Selection of the Dominant Follicle in Cattle: Role of Estradiol1

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samples were taken every 4 h from the caudal vena cava cranial to the junction with the ovarian veins in heifers with the largest follicle intact (controls) or ablated ...
BIOLOGY OF REPRODUCTION 63, 383–389 (2000)

Selection of the Dominant Follicle in Cattle: Role of Estradiol1 O.J. Ginther,2 D.R. Bergfelt, L.J. Kulick, and K. Kot Department of Animal Health and Biomedical Science, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT

in diameter between the two largest follicles at the beginning of deviation apparently allows adequate time (,8 h) for the largest follicle to establish dominance before the next largest follicle reaches a similar diameter. This establishment of dominance at the beginning of deviation involves inhibition of the smaller follicles, considering that all growing follicles of the wave are capable of dominance until such inhibition is complete [4, 7–9]. It has been postulated that the essence of follicular deviation is a close, two-way functional coupling between changing FSH concentrations and follicular growth and development, leading to inhibition of the smaller follicles through FSH deprivation [5]. Emergence of a follicular wave is stimulated by an FSH surge that reaches its peak when the largest follicle of the wave is approximately 4.0 mm [6]. By the time the follicles reach 5.0 mm, they develop an FSH-suppressing ability [10], and all the growing follicles of 5.0 mm or larger contribute to the decline in the FSH surge [7]. Despite the negative effect of these follicles on FSH, the declining FSH concentrations continue to exert a required, positive effect on the follicles [5]. The continuing dependence of the follicles on FSH sets the stage for follicular deviation. When the largest follicle reaches a mean diameter of approximately 8.5 mm (the expected beginning of deviation), it plays a major role in the continued decline of FSH concentrations [5]. Deviation begins when the FSH concentrations are temporarily maintained at less than the concentrations required by the smaller follicles. Deviation through FSH deprivation can occur in less than 8 h [4], satisfying the assumption that the process must be established in a time equivalent to the difference in diameter between the two largest follicles. This conclusion is supported by the findings of increased circulating FSH concentrations beginning 5 h after ablation of the largest follicle at the expected beginning of deviation and reduced diameter of follicles within 6 h after experimental suppression of FSH concentrations [4]. Based on blood sampling at hourly intervals, FSH concentrations decreased for 10 h [4] or 16 h [5] after the largest follicle reached the expected beginning of deviation at 8.5 mm or larger. The low FSH concentrations at deviation, although inadequate for the smaller follicles, are a requirement for continued growth and development of the largest follicle, as indicated by inhibited growth of the largest follicle when FSH was experimentally depressed to less than the control concentrations [4]. In addition, the dominant follicle also apparently begins to utilize LH for at least part of its gonadotropin stimulation, beginning at an unknown point in relation to the beginning of deviation [4, 6, 11]. Follicular factors likely are responsible for the depressed FSH concentrations during the declining portion of the FSH surge that stimulated wave emergence [5] and for those during deviation [4, 5]. The identity of such factors has not been demonstrated, but estradiol and inhibin are candidates [6]. In a recent study [10], 5-mm follicles were temporally associated with suppressed FSH but not with increased circulating estradiol levels. Estradiol has not been demonstrat-

