeding status for any end point. Therefore, combined data for the two statuses are presented. Ovulation occurred from the dominant follicle of Wave 1 in all heifers treated on Days 5 or 8 and from the dominant follicle of Wave 2 in all heifers treated on Day 12 ( P < 0.005; Table 1 ). Wave 2 was first detected on Days 8, 9, 9, and 9, respectively, in the four Day-12 heifers, but was not detected in any of the six Day-8 heifers. Data have been tabulated and significant differences are shown for diameter of ovulatory follicle on day of treatment, diameter of ovulatory follicle on day TABLE1 Ovarian responses (mean_+ s.e.m.) to PGF2,~ treatment on Days 5, 8, or 12 Item
Day of treatment a 5
No. heifers ovulating from Wave 1 Diameter of ovulatory follicle on day of treatment ( m m ) Diameter of ovulatory follicle on day prior to posttreatment ovulation ( m m ) Increase in diameter of ovulatory follicle from treatment to posttreatment ovulation ( m m ) Length of interval from treatment to posttreatment ovulation (days)
Difference among days
8 5/5
12 6/6
0/4
P<0.005
13.8b+ 1.0
17.3c+_0.6
11.8b_+0.8
P<0.002
16.0b+0.6
19.5¢_+ 1.1
16.4b_+0.5
P<0.04
2.2b+ 0.4
2.2b+ 0.8
4.6c+0.4
P<0.05
4.5d_+ 0.3
P < 0.002
3.0b+ 0.0
3.7¢_+0.2
aDay 0 is the day of pretreatment ovulation. b'c'd'Within each row, means with a different superscript letter are different ( P < 0.05 ).
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J.P. K A S T E L I C E T AL.
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Fig. 1. Mean diameters of the dominant follicles of Wave 1 for heifers treated with prostaglandin F2,, on Days 5, 8, or 12 and mean diameters of the dominant follicle of Wave 2 for heifers treated on Day 12. Day before ovulation refers to the day before the first heifer in a group ovulated. Day effect ( P < 0.0001 ) for each profile. There was an increase in the diameter of the ovulatory follicle from the day of treatment to the day prior to posttreatment ovulation ( P < 0.004, P<0.04, and P<0.001 for heifers treated on Days 5, 8, and 12, respectively). 7"
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Fig. 2. Mean plasma progesterone concentrations from day of pretreatment ovulation to 3, 3, and 4 days after treatment for heifers treated with prostaglandin F2,~ (arrow) on Days 5, 8, or 12, respectively. For each group (treatment on Days 5, 8, or 12), there was a day effect ( P < 0.0001 ) and plasma progesterone concentrations decreased significantly between the day of treatment and the following day.
prior to posttreatment ovulation, increase in diameter o f ovulatory follicle from day of treatment to day prior to posttreatment ovulation, and length o f interval from day o f treatment to day o f posttreatment ovulation (Table 1 ). The diameter o f the dominant follicle of Wave l was 17.3 ab mm and 15.2 b mm on Day 8 (Day-8 and Day-12 group, respectively) and was 19.5 a mm on the day prior to posttreatment ovulation (Day-8 group; means without a common superscript are significantly different). Profiles for mean diameters of the dominant follicle o f Waves l and 2 and results o f statistical analyses
EFFECT OF PROSTAGLANDIN
TREATMENT
DAY ON OVULATORY FOLLICLE
l 75
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Fig. 3. Dominant follicle diameters and plasma progesterone concentrations in four heifers with a prolonged ( > 10 days) interval from day of prostaglandin F2,~treatment (dashed arrow) to day of posttreatment ovulation (open circle). Heifer A: plasma progesterone concentration decreased precipitously following treatment and remained near baseline. The dominant follicle of Wave 1 reached a diameter of 17 mm, but failed to ovulate. Ovulation from two follicles of Wave 2 was detected on Day 16. Heifer B: two follicles of Wave 1 reached diameters of 20 and 17.5 mm, respectively and developed thick walls (apparent luteinization). Ovulation was not detected by Day 21 (end of observation period). Plasma progesterone concentrations declined following treatment and then increased when the follicles developed thick walls. Heifer C: plasma progesterone concentrations indicated delayed luteal regression. The dominant follicle of Wave 1 regressed and ovulation from the dominant follicle of Wave 2 was detected on Day 18. This heifer developed rectal irritation and follicles were identified but diameters were not determined after Day 12. Heifer D: dominant follicle of Wave 1 regressed. Dominant follicle of Wave 2 reached a diameter of 32 mm with a thick wall (apparent luteinization) and ovulation was not detected by Day 26 (end of observation period). Plasma progesterone concentrations decreased to baseline following treatment and then increased when the follicle developed a thick wall. are g i v e n in Fig. 1. Profiles for m e a n p l a s m a p r o g e s t e r o n e c o n c e n t r a t i o n s a n d results o f statistical analyses are given in Fig. 2. F o u r heifers h a d a p r o l o n g e d ( > 10 d a y s ) interval f r o m day o f t r e a t m e n t to d a y o f p o s t t r e a t m e n t o v u l a t i o n ; d i a m e t e r s o f d o m i n a n t follicles a n d p l a s m a p r o g e s t e r o n e c o n c e n t r a t i o n s are s h o w n (Fig. 3). In a n o t h e r heifer ( n o t s h o w n ) , there were t w o d o m i n a n t W a v e - 1 follicles; this heifer was t r e a t e d o n D a y 5 a n d b o t h follicles o v u l a t e d o n D a y 10.
