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a Departments of Animal Health and Biomedical Sciences, and
b Dairy Science, University of Wisconsin, Madison, Wisconsin 53706
c Department of Animal Science, Federal University of Viçosa, Viçosa, MG 31570-000, Brazil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 mm, whereas the other follicles of the wave (subordinate follicles) regress. The diameter relationships of the two largest follicles have been studied with a two-follicle model, wherein other follicles were ablated to allow precise tracking of the two largest follicles by transrectal ultrasonography [1]. The future dominant follicle emerged at 6 mm a mean of 1 day earlier than the future subordinate follicle, the growth rates for the two follicles between emergence and deviation (6 days later) did not differ, and the future dominant follicle was a mean of 3 mm larger than the future subordinate follicle at the beginning of deviation (23 vs. 20 mm). These results supported the hypothesis that the follicle destined to become dominant has a size advantage and is the first to reach a critical stage at approximately 23 mm. At that time, the selected follicle is involved in a deviation mechanism that inhibits the other follicles before the second-largest follicle reaches a similar diameter an average of 1 day later. Similar results have been reported in cattle, except for the differences between species in follicle diameters [2, 3]. The specific mechanism for continued growth of one follicle and inhibition of the others is not known in either species, although estradiol has been suggested as a potential facilitator of deviation in cattle [2]. On the basis of in vivo sampling of follicular fluid in heifers, estradiol concentrations were not higher in the dominant follicle than in the largest subordinate follicle until the day after the two follicles began to deviate in growth rates [4]. These observations indicate that the future dominant follicle cannot be identified in cattle reliably by estradiol production before the deviation in growth rates between the two largest follicles.
The experiments reported here were designed to evaluate the interrelationships among changing diameters of the larger and smaller follicles, day of deviation, and intrafollicular and circulating estradiol concentrations using the two-follicle model in mares. In order to perform this research, an in vivo method was developed and validated for sampling follicular fluid before deviation in growth rates between the two follicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonlactating cyclic pony mares (n = 37), 4–13 yr and 218–431 kg, were used from October to November of Year 1 (experiment 1) and from May to June of Year 2 (experiment 2). The mares were kept in partially sheltered outdoor paddocks under artificial light (15 h per day) from December to March preceding the studies. They were maintained on alfalfa/grass hay and had access to water and trace-mineralized salt. The ultrasound scanner was equipped with a 5-mHz linear-array transrectal transducer (Tokyo Keiki LS300; Products Group International, Lyons, CO) for examinations of the ovaries and uterus. An ultrasound scanner (Aloka SSD-500V; Aloka, Wallingford, CT) equipped with a 5-mHz convex-array transvaginal transducer (Aloka UST974V–5) was used for follicle ablations and follicular fluid sampling. All mares were determined to be in the ovulatory season by ultrasonic examinations for detection of ovulation and the formation of a corpus luteum [5]. Mares with indications of ovarian or uterine abnormalities were not used. To minimize the potential for obscuring the results, criteria were established for removing mares from the experiments. The criteria are given in the Results section for each experiment.
Before the start of the study, mares were synchronized with prostaglandin-F2
(Lutalyse; Pharmacia & Upjohn Co., Kalamazoo, MI). Follicles 30 mm in diameter were monitored daily until ovulation. The experiments were started on Days 10 and 9 in experiments 1 and 2, respectively. Initially, before the establishment of the two-follicle model, the average of height and width of the antrum at the maximal area from a single image was used for follicle diameter.
Both experiments used the two-follicle model, prepared as described [1]. Briefly, on Days 9 or 10, follicles were ablated by transvaginal aspiration of follicular contents. Follicles 5 mm were ablated in experiment 1, but to simplify the procedure, follicles 8 mm were ablated in experiment 2. Growing follicles ( 4 mm) of the new or post-ablation wave were tracked daily [5]. The relative location of follicles, corpus luteum, and follicle ablation sites (echoic areas) were used as references for identifying and tracking. Subsequent ablation sessions were done for ablating follicles of the new wave when the largest follicle reached 15 mm; all follicles 5 mm (experiment 1) or 8 mm (experiment 2) were ablated except the two largest. Ablation sessions were repeated whenever a new follicle reached 15 mm or an ablated follicle refilled, and this continued until the larger of the two retained follicles reached 25 mm. Subsequent to the Day 9/10 ablation session, an average of 1.2 ± 0.1 ablation sessions for establishing the model were needed per mare. The two retained follicles were monitored daily until ovulation or Day 30. Height and width of the two follicles were taken from three images during a scanning session, and the average of the six measurements per follicle was used as the follicle diameter for each day. The diameter of the corpus luteum was evaluated daily as described [5].