Involvement of estradiol in the deviation in growth rates between the two largest follicles of a wave was studied in 39 heifers. In experiment 1, the largest follicle remained intact in a control group and was ablated in five estradiol-treated groups when the largest follicle reached 8.5 mm or larger (expected beginning of deviation; Hour 0). The ablation groups were given a single injection of 0, 0.004, 0.02, 0.1, or 0.5 mg of estradiol. Blood samples were taken from a jugular vein every hour at Hours 0 to 16. By Hour 8, FSH concentrations were greater (P , 0.05) in the ablation group that received 0 mg of estradiol than in the controls. Among the estradiol groups, that receiving 0.02 mg had the lowest detectable increase in estradiol. In this group, FSH concentrations were not suppressed below the control concentrations, but the increase in FSH concentrations following ablation of the largest follicle was delayed for 2 or 3 h. This delay in the increase of FSH concentrations corresponded to the hours that estradiol was maximal. In experiment 2, blood samples were taken every 4 h from the caudal vena cava cranial to the junction with the ovarian veins in heifers with the largest follicle intact (controls) or ablated at 8.5 mm or larger (Hour 0). Averaged over Hours 4 to 48, estradiol concentrations were higher (P , 0.04) in the controls than in the ablation group. During Hours 0 to 12, estradiol concentrations increased (P , 0.05) in the controls, whereas FSH concentrations decreased (P , 0.05). In the ablation group, estradiol concentrations were lower than in the controls by Hour 4, and FSH concentrations increased (P , 0.05) between Hours 4 and 12. These results support the hypothesis that the largest follicle releases increased estradiol into the blood at the beginning of follicular deviation, and that the released estradiol is involved in the continuing depression of FSH concentrations to below the requirement of the smaller follicles.

estradiol, FSH, follicle, follicular development

INTRODUCTION

In cattle, on average, the largest follicle of a follicular wave emerges at 3 or 4 mm and 6 h [1] or 7 h [2] before the second-largest follicle. The two largest follicles grow at a similar rate for a few days and begin to deviate in their growth rates when the largest follicle reaches a mean of approximately 8.5 mm, as indicated by several studies that used an 8-h interval between examinations [2–5]. At deviation, the largest follicle continues to grow and becomes the dominant follicle, but the smaller follicles begin to regress and become subordinate follicles [6]. The difference Supported by the University of Wisconsin, Madison; U.S. Department of Agriculture grant 99-35203-7669; and by Equiservices Publishing and the Eutherian Foundation, Cross Plains, WI. 2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Science, 1656 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; e-mail: [email protected] 1

Received: 7 December 1999. First decision: 25 February 2000. Accepted: 8 March 2000. Q 2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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ed to be involved in the initial declining portion of the FSH surge, but temporal considerations indicate that it may be at least one of the follicular factors involved in the FSH suppression associated with deviation [6, 12]. Estradiol could also act as a local facilitator for a change from FSH to LH responsiveness by the largest follicle [6, 11]. Regarding events other than deviation, follicular-fluid estradiol concentrations in the largest follicle of a wave were elevated when its mean diameter was 8 to 9 mm [13–16]. Concentrations of estradiol in systemic venous blood or the vein ipsilateral to the largest follicle began to increase 3 days after the LH surge [17] and 2 to 2.5 days after ovulation [13, 18] or emergence of follicles at 4 mm [2]. These detected increases in estradiol concentrations apparently were close to the expected beginning of deviation based on the average time of deviation reported in other studies [2–4, 19]. In studies using deviation as a reference point, systemic estradiol [2] and intrafollicular-fluid estradiol levels of the largest follicle [19] began to increase at the beginning of deviation. That is, an increased difference between the two largest follicles in estradiol concentrations was not detected until the day of an increased difference in diameter. Thus, results of studies concerning the temporal relationships between changes in systemic and follicular-fluid estradiol concentrations and the time of expected or demonstrated occurrence of deviation indicate that the two events begin at approximately the same time in cattle. The temporal relationships are compatible with the concept that estradiol plays a role in the deviation mechanism, but other approaches are needed to demonstrate functional involvement. The present experiments tested the hypothesis that increased estradiol is released into the blood by the largest follicle at the beginning of follicular deviation, and that this released estradiol causes or contributes to continuing suppression of circulating FSH concentrations to less than the requirements of the smaller follicles. Support for this hypothesis depends on the following: 1) the ability of exogenous estradiol to delay the increase in FSH concentrations occurring after ablation of the largest follicle at the expected beginning of deviation (experiment 1) and 2) an increased output of estradiol in association with decreasing concentrations of FSH at the expected beginning of deviation, when the largest follicle is intact but not when ablated (experiment 2). MATERIALS AND METHODS