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J.P. KASTELIC ET AL.
DISCUSSION
There were no significant effects of breeding status on any end point. Therefore, the hypothesis that the dominant follicle of Wave 1 is altered when a conceptus is present was not supported. The diameter of the dominant follicle of Wave 1 was not different between bred and nonbred heifers on Day 8, and the previously reported tendency for a lower plateau in the follicle profile of pregnant heifers was not confirmed. It cannot be stated unequivocally that the bred heifers in the present experiment were pregnant on the day of PGF treatment because luteolysis was induced before reliable ultrasonic pregnancy diagnosis could be made (Kastelic et al., 1989 ). However, in contemporary studies, pregnancy was confirmed on Day 24 in 5 of 6 (83%) heifers from the same herd and bred with the same semen by the same technician. In all heifers treated with PGF on Day 5, the dominant follicle of Wave 1 had not yet reached its static phase, and in all heifers treated on Day 12, Wave 2 was detected before PGF was given. For both days of treatment, therefore, a dominant follicle was in its growing phase on the day of treatment. It is concluded that, for both waves, the dominant follicle became the ovulatory follicle when luteolysis was induced during the growing phase of the dominant follicle. This result is consistent with the report (G.inther et al., 1989b) that the viable dominant follicle present at the onset of spontaneous luteolysis became the ovulatory follicle for both two-wave and three-wave estrous cycles. The rhythmic emergence of follicular waves during early pregnancy (Ginther et al., 1989c) is consistent, therefore, with the absence of luteolysis. After induced luteolysis, ovulation occurred from only the dominant follicle of a wave, indicating that subordinate follicles lost their capability for growing and ovulating. In previous studies (Sirois and Fortune, 1988; Ginther et al., 1989a; Knopf et al., 1989 ), subordinate follicles ceased growing in an average of 3 days after detection of a wave (follicle diameter at wave detection, 4-5 mm). Wave-2 follicles did not reach a detectable size (diameter, 4-5 m m ) in any of the Day-8 heifers with rapid and complete luteal regression. In a previous study, the average day of detection of Wave 2 was Day 9.8 (range, days 8-11; Ginther et al., 1989a). In the Day-8 group, the dominant follicle of Wave 1 was its static phase on the day of treatment, but was viable as indicated by its ovulatory capability. The diameter of the dominant follicle of Wave 1 increased an average of 2.2 m m after treatment, even though it was in its static phase and had apparently reached m a x i m u m diameter. These results indicate that the Day-8 static-phase dominant follicle of Wave 1 in these heifers was not only capable of ovulating, but was also capable of further growth and that a stimulus for growth was present immediately after luteolysis. In this regard, Schallenberger et al. (1984) showed an increased frequency of FSH pulses after luteolysis.