Emergence of a follicle was defined as occurring on the day before the follicle first exceeded 6 mm. The dominant follicle (one that grew to a preovulatory size of 30 mm) and subordinate follicle (one that grew to a moderate size and regressed) were chosen retrospectively according to the maximum attained diameters. The beginning of deviation between the future dominant and subordinate follicles was defined as the day the two follicles began to differ in growth rates [1]. Thus, the beginning of deviation refers to the examination preceding the first change in differences in diameters between the two follicles.
Follicle Ablation and Sampling
Mares were held in a padded squeeze stock to prevent excessive movement. Sedation and analgesia were induced with detomidine hydrochloride (Dormosedan, 0.02–0.04 mg/kg i.v.; Pfizer Animal Health, West Chester, PA) and butorphanol tartrate (Torbugesic, 0.05 mg/kg i.v.; Fort Dodge Laboratories, Fort Dodge, IA). Rectal relaxation was induced with hyoscine N-butyl bromide (Buscopan, 0.2 mg/kg i.v.; Sigma Chemical Co., St. Louis, MO). A tail bandage was applied, and the perineal area was aseptically prepared.
Follicle ablation was done transvaginally by ultrasound-guided follicle entry similar to the procedure described in cattle [6]. The face of the convex transducer was applied to the wall of the vaginal fornix. The ovary containing the targeted follicle was positioned transrectally against the vaginal wall over the transducer face, so that the follicle was transected by the built-in line on the ultrasound monitor representing the projected needle path. The needle path to the follicle was positioned so that it transected the ovarian stroma but not other detected follicles or luteal tissue. For ablation, a 17-gauge needle (outer diameter, 1.5 mm; inner diameter, 1 mm; length, 55 cm) was used to puncture individual follicles. Follicular contents were removed using a vacuum pump (250–300 mm Hg). Follicle ablation was defined as collapse of the antral follicle after evacuation of follicular contents.
The procedure for ultrasound-guided transvaginal sampling of follicular fluid was similar to that used for cattle [4]. The larger and smaller follicles were sampled once per mare. A double-channel needle system (RAM IVF Supply, Madison, WI) was used for the sampling and consisted of a 20-gauge outer needle (outer diameter, 0.91 mm; inner diameter, 0.55 mm; length, 53 cm) and a 25-gauge inner needle (outer diameter, 0.52 mm; inner diameter, 0.24 mm; length, 57 cm). The inner needle was filled with physiological saline, and the syringe was left attached. The inner needle was inserted into the outer needle until the tip was approximately 1 cm from the tip of the outer needle, and the needle set was then inserted into the needle-guide handle of the transvaginal probe. When the ovary and targeted follicle were in position, the needles were advanced by a second operator until the image of the tip of the outer needle became visible on the scanner screen, indicating that the vaginal wall and peritoneum were penetrated. The inner needle was then advanced until the image of the inner needle tip was centered within the targeted follicle. Sampling of follicular fluid was done by the second operator with a 100-µl syringe (Hamilton Co., Reno, NV) preset to the desired sample volume (20 µl). Successful sampling required continuous observation of the needle tip. The needles and probe were withdrawn immediately after sampling to avoid exerting continued pressure on the newly sampled follicle. The 15-µl portion of the 20-µl sample closest to the needle tip was inserted into tapered microtubes and stored at -20°C.
Estradiol Assays
Follicular fluid estradiol-17ß concentrations were evaluated with specific ELISA as previously described [7] and modified for direct use with follicular fluid [8]. Briefly, follicular fluid samples were either diluted 1:100 or 1:200 in assay buffer and analyzed directly with the ELISA. The standard curve was made in assay buffer containing a 1:100 or 1:200 dilution of charcoal-treated equine follicular fluid with estradiol concentrations from 31 to 2000 pg/ml. The intraassay and interassay coefficients of variation were 13.0% and 14.0%, respectively. The sensitivity (3 standard deviations from maximum bound) was equivalent to a concentration of 0.96 pg/ml.