Animals and Ultrasound Scanning

Experiments were conducted during the wave that emerges in the periovulatory period (wave 1). Animals were Holstein heifers between 24 and 36 mo of age. The feeding program and protocol for inducing luteolysis to schedule ovulation have been described elsewhere [4]. An ultrasound scanner (Aloka SSD-500V Micrus; Aloka Co., Wallingford, CT) equipped with a 7-5 MHz, linear-array intrarectal transducer was used to monitor the ovaries [20]. This scanner measures in increments of 0.1 mm, and measurements were made as described elsewhere [4, 20]. Scanning was performed every 24 h beginning on the day of induced luteolysis and continued until the largest follicle of a new wave reached a diameter of 6.0 mm or larger (experiment 2) or 7.0 mm (experiment 1). Thereafter, scanning was performed every 8 h until the largest follicle of wave 1 reached 8.5 mm or larger (expected beginning of deviation; Hour 0). Ablation of the largest follicle was done at

Hour 0 by ultrasound-guided transvaginal aspiration of the follicular contents as described elsewhere [4, 20]. Experiment 1

Heifers were randomized by replicate into six groups (n 5 5/group). The five largest follicles at Hour 0 were defined as follicles 1, 2, 3, 4, and 5 according to descending diameter to retrospectively identify whether follicles 1, 2, 3, 4, or 5 became dominant. When follicle 1 reached 8.5 mm or larger (Hour 0), it remained intact in a control group and was ablated in the remaining five groups. Each heifer in the five ablation groups was given either 0 (safflower-oil vehicle), 0.004, 0.02, 0.1, or 0.5 mg of estradiol-17b at Hour 0 (just after ablation). The groups were designated as control and 0-, 0.004-, 0.02-, 0.1-, and 0.5-mg groups. Estradiol was prepared by dissolving 50 mg of estradiol-17b in 20 ml of benzyl alcohol and then adding safflower oil to make a total volume of 100 ml. This initial preparation served as the highest treatment dose, from which lower treatment doses were made by taking 10, 2, and 0.4 ml and adding safflower oil to make a total volume of 50 ml. Concentrations were calculated so that each heifer of the respective treatment groups received a single injection of 1 ml of safflower oil containing the appropriate dose of estradiol. The single injection was given i.m. in the gluteal area. Color-coded vials were used so that the scanner operator was unaware of the dose. After Hour 0, ultrasound scanning of the follicles was performed every 8 h until Hour 24, after which daily measurements of the dominant follicle were made until the second day of wave 2. Follicular end points were identification of the follicle that became dominant; maximum diameter of the follicle that attained the largest diameter over Hours 28 to 168 regardless of its identity as follicle 1, 2, 3, 4, or 5; and day of emergence of wave 2. Blood samples were collected every hour from Hours 0 to 16 through an indwelling catheter placed into a jugular vein [4] when the largest follicle reached 8.0 mm or larger. Hormonal end points were plasma concentrations of estradiol and FSH over Hours 0 to 16. Experiment 2

Heifers were randomized into two groups when the largest follicle reached 8.5 mm or larger (Hour 0). The groups were a control group (n 5 7) and an ablation group with the largest follicle being ablated at Hour 0 (n 5 7). Ultrasound scanning was done at 8-h intervals beginning when the largest follicle reached 6.0 mm or larger and ending at Hour 0. When the largest follicle reached 6.0 to 6.9 mm, a catheter was placed into the caudal vena cava as described elsewhere [12, 18]. Heifers were sedated with detomidine hydrochloride (Dormosedan, 0.02 mg/kg i.v.; Pfizer Animal Health, West Chester, PA). Local anesthesia was induced with an s.c. injection of 2% lidocaine hydrochloride in the cannulation area of the medial saphenous vein on the inner aspect of the left or right thigh, approximately 23 cm below the inguinal region. The catheter was a 140-cm-length of Teflon tubing (TFT 15 oc; inside diameter, 1.4986 mm; outside diameter, 2.1082 mm; Atlantic Tubing Co., Chestnut Ridge, NY). A thin-wall, 12-gauge needle was used to puncture the vein. Approximately 95 cm of tubing was inserted (length based on results of preliminary trials). A metal intraluminal guide was not used, although procedures reported elsewhere have apparently required a guide [18].