EFFECT OF PROSTAGLANDIN TREATMENT DAY ON OVULATORY FOLLICLE
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At the time of PGF treatment, the dominant follicle of Wave 1 was in its late growing and early static phase in heifers treated on Days 5 and 8, respectively, and the dominant follicle of Wave 2 was in its early growing phase in heifers treated on Day 12. Therefore, at the time of treatment, the diameter of the ovulatory follicle was smallest in Day-12 heifers, intermediate in Day5 heifers, and largest in Day-8 heifers. In all three treatment groups, the diameter of the ovulatory follicle increased significantly from the day of treatment to the day prior to posttreatment ovulation. This increase was significantly greater in the heifers treated in Day 12 than in heifers treated on Day 5 or 8, consistent with the diameter of the ovulatory follicle on the day of treatment. The dominant follicle of Wave 2 (treatment on Day 12 ) was an average of 2.0 mm smaller (not significant) on the day of treatment and had a 2.4-mm greater increase in diameter after treatment (significant) than the dominant follicle of Wave 1 (treatment on Day 5 ). The diameter of the ovulatory follicle on the day prior to posttreatment ovulation, however, was not significantly different between these two groups. Therefore, the interval from treatment to posttreatment ovulation was longer for heifers treated on Day 12 than for heifers treated on Day 5 (difference, 1.5 days). The longer intervals from treatment to ovulation in the Day-12 group than in the Day-5 group were consistent with the results of previous studies (King et al., 1982; Stevenson et al., 1984) and with the proposal (Scaramuzzi et al., 1980; Macmillan and Henderson, 1984) that the size of the largest nonatretic follicle at the time of treatment is a determining factor in the time required for a follicle to reach ovulatory size. Several reports (King, et al., 1982; Macmillan and Henderson, 1984; Stevenson, et al., 1984 ) have concluded that the interval from PGF treatment to luteolysis is similar when PGF is given to cattle with a mature corpus luteum, regardless of day of treatment. Results of the present study are consistent with these reports in regard to the uniformity of luteolysis for the various days of treatment, as indicated by the profiles of plasma progesterone concentration. In Heifer A and another heifer treated on Day 5, two dominant follicles developed and ovulated. The incidence of twin ovulations in this experiment was consistent with the reported incidence of twin ovulations in Holstein cattle ( 5-13%; Kidder et al., 1952; Labhsetwar et al., 1963 ). Our working hypothesis is that an emerging dominant follicle initiates or is an important factor in the regression of its subordinates and the previous dominant follicle (Ginther et al., 1989c). In Heifer A, development and ovulation of the Wave-2 follicles was not accompanied by a decrease in diameter of the dominant follicle of Wave 1. It is not clear why the dominant follicle failed to cause regression of the previous dominant follicle in this heifer. In Heifers A, B, and D, at least one dominant follicle reached preovulatory diameter after luteolysis, but ovulation was not detected. Prolonged intervals
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( > 8 days) of low peripheral progesterone concentrations following treatment with a PGF analogue have been reported previously (Jackson et al., 1979; Baishya et al., 1980). However, ovarian structures were not monitored in these studies. Impairment of positive feedback of estradiol on LH release has been suggested as the cause of anovulatory (cystic) ovarian follicles in postpartum cows (Kesler et al., 1979) and may have been the cause of ovulation failure in the present experiments. In Heifer A, the impairment was apparently short-lived, as ovulation occurred from two dominant follicles of Wave 2. In Heifers B and D, ovulation was not detected during the study period; perhaps a preovulatory LH surge was suppressed by the increased plasma progesterone concentrations associated with follicle luteinization. In Heifer D but not in Heifer B, there was emergence of other follicles in the presence of large, anovulatory follicles. In a previous study (Kesler et al., 1980 ) anovulatory follicles regressed and were replaced by other anovulatory follicles in 6 of 8 cystic cows. In Heifer C, plasma progesterone concentrations declined following PGF treatment, but remained above baseline for approximately 1 week. In this heifer, the interval from treatment to posttreatment ovulation was prolonged, but the viable dominant follicle present at the time of luteolysis became the ovulatory follicle. In conclusion, growth of the dominant follicle of Wave 1 and response to PGF treatment was not different between bred and nonbred heifers. In heifers with rapid and complete luteal regression, ovulation occurred from the dominant follicle of Wave 1 in all heifers treated on Days 5 and 8 and from the dominant follicle of Wave 2 in all heifers treated on Day 12. In all groups, the ovulatory follicle increased in diameter from the time of treatment to the day before ovulation. Therefore, the viable dominant follicle present at the time of luteolysis increased in diameter and became the ovulatory follicle. ACKNOWLEDGEMENTS
Supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison and USDA Grant 87-CRSR-2-3158, and by an NIH Biomedical Research Support Grant to the School of Veterinary Medicine and Department of Veterinary Science, University of Wisconsin-Madison. We thank Lisa Kulick for graphic and computer assistance, Inge Redl for technical assistance, Sophie Yap for statistical assistance, Sharon Schade and Maria Westphal for manuscript preparation, and Dr. E. Schallenberger for performing the progesterone assays. Lutalyse and bull semen were provided by The Upjohn Company, Kalamazoo, MI and American Breeders Service, De Forest, WI, respectively. J.K. is supported by a fellowship from the Medical Research Council of Canada. L.K. is Research Fellow of the Deutsche Forschungsgemeinschaft and the
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H.W. Schaumann Stiftung and is on leave from Department of Physiology, Technical University Munich, Weihenstepan, Federal Republic of Germany.
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