Systemic estradiol-17ß concentrations were measured by a sensitive RIA procedure that we have modified for use with equine plasma. The standard curves were made in charcoal-treated equine plasma and contained estradiol concentrations from 1.25 to 40 pg/ml. All standard curve, quality control, and unknown samples had estradiol extracted from plasma using diethyl ether. Briefly, 500 µl of plasma was combined with 2.5 ml of diethyl ether and vortexed for 2 min. Samples were then frozen in dry ice/methanol, and the ether fraction was poured into a glass assay tube (12 x 75 mm). The extraction procedure was repeated, and ether was evaporated overnight in a ventilated hood. Dried samples were resuspended in 100 µl of assay buffer and were directly evaluated with a commercially available RIA (Ultra-sensitive estradiol DSL-4800; Diagnostic Systems Laboratory, Webster, TX). For the assay, we used 30 µl of antiserum, 50 µl of 125I-estradiol, and 1 ml of precipitating reagent. According to the manufacturer, the assay has low cross-reactivity with estrone (2.4%), estriol (0.65%), estrone-3-sulfate (0.01%), equilin (0.34%), and estradiol-3-sulfate (0.17%). All samples were extracted and evaluated in duplicate in a single assay. The standard curve was extracted in an identical manner to those of unknown samples in order to account for extraction efficiency in the assay. The assay had a sensitivity of 0.31 pg/ml (3 standard deviations from maximum bound), an 80% point of 1.41 pg/ml, a 50% point of 7.39 pg/ml, an intraassay coefficient of variation of 6.6% (calculated from quality controls), and a mean intraassay coefficient of variation for samples of 14.3% (calculated from duplicate extractions of samples).
Experiment 1. Follicular Fluid Estradiol Concentrations before Deviation
Mares were randomized into a control group (unsampled, n = 9) and a sampled group (20 µl of follicular fluid removed from each of the 2 follicles, n = 10). Sampling of the two follicles was done once when the larger follicle first exceeded 14.9 mm (defined as 15 mm; actual diameters ranged from 15.2 to 17.8 mm).
Experiment 2. Follicular Fluid and Systemic Estradiol Concentrations Associated with Deviation
Mares (n = 6 per group) were randomized into three groups for sampling of 20 µl of follicular fluid from each of the two follicles once when the larger follicle first exceeded 14.9 mm (15-mm group; before deviation), 19.9 mm (20-mm group; approximately at deviation), or 24.9 mm (25-mm group; after deviation). Actual diameters for the three groups were 15.0–18.0 mm, 20.8–23.5 mm, and 25.0–26.8 mm, respectively. Blood samples were collected daily, beginning on Day 10 and ending on the day of the next ovulation. Samples were collected by jugular venipuncture into heparinized tubes and held for 1–2 h at 4°C until sedimentation. Plasma was decanted and placed in vials for cold storage (-20°C) until estradiol assay.
Combined Data
The association between follicle diameters and estradiol concentrations was determined using all sampled follicles of the two experiments. To confirm previously reported [1] relationships between future dominant and subordinate follicles, the control group of experiment 1 (no sampling) and the 25-mm group of experiment 2 (deviation before sampling) were combined (n = 11 follicular waves) and studied from emergence of the dominant follicle until ovulation.
Statistical Analyses
Data were normalized to day of follicular fluid sampling, day of emergence of the dominant follicle, and day of the beginning of deviation. Growth profiles of the larger and smaller follicles at the time of sampling were analyzed by a group-by-day factorial ANOVA for sequential data. One-way ANOVA was used to compare groups within the same end points. Paired and unpaired t-tests were used to compare various characteristics between days within a follicle, between follicles within end points, or between days. A two-sample t-test applied to the ranks [9] was used to compare the diameters of the dominant and subordinate follicles on the day of emergence of the dominant follicle; the ranking test was used because actual diameter of the subordinate follicle was not available for the day of emergence of the dominant follicle in 5 of 11 mares. Pearson correlation analyses were used to compare follicle diameters just before sampling and intrafollicular estradiol concentrations. Significance was defined as p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiment 1. Follicular Fluid Estradiol Concentrations before Deviation
Mares were removed from the experiment because of an anovulatory wave with no follicle reaching > 27 mm (3 control and 2 sampled mares); subsequent monitoring indicated that these mares had entered the anovulatory season. Thus, a total of 6 control and 8 sampled mares were available for statistical analyses.