FOLLICULAR DEVIATION AND ESTRADIOL

Transrectal ultrasonography was used to position the catheter tip in the vena cava near the cranial pole of the left kidney. After removing the 12-gauge needle, the catheter was secured in-place at the external entrance by a simple suture on either side of the cannula and protected with an elastic/adhesive bandage. The catheter tip was fitted with a 16-gauge blunt needle, and the catheter was filled with a heparin/saline solution and capped. Blood samples were taken from the caudal vena cava by withdrawing 3 ml of fluid from the indwelling catheter for discard and then 20 ml for the experimental sample. At the end of each sampling, the catheter was filled with 3 ml of a heparin/saline solution and capped. Blood samples (20 ml) were collected from the indwelling catheter every 4 h from the time the largest follicle reached 7.0 mm or larger to Hour 48. The end points were plasma concentrations of estradiol and FSH. Hormone Assays

Blood samples were placed into heparinized tubes and refrigerated (48C) until centrifugation. Within 24 h of collection, plasma was separated, decanted into storage vials, and frozen (2208C) until assay. Plasma concentrations of FSH were determined using validated radioimmunoassays for cattle [21]. Details on the methodology as used in this laboratory have been reported elsewhere [22]. Hormone sensitivity, which was calculated as 2 standard deviations below the mean counts per minute at maximum binding, was 0.20 ng/ml for experiment 1 and 0.36 ng/ml for experiment 2. Coefficients of variation for within and between assays for experiment 1 were 5.2% and 9.3%, respectively; the coefficient of variation for within assay of experiment 2 was 6.8%. Determinations were made from pooled bovine plasma collected during diestrus. Plasma concentrations of estradiol were determined using modifications of a commercially available radioimmunoassay kit (Second Antibody Estradiol; Diagnostic Products Corp., Los Angeles, CA), which has been validated for use in cattle [23]. Details of the methodology as used in this laboratory have been reported elsewhere [5]. Triplicate aliquots of 500 ml of bovine plasma were extracted with 3 ml of diethyl ether, frozen in a dry-ice/methanol bath, and decanted into assay tubes. After drying, both samples and standards were resuspended with 100 ml of assay buffer. Recovery of radiolabeled estradiol added to plasma from ovariectomized heifers was 75.3 6 0.3%, and the slopes of plasma pools collected during estrus and diestrus (200 to 600 ml before extraction) were similar to the slope of the estradiol standard. Plasma concentrations of estradiol were expressed relative to the standard curve, and no correction was made for recovery. Assay sensitivity was 1.40 pg/ml for experiment 1 and 1.45 pg/ml for experiment 2, as determined by 2 standard deviations below the mean counts per minute at maximum binding. Coefficients of variation for within and between assays for estradiol were 11.4% and 16.8%, respectively, for experiment 1 and 14.1% and 14.3%, respectively, for experiment 2. Determinations were made from pooled bovine plasma collected during diestrus. Statistical Analyses

Concentrations of FSH in experiment 1 and of estradiol in experiment 2 were converted to percentage changes from Hour 0. Percentages were used because of a disparity among group means at Hour 0. It was thought that the disparity, although not significant at Hour 0, could contrib-

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ute to significant differences at other hours. For percentage data, Hour 0 was not included in the statistical analyses because of the absence of variation. For sequential data, split-plot ANOVA was used for determining main effects of group and hour as well as the interaction of group by hour. Variation from sequential data was accounted for by using heifer as the error term to test the effect of group. If a significant effect of hour or an interaction of group by hour was indicated, Duncan’s multiple range tests or unpaired Student’s t-tests were used to locate mean differences among groups within hours, and paired Student’s t-tests were used between hours within a group. In experiment 1, a significant group-by-hour interaction for hormones was further examined by comparing the 0-dose ablation group with each of the other five groups in separate ANOVAs. In experiment 2, the pretreatment hormonal changes (before Hour 0) were analyzed by ANOVA. Data are presented as the mean 6 SEM unless otherwise indicated. Significance was indicated by a probability of P , 0.05, and a probability that approached significance was defined as P , 0.1 to P , 0.06. RESULTS

Experiment 1

The main effects (group and hour) and their interaction for estradiol concentrations were significant (Fig. 1). When analyzed separately, no group effect or interaction between controls and the 0-mg group or between controls and the 0.004-mg group was found. Progressively higher concentrations were present at the first postinjection sampling (Hour 1) for the 0.02-, 0.1-, and 0.5-mg groups. Regarding FSH concentrations, the hour effect and the group-by-hour interaction for percentage changes from Hour 0 were significant (Fig. 1). When each group was compared separately with the 0-mg ablation group, a groupby-hour interaction (P , 0.001) was found for the comparison with controls and the 0.02-, 0.1-, and 0.5-mg groups; however, the interaction was not significant for controls versus the 0.004-mg group. Concentrations were greater (P , 0.05) in the 0-mg group than in the control group by Hour 8. Compared with the 0-mg group, concentrations were lower (P , 0.05) at Hour 8 in the 0.02-mg group, at Hours 4 to 8 in the 0.1-mg group, and at Hours 4 to 13 in the 0.5-mg group. Concentrations were higher (P , 0.05) in the 0.1-mg group than in the 0-mg group at Hours 14 to 16. Compared with controls, concentrations were lower (P , 0.05) at Hours 2 to 5 in the 0.1-mg group and at Hours 4 to 8 in the 0.5-mg group. Within the control group, the concentrations decreased (P , 0.05) between Hours 6 and 10. Follicle 1 became a dominant follicle in five of five control heifers. In the 25 heifers in the five groups with ablation of follicle 1, follicle 2 became dominant in 60% of heifers; follicles 3, 4, or 5 became dominant in 24%; and a dominant follicle larger than 10 mm did not develop during wave 1 in 16% (4 heifers). The four heifers that did not develop a dominant follicle during wave 1 were in the 0.1and 0.5-mg groups and were included in the analyses of follicular diameters and hormonal concentrations. The maximum diameter of the largest follicle of wave 1 differed among the six groups (P , 0.05), primarily because a dominant follicle (.10 mm) did not develop in two heifers in each of the 0.1- and 0.5-mg groups. The mean maximum diameters (mm) were as follows: controls, 16.6 6 0.8; 0mg group, 16.2 6 0.8; 0.004-mg group, 15.1 6 1.0; 0.02-

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FIG. 1. Experiment 1: circulating concentrations (mean 6 SEM, n 5 5 heifers/group) of estradiol and percentage changes in FSH concentration in controls (follicle intact, no estradiol) and follicle-ablated heifers given a single injection of estradiol (E2). Ablation of the largest follicle and injection of estradiol were done when the largest follicle reached 8.5 mm or larger (Hour 0). Main effects (group, hour; P , 0.006) and an interaction (P , 0.0001) were observed for each hormone. The lowest dose of E2 that produced an increase in estradiol concentration (Hours 1 and 2; P , 0.05) was 0.02 mg. At Hour 8, percentage changes in FSH concentrations were greater (P , 0.05) in the 0-mg ablation group than in controls of the 0.02-, 0.1-, and 0.5-mg groups.

mg group, 15.7 6 0.4; 0.1-mg group, 11.8 6 2.0; and 0.5mg group, 12.7 6 1.8. Wave 2 emerged (4-mm follicle) 2 or 3 days after Hour 0 in the four heifers with no dominant follicle. In comparison, the mean number of days to emergence of wave 2 in the remaining heifers was 7.1 6 0.4. Experiment 2