No significant differences between the control and sampled groups were found for any of the following end points: 1) length of intervals between emergence and deviation, sampling and deviation, sampling and ovulation, and Day 10 to ovulation (Table 1); 2) follicle diameters at sampling and at deviation (Table 1); 3) day of follicle deviation (controls, 18.2 ± 0.5; sampled, 17.2 ± 0.4); 4) difference in diameter between larger and smaller follicles at sampling (controls, 1.7 ± 0.4 mm; sampled, 1.8 ± 0.6 mm); and 5) number of larger follicles at 15 mm that became dominant follicles (controls, 6 of 6; sampled, 5 of 8). The growth rates from the day before sampling to the day of sampling and from 1 day after to 2 days after sampling were not different between groups within follicles (Table 1). However, the growth rate between the day of sampling and the next day was slower (p < 0.01) in the sampled group than in the control group. The subsequent four daily growth rates were not different between groups. For the diameter of the larger follicle, the interaction of group and day was significant (p < 0.03; Fig. 2a). For the smaller follicle, there was no significant group effect nor an interaction (Fig. 2c). Intrafollicular estradiol concentrations were higher (p < 0.007) in the larger (460 ± 67 ng/ml) than in the smaller (322 ± 50 ng/ml) follicles.
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Experiment 2. Follicular Fluid and Systemic Estradiol Concentrations Associated with Deviation
Mares were removed from the experiment for the following reasons: loss of identity of the smaller follicle after sampling (one mare in the 15-mm group); larger follicle regressed after reaching 27.5 mm in the presence of a maintained corpus luteum (anovulatory wave, one mare in the 20-mm group); and double ovulations (codominant follicles, one mare in the 25-mm group). Thus, five mares per group were available for statistical analyses.
There were no significant differences among the three groups (15-mm, 20-mm, and 25-mm) for day at the beginning of deviation (Day 16.8 ± 0.4, 16.4 ± 0.5 and 16.8 ± 0.8, respectively), interval from Day 10 to ovulation (Table 1), and diameters of larger and smaller follicles at the beginning of deviation (Table 1). Diameter of the preovulatory follicle on day before ovulation was smaller (p < 0.05) in the 25-mm group than in the 20-mm group. The growth rates of the larger follicle for the 3 days encompassing deviation did not differ among days or between groups (Table 1), but a difference (p < 0.02) in growth rates during the three intervals was observed between the smaller follicle of the 25-mm group and the other two groups (Table 1). Growth profiles and statistical data for the two follicles when normalized to day of sampling are shown (Fig. 2, b and d). As for experiment 1, a transient decrease in growth rates of the larger follicle occurred immediately after sampling in all groups.
Mean follicle diameters and follicular fluid and systemic estradiol concentrations of the larger and smaller follicles on the day of sampling for the three groups are shown (Fig. 3, a–c). The diameter of the larger follicle increased (p < 0.0001) progressively over the three groups, whereas the diameter of the smaller follicle increased (p < 0.04) only between the 15-mm and 20-mm groups. The difference in diameters between the larger and smaller follicles was similar (p > 0.05) for the 15-mm and 20-mm groups, but the difference was greater (p < 0.004) for the 25-mm group than for either of the other 2 groups. Estradiol concentrations were not different between the larger and smaller follicles in the 15-mm group (493 ± 91 vs. 480 ± 106 ng/ml) but were for the 20-mm (1353 ± 91 vs. 631 ± 168 ng/ml; p < 0.01) and 25-mm (2220 ± 328 vs. 346 ± 176 ng/ml; p < 0.009) groups. The differences in estradiol concentrations between the larger and smaller follicles increased (p < 0.0001) progressively over the 15-mm, 20-mm, and 25-mm groups (Fig. 3b). The differences were significant between the 15-mm and 20-mm groups and between the 20-mm and 25-mm groups. For systemic estradiol, the difference among the three groups on the day of sampling was not significant (p < 0.09; Fig. 3c). When the systemic daily estradiol concentrations were normalized to the beginning of deviation, the day effect was significant (p < 0.0001), but the group effect and interaction were not (Fig. 4a). The first significant (p < 0.008) increase in daily circulating estradiol concentrations occurred between Day -1 (day before the beginning of deviation) and Day 0 (Fig. 4b).
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Combined Data
Totaled over all experiments, intrafollicular estradiol concentrations were positively and significantly correlated to diameter of the larger follicles (r = 0.92; p < 0.0001) and to diameter of the smaller follicles (r = 0.59; p < 0.004). Mean day-to-day changes in diameter of the future dominant and subordinate follicles of the control group of experiment 1 and the 25-mm group of experiment 2 are shown, using as reference points the day of emergence of the future dominant follicle at 6 mm (Fig. 5a) and the day at the beginning of deviation (Fig. 5b). Data were truncated to the days when observations were available for all mares. The mean day of follicle deviation was 6.2 ± 0.3 days after emergence of the dominant follicle or on Day 17.4 ± 0.4 after ovulation. The future dominant follicle emerged earlier than the future subordinate follicle and was larger than the future subordinate follicle on the day of emergence of the dominant follicle and on the day at the beginning of deviation (Table 2). The growth rates of the future dominant and subordinate follicles between emergence and deviation (6 days later) did not differ between follicles.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The diameter advantage of the future dominant follicle over the future subordinate follicle, beginning on the day of emergence at 6 mm, is consistent with a previous study [1]. The future dominant follicle emerged an average of 1 day earlier than the future subordinate follicle and was larger on the day of emergence and on the day of deviation. The two follicles, on average, grew in parallel as indicated by the absence of a significant difference in growth rates between the days of emergence and deviation. Several studies in cattle have indicated that removal of the largest follicle before or soon after the beginning of deviation allows the second-largest follicle to become dominant [2]. Apparently, selection of the dominant follicle is not complete until deviation occurs. Results of the present study and previous reports in ponies [1] and cattle [2, 3] indicate the need for focusing on the deviation mechanism. In a previous study in cattle [4], intrafollicular but not systemic estradiol concentrations were examined in relationship to time of deviation. In the present studies in mares, the relationships between follicle deviation and intrafollicular and systemic estradiol concentrations were considered.
Ultrasound-guided follicular entry for injection of substances has been done in mares for eCG [13], in heifers for hCG [14], and in cattle for sampling follicular fluid 1–4 days after follicular-wave emergence [4]. In the present studies, sampling of follicular fluid by the ultrasound-guided technique was effective for 10- to 27-mm follicles in mares that were accustomed to the padded squeeze stock and transrectal and transvaginal procedures. The volume of follicular fluid sampled (20 µl) was approximately 1.1%, 0.5%, and 0.2% of the fluid volume of a spherical follicle with an antral diameter of 15, 20, and 25 mm, respectively. Usable samples and follicle data were obtained from 25 of 28 (89%) pairs of targeted follicles. The undesirable effects of follicular fluid sampling were similar to those reported for cattle [4]. Extensive blood contamination of the samples and blood clot formation in the antrum of targeted follicles were considered evidence of unsuccessful sampling attempts. The failures seemed associated with mare movements or technique problems during the follicle puncture. The frequency of failures seemed to decrease as the experiments progressed. Better maintenance of the needle tip in the center of the follicle and better coordination between the two operators may account for some of the improved success with increased experience. The results of a trial in cattle [4] indicated that aspiration of a consistent volume of follicular fluid required that the inner needle (25-gauge) be filled with fluid (saline). However, some mixing of the follicular fluid with the saline occurred, as indicated by a 3–11% reduction in estradiol in the sampled fluid closest to the saline. In the present study, altered concentrations because of mixing of fluids presumably was minimized by using the 15-µl portion of the sample closest to the needle tip of an aspirated volume of 20 µl.