The percentage changes in estradiol concentrations were not significant during Hours 216 to 24 (Fig. 2). The ANOVA for Hours 4 to 48 indicated significant main effects of group and hour but no interaction. The group effect resulted from lower concentrations in the ablation group than in controls. The higher concentrations in the control group were characterized by three surges in the mean concentrations, with peaks at Hours 12, 28, and 48. Pretreatment FSH mean concentrations did not differ over Hours 216 to 0 according to ANOVA (Fig. 2), but based on paired Student’s t-tests, these concentrations decreased (P , 0.05) from the beginning to the end of the pretreatment period. The interaction of group and hour was significant for Hours 0 to 48. The means were different (P , 0.05 to P , 0.004) between groups for each of Hours 4 to 48. In controls, the FSH concentrations decreased (P ,

FIG. 2. Experiment 2: mean 6 SEM percentage changes from Hour 0 in concentrations of estradiol and actual concentrations of FSH among seven heifers with an intact follicle (controls) and seven heifers with the largest follicle ablated when it reached 8.5 mm or larger (Hour 0). For estradiol at Hours 4 to 48, the effect of group was significant (P , 0.04). For FSH at Hours 0 to 48, an effect of group (P , 0.004) and a group-by-hour interaction (P , 0.008) were observed. Concentrations of FSH were lower (P , 0.05) in the control group than in the ablation group at each of Hours 8 to 48. A star indicates a subsequent increase or decrease (P , 0.05) in concentrations, and a pound mark indicates a subsequent increase or decrease that approached significance (P . 0.1 to P , 0.06).

0.05) between Hours 0 and 16. Thereafter, the mean concentrations progressively increased between Hours 24 and 48, although not significantly. In the ablation group, the concentrations increased (P , 0.05) between Hours 4 and 8 and, thereafter, did not change significantly. DISCUSSION

An earlier study [24] demonstrated increased FSH concentrations 24 h after surgical cauterization of the largest follicle 3 or 5 days after ovulation. In the present experiments, transvaginal ultrasound-guided aspiration of follicular contents was used. The effectiveness of the functional ablation of follicles by this technique has been discussed elsewhere [4]. Ablation of the largest follicle when it first reached 8.5 mm or larger (Hour 0) produced increased FSH concentrations after Hour 4 (sampling every 4 h) and Hour 5 (sampling every 1 h) in experiments 2 and 1, respectively. The concentrations continued to increase until Hours 10 or 12. These results are similar to those of previous studies [4, 5]. The postablation increase in FSH concentrations was utilized to test the hypothesis that estradiol is involved in the FSH suppression associated with follicular deviation. Experiment 1 examined the ability of exogenous estradiol to prevent the postablation increase in FSH concentrations.