The mean growth rate of the larger sampled follicles (15, 20, and 25 mm) was reduced on the day immediately after sampling, but thereafter the follicles grew at a rate similar to the rate in controls or the rate before sampling. The effects of sampling on diameter of the smaller follicle were confounded by regression in association with deviation. As indicated by the comparisons between the sampled and control groups in experiment 1, sampling at 15 mm did not significantly alter the subsequent follicle characteristics associated with deviation (interval from sampling to the beginning of deviation, diameter of follicles at deviation). The larger follicle at sampling was on average significantly larger at deviation; however, in 3 of 8 mares in the sampled group, compared to 0 of 6 in the control group, the larger follicle at sampling became the subordinate follicle after deviation. These ratios were not significantly different, but the number of mares was small. When data were combined for the control group of experiment 1 and the 25-mm group of experiment 2 (dominance established before sampling), the larger follicle at 15 mm became the dominant follicle in 11 of 11 mares. Combined data for mares with follicles sampled at 15 mm in the 2 experiments showed that the follicle that was larger when sampled became the dominant follicle in 8 of 13 mares (11 of 11 vs. 8 of 13, p < 0.07). On this basis, an effect of sampling on the future status of a follicle (dominant vs. subordinate) was considered suggestive or at least equivocal. For this reason, no attempt was made to reclassify the larger and smaller follicles at sampling on the basis of their eventual status as dominant and subordinate follicles.
There were indications that follicle sampling or puncture hastened the time of ovulation after puncture at 15 mm in experiment 1. The interval from attainment of a diameter of 25 mm to ovulation was reduced by a mean of 2.3 days compared to that in the controls. The corresponding intervals for the 15, 20, and 25-mm sampled groups in experiment 2 were similar to that of the 15-mm sampled group in experiment 1. In addition, the diameter of the follicle on the day before ovulation was reduced by a mean of 5 or 6 mm in the 25-mm group compared to the other 2 groups. The reason for the ovulation effect is not known. Ovulation is associated with an inflammatory reaction in many species [15], and speculatively the inflammation or healing associated with puncture of the follicular wall in mares may have hastened ovulation.
In experiment 2, follicle deviation had not begun on the day of sampling in the 15-mm group, but it had begun in the 25-mm group on the basis of changes in the differences in diameters between the larger and smaller follicles. The beginning of deviation, defined as the day before a change in the differences in diameters between the two follicles, was on the day of sampling for each mare in the 20-mm group, as indicated by examination of the growth profiles of the two follicles for each mare. Furthermore, there was close agreement between mean diameter of the larger follicle on the day of sampling in the 20-mm group with the mean diameter of the larger follicle on the day at the beginning of deviation in nonsampled mares. The larger follicle at the time of sampling was a mean of 22 mm, which was similar to the diameter at the beginning of deviation in the controls of experiment 1 (22 mm), in the 25-mm group of experiment 2 (deviated before sampling; 22 mm), and in unsampled follicles of a previous study (22–23 mm in various groups) [1].
The low systemic estradiol concentrations of 1–5 pg/ml found in this study agree with some findings [12, 16], but were much lower than others [17, 18]. An explanation for the apparent differences in systemic estradiol concentrations in the literature could be the different types of assays used and the cross-reactivity of each assay with other estrogens (e.g., estrone). According to the manufacturer, the assay used in the present study has low cross-reactivity with other estrogens.
The most novel aspect of this study involved the assessment of changes in both intrafollicular and systemic concentrations of estradiol in relation to changes in follicle diameters. In experiment 2, the progressive increase (not significant, p < 0.09) in systemic concentrations of estradiol in groups 15 mm, 20 mm, and 25 mm was based on blood samples collected on the day of follicular fluid sampling. More detailed information was obtained by normalizing all mares to the day of the beginning of diameter deviation and using daily blood samples. The absence of a difference among groups or an interaction of group and day indicated that the concentrations followed a similar pattern in the three groups. The significant day effect resulted from progressive increases from the day before the beginning of deviation to the last day considered (2 days after the beginning of deviation). Thus, an increase in systemic estradiol was detected the day before the detection of follicle deviation, suggesting that follicular estradiol production changed before the diameter manifestation of follicle deviation in the mare.
In experiment 1, estradiol concentrations were higher in the larger follicle when the larger and smaller follicles were a mean of 16.4 mm and 14.6 mm. However in experiment 2, there was not a significant difference between follicles in the 15-mm group. This apparent contradiction can be attributed, at least partly, to more mares in experiment 1 (n = 8) than in experiment 2 (n = 5) and to differences in season (late and early in the ovulatory season for experiments 1 and 2, respectively) [12]. In experiment 2, intrafollicular estradiol concentrations were analyzed at times encompassing the beginning of deviation. The difference in follicular fluid estradiol concentrations between the larger and smaller follicles increased progressively for the 15-mm, 20-mm, and 25-mm groups. In mares in which follicles were sampled at the beginning of deviation (all in the 20-mm group), the difference between the two follicles in estradiol concentrations was greater than for the 15-mm group. Thus, differences in intrafollicular estradiol concentrations between the two follicles occurred before a change in the differences in diameter. This result and the observed changes in systemic estradiol concentrations are compatible with the conclusion that intrafollicular estradiol production began to increase in the larger follicle before detection of the beginning of deviation in diameter. This temporal relationship encourages further study focused on the possible functional relationship between an increase in intrafollicular estradiol in the largest follicle and the mechanism of diameter deviation. Speculation on the potential manner in which estradiol could be involved has been reviewed [2].
In conclusion, the results indicated that follicle sampling did not alter the day of deviation or follicle diameters on the day of deviation. The follicular fluid sampling technique developed in these studies may be useful in relating other intrafollicular factors to the mechanism of follicle selection in mares. The detection of a change in the differences in intrafollicular estradiol concentrations between the two follicles before the detection of a change in the differences in diameter suggested, on a temporal basis, that estradiol is a candidate for involvement in the mechanism that leads to follicle-diameter deviation in mares.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Drive, Madison, WI 53706. FAX: 608 262 7420; ojg{at}ahabs.wisc.edu
Accepted: February 2, 1999.
Received: December 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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O.J. Ginther, D.R. Bergfelt, M.A. Beg, C. Meira, and K. Kot In Vivo Effects of an Intrafollicular Injection of Insulin-Like Growth Factor 1 on the Mechanism of Follicle Deviation in Heifers and Mares Biol Reprod, January 1, 2004; 70(1): 99 - 105. [Abstract] [Full Text] [PDF]
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A. R. Baerwald, G. P. Adams, and R. A. Pierson Characterization of Ovarian Follicular Wave Dynamics in Women Biol Reprod, September 1, 2003; 69(3): 1023 - 1031. [Abstract] [Full Text] [PDF]
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O.J. Ginther, C. Meira, M.A. Beg, and D.R. Bergfelt Follicle and Endocrine Dynamics During Experimental Follicle Deviation in Mares Biol Reprod, September 1, 2002; 67(3): 862 - 867. [Abstract] [Full Text] [PDF]
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F.X. Donadeu and O.J. Ginther Changes in Concentrations of Follicular Fluid Factors During Follicle Selection in Mares Biol Reprod, April 1, 2002; 66(4): 1111 - 1118. [Abstract] [Full Text] [PDF]
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M.A. Beg, D.R. Bergfelt, K. Kot, and O.J. Ginther Follicle Selection in Cattle: Dynamics of Follicular Fluid Factors During Development of Follicle Dominance Biol Reprod, January 1, 2002; 66(1): 120 - 126. [Abstract] [Full Text]
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O.J. Ginther, M.A. Beg, D.R. Bergfelt, F.X. Donadeu, and K. Kot Follicle Selection in Monovular Species Biol Reprod, September 1, 2001; 65(3): 638 - 647. [Abstract] [Full Text] [PDF]
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D.R. Bergfelt, E.L. Gastal, and O.J. Ginther Response of Estradiol and Inhibin to Experimentally Reduced Luteinizing Hormone During Follicle Deviation in Mares Biol Reprod, August 1, 2001; 65(2): 426 - 432. [Abstract] [Full Text] [PDF]
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O.J. Ginther, D.R. Bergfelt, M.A. Beg, and K. Kot Follicle Selection in Cattle: Role of Luteinizing Hormone Biol Reprod, January 1, 2001; 64(1): 197 - 205. [Abstract] [Full Text]
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E.L. Gastal, D.R. Bergfelt, G.P. Nogueira, M.O. Gastal, and O.J. Ginther Role of Luteinizing Hormone in Follicle Deviation Based on Manipulating Progesterone Concentrations in Mares Biol Reprod, December 1, 1999; 61(6): 1492 - 1498. [Abstract] [Full Text]
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E.L. Gastal, M.O. Gastal, and O.J. Ginther Experimental Assumption of Dominance by a Smaller Follicle and Associated Hormonal Changes in Mares Biol Reprod, September 1, 1999; 61(3): 724 - 730. [Abstract] [Full Text]
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