FOLLICULAR DEVIATION AND ESTRADIOL

Experiment 2 examined the temporal relationships at the expected time of deviation between an increase in estradiol and a decrease in FSH concentrations when the largest follicle was retained and a decrease in estradiol and an increase in FSH concentrations when the follicle was ablated. The present experiments were based on reports that the diameter deviation between the two largest follicles begins, on average, when the largest follicle first reaches 8.5 mm or larger [2–4, 19]. In the reported studies, the examination at the beginning of deviation in individual waves was identified as the examination before a change occurred in the differences in diameter between the two largest follicles [1]. Therefore, diameter deviation could have begun at any time between the identified examination and the next examination. When the largest follicle reached 8.5 mm or larger, the actual diameters of the two largest follicles were 8.7 6 0.04 mm and 7.6 6 0.1 mm in experiment 1 and 8.8 6 0.1 mm and 7.6 6 0.2 mm in experiment 2. The difference in diameter between the means for the two largest follicles at the expected time of deviation was close to the reported mean differences in diameter of 0.9 mm [3] and 1.3 mm [19] when the beginning of deviation was assessed by inspection of follicle profiles. Thus, on average, the criterion of 8.5 mm or larger for the expected beginning of deviation was appropriate for these experiments. A change in circulating concentrations of endogenous estradiol in the systemic circulation (jugular vein) in experiment 1 during Hours 0 to 16 was not detected in the control group or the 0-mg ablation group, either in the overall analysis or when analyzed separately. Failure to detect changes in endogenous systemic estradiol may have resulted from low assay sensitivity (sensitivity, 1.40 ng/ml; mean concentration at Hour 0, 1.49 ng/ml). A change in estradiol concentrations was detected under similar conditions in the samples from the caudal vena cava in experiment 2. Before Hour 0, the concentrations of FSH appeared to decrease progressively in experiment 2 and reached minimal concentrations in controls by Hour 14 (experiment 1) or Hour 20 (experiment 2). In experiment 2, the concentrations increased progressively, although not significantly, between Hours 20 and 48. These results agree with those of a previous study [4] in which the mean FSH concentrations decreased progressively over Hours 216, 28, and 0; continued to decrease in hourly samples until Hour 10; and then increased in 8-h samples between Hours 16 and 48. The results of both the present and previous studies indicate that, on average, the FSH nadir is reached less than 24 h after the expected beginning of follicle deviation and, thereafter, gradually increases. In experiment 1, a detectable mean increase in circulating estradiol concentrations occurred in all estradiol-treated groups, except the 0- and 0.004-mg groups. After a single i.m. injection of 0.02, 0.1, or 0.5 mg of estradiol, the mean concentrations had already reached maximum by Hour 1 and progressively declined to control concentrations over Hours 1 to 12, except that concentrations had not reached baseline by the end of blood sampling (Hour 16) in the 0.5mg group. No difference in FSH concentrations was detected between the follicle-ablation groups receiving 0.004 mg versus 0 mg of estradiol. However, the lowest dose of estradiol (0.02 mg) associated with a detectable change in circulating estradiol concentrations was also associated with a reduction in FSH concentrations compared with the 0-mg group. Thus, the increased FSH concentrations that followed ablation of the largest follicle were delayed for 2 or 3 h by a

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single 0.02-mg dose of estradiol. The delay corresponded to the number of hours (Hours 1 and 2) that estradiol was at its highest mean concentrations. The 0.02-mg dose did not reduce the FSH concentrations to less than control concentrations at any hour. These results supported the hypothesis that estradiol is involved in follicle deviation, as indicated by the ability of a minimal, single injection of estradiol to delay the increase in FSH concentrations that occurs after ablation of the largest follicle at the expected beginning of deviation. In experiment 2, samples of venous blood were collected from the caudal vena cava cranial to the entry of the ovarian veins. This approach was used because of the absence of detected changes in systemic blood during experiment 1 and a report that samples collected from the vena cava had a mean concentration of ovarian hormones 6.8-fold greater than that in samples collected from the jugular vein [18]. In the present experiments, the mean estradiol concentration at Hour 0 was 1.49 6 0.47 pg/ml for experiment 1 (jugular samples) and 7.30 6 1.38 pg/ml, or 4.9-fold greater, for experiment 2 (vena cava samples). Concentrations of estradiol in the vena cava of the follicle-intact group began to increase at the expected beginning of deviation. This agrees with results from previous studies of changes in systemic [2] and follicular-fluid [19] estradiol concentrations using the beginning of deviation as a reference point. The results of many other studies are also compatible, although other events were used as a reference point. The estradiol increase in controls occurred at the time FSH concentrations were continuing a gradual decline after the expected hour of the beginning of deviation. Concentrations of estradiol remained high, and concentrations of FSH low, until the end of the experiment at Hour 48. When the largest follicle was ablated at 8.5 mm or later, concentrations of estradiol were lower at the first postablation sample (Hour 4) than in controls. This result indicates that the largest follicle and not the others was the primary, if not the only, source of venous estradiol at the expected beginning of deviation. Furthermore, a relationship between estradiol and FSH concentrations was indicated in the ablation group by the beginning of an increase in FSH concentration at Hour 4 in association with the low estradiol concentrations. Apparently, the close two-way functional coupling between developing follicles and FSH at this time [5] involves additional FSH suppression from the release of follicular estradiol into the circulation, at least in part. The low FSH concentrations cause the demise of the smaller follicles, whereas the largest follicle is able to continue utilizing the low FSH concentrations [5]. The high doses of estradiol (0.1 and 0.5 mg) in experiment 1 delayed the postablation increase in FSH concentrations for progressively longer intervals according to dose. Unlike the 0.02-mg dose, the high doses decreased the FSH concentrations to less than those in controls over Hours 4 and 5 (0.1-mg group) and 4 to 8 (0.5-mg group). In the 0.1-mg group, FSH concentrations rebounded to concentrations greater than those in the 0-mg group at Hours 14 to 16. In the 0.5-mg group, the experiment ended (Hour 16) before the extent of the rebound could be assessed. The high doses of estradiol and the prolonged depression in FSH concentrations were associated with follicle inhibition, as indicated by 1) the reduced maximum diameter of the follicle attaining the largest diameter over Hours 48 to 168, 2) the failure of any follicle of wave 1 to become dominant in 40% of the heifers, and 3) the shorter interval to emergence of wave 2 in the heifers that did not develop a dom-

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inant follicle during wave 1. These three responses to the 0.1- and 0.5-mg doses reflect the lack of development of a dominant follicle in some of the heifers. In those that developed a dominant follicle, these end points were not different from those in the 0-mg group. The follicular effect with the 0.1- and 0.5-mg doses is attributable to the prolonged depression in FSH concentrations to less than those in controls. The possibilities that the follicular effect was exerted through altered LH concentrations or directly between estradiol and the follicles were not studied in these experiments. Concentrations of LH are involved in growth of the dominant follicle [6, 11], but when this effect occurs in relation to the beginning of deviation in cattle is not known. In a recent study [5], the 0.1-mg dose of estradiol inhibited both FSH and the largest follicle without a detected effect on LH. Those authors concluded that the low FSH concentrations after the beginning of deviation are utilized by the dominant follicle for continued growth. In another study [5], concentrations of LH were not altered significantly during the increase in FSH concentrations after ablation of the largest follicle at 8.5 mm or larger. In another recent study of mares [25], LH concentrations were reduced by progesterone treatment 2 days before follicular deviation. These depressed LH concentrations did not alter the diameter of the second largest follicle, suggesting that the time of onset of follicle deviation was not altered. The growth of the largest follicle was not reduced significantly until approximately 1 day after deviation had begun in controls. To our knowledge, similar studies have not been done in cattle, and the role of LH in follicle deviation remains unknown. Estradiol appeared to be released in surges, despite the limitations of sampling at 4-h intervals. However, demonstration of surges was not part of the planned experiment, and this observation should not be accepted unless it can be confirmed in future studies. In addition, mean concentrations seemed to be a poor representation of apparent surges in individuals. Mean increases in estradiol concentrations during the growing phase of the dominant follicle have been reported to be preceded by a release of LH [18]. That report, and the suggestion of surges in experiment 2 of the present study, indicate a need for further examination utilizing more frequent sampling and determination of LH as well as estradiol concentrations during the deviation phenomenon. In conclusion, the increase in FSH concentrations that occurred after ablation of the largest follicle when it reached 8.5 mm or larger (the expected beginning of deviation) was delayed by the lowest dose of a single injection of estradiol that produced a detectable increase in circulating estradiol concentrations. As determined by blood samples taken from the caudal vena cava in controls and follicle-ablated heifers, estradiol was produced by the largest follicle and released into the circulation at the beginning of deviation. The increase in estradiol concentrations among follicle-intact heifers was accompanied by continuing depression of FSH concentrations, and the absence of an increased estradiol concentration in follicle-ablated heifers was accompanied by an immediate increase in FSH concentrations. These results support the hypothesis that estradiol is produced by the largest follicle at the beginning of follicular deviation, and that it is involved in the continuing depression of FSH concentrations to less than the requirements of the smaller follicles. However, an associated role for other follicular substances has not been eliminated.

ACKNOWLEDGMENTS The authors thank the Pharmacia and Upjohn Company for a gift of lutalyse and the U.S. Department of Agriculture’s Animal Health Program for providing antigens and the primary antisera for the gonadotropin assays.

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