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BOR - Papers in Press, published online ahead of print June 9, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030726
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BIOLOGY OF REPRODUCTION 71, 1244–1251 (2004)
DOI: 10.1095/biolreprod.104.030726
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Developmental Pattern of Small Antral Follicles in the Bovine Ovary1

R.S. Jaiswal, J. Singh, and G.P. Adams2

Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The study was designed to characterize the developmental pattern of 1- to 3-mm follicles and to determine the stage at which the future dominant follicle first attains a size advantage among its cohorts. In experiment 1, heifers (n = 18) were examined every 24 h by transrectal ultrasonography for one interovulatory interval (IOI). In experiment 2, cows (n = 9) were examined every 6 h from 5 to 13 days after ovulation to monitor precisely the diameter changes of individual follicles ≥1 mm during emergence of wave 2. Results revealed a change over days (P < 0.05) in the number of 1- to 3-mm follicles, with a maximum (P < 0.05) 1 or 2 days before wave emergence (conventionally defined as the time when the dominant follicle is first detected at 4 mm), followed 3–4 days later by a maximum (P < 0.05) in the number of ≥4-mm follicles. The profiles of small (1–3 mm) and large (≥4-mm) follicles were inversely proportional (r = –0.79; P = 0.01). The profile of the number of 1- to 3-mm follicles during wave emergence was similar (P = 0.63) between waves in two-wave IOI, but differed (P < 0.01) among waves in three-wave IOI as a result of a greater number of follicles in the ovulatory wave (P < 0.04). As well, the number of follicles in the ovulatory wave tended to be greater (P < 0.06) in three-wave IOI than in two-wave IOI. The future dominant follicle was first identified at a diameter of 1 mm and emerged 6–12 h earlier than the first subordinate follicle (P < 0.01). After detection of the dominant follicle at 1 mm (0 h), its diameter differed from that of the first and second subordinate follicles at 24 h (P = 0.04) and 12 h (P = 0.01), when the dominant follicle was 2.4 ± 0.17 mm and 1.7 ± 0.14 mm, respectively. The growth rate of the dominant follicle differed from that of the first and second subordinate follicles at 120 h (P = 0.03) and 108 h (P = 0.02), when the dominant follicle was 9.5 ± 0.30 mm and 8.8 ± 0.49 mm, respectively. Emergence of the future dominant (r = 0.71), first (r = 0.73), and second (r = 0.76) subordinate follicles was temporally associated (P < 0.01) with a rise in circulating concentrations of FSH. Transient, nocturnal elevations in plasma FSH concentration were followed within 6 h by an increase in the growth rate of 1- to 3-mm follicles. We conclude that 1) 1- to 3-mm follicles develop in a wave-like manner in association with surges in plasma concentrations of FSH, 2) 1- to 3-mm follicles are exquisitely responsive to transient elevations in FSH, and 3) selection of the dominant follicle is manifest earlier than previously documented and is characterized by a hierarchical progression over a period encompassing the entire FSH surge (5 days).

diurnal gonadotropin secretion, follicle dynamics, follicular development, follicular dominance and selection, follicle-stimulating hormone, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The wave-like developmental dynamics of follicles ≥4 mm have been well documented in cattle [1–7], but the dynamics of smaller follicles have not. The majority of bovine estrous cycles (>95%) consist of two or three waves of follicular development [8], each of which is characterized by a surge in circulating concentration of FSH [9] followed by a sudden (within 1–2 days) appearance of several follicles 4- to 6-mm in diameter, as detected by serial ultrasonography [4]. Follicles of the cohort grow at a similar rate for about 2 days, followed by preferential growth of one (dominant) follicle over the others (subordinates) in a process referred to as selection. The dominant follicle suppresses growth of its subordinates [10–14] and continues to grow for about 6 days [4]. In the presence of a functional corpus luteum, the dominant follicle stops growing and enters a static phase followed by a regressing phase like its subordinates. If luteal regression occurs during the growing or early static phase, the dominant follicle ovulates [15–17]. In either instance, a new follicular wave starts [18–20].

As opposed to the known wave-like pattern of follicles ≥4 mm, information is lacking about the developmental pattern of follicles <4 mm. Primordial follicles in the fetal bovine ovary constitute the lifetime reservoir of follicles (approximately 150 000 at birth), a reservoir that is progressively depleted throughout the life span of a cow [21]. The mechanism controlling recruitment of primordial follicles into the growing pool, and the stage at which growing follicles conform to the wave pattern of development, are unknown. However, consistency in the number of follicles ≥4 mm recruited into a follicular wave from one wave to the next [22, 23] suggests that follicular development may be entrained to waves before follicles become ultrasonographically detectable. Mean growth rates of follicles from early to ovulatory stages of development have been estimated in cows [24, 25], rodents [26–28], sheep [29], and women [30]. However, these estimations do not shed light on the dynamics of small follicle development relative to wave emergence or the relationship to changes in circulating concentrations of gonadotropins.

Granulosa cells of follicles as early as the primary stage of development (i.e., immediately after activation from the primordial pool) possess FSH receptors [31, 32], and in vivo and in vitro studies have documented the stimulatory effects of FSH on these follicles [31–35]. These observations, plus the known phenomenon of periodic surges in the circulating concentrations of FSH during the estrous cycle [9], provide rationale for the hypothesis that small follicles (<4 mm) develop in a wave-like manner.

The objective of this study was to characterize the developmental pattern of small antral follicles (1–3 mm) in cattle using ultrasonography. Our hypotheses were 1) small antral follicles (1–3 mm) develop in a wave-like manner, 2) the future dominant follicle of a wave maintains a size advantage over future subordinates from its earliest detection (i.e., 1 mm), and 3) emergence of dominant and subordinate follicles at the diameter of 1 mm is associated with rising plasma concentrations of FSH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiment 1: Developmental Pattern of 1- to 3-mm Follicles During One Interovulatory Interval

Animals Sexually mature Hereford-cross heifers (n = 18) 18–24 mo of age and weighing 450–550 kg were selected from a group of 28 on the basis of physical and reproductive fitness (normal reproductive tract, presence of corpus luteum, nonpregnant) as judged by two ultrasound examinations 10 days apart. The heifers had not been treated during the previous 6 mo with hormones that may be expected to influence ovarian function (e.g., growth promotants, ovarian synchronization or superovulation treatment). The experiment was done between January and April and heifers were maintained in a single outdoor corral at the University of Saskatchewan Goodale Research Farm (52°N and 106°W) and fed alfalfa grass hay and grain to gain approximately 1.3 kg in weight per day.

Ovarian ultrasound examinations Ovarian follicular development was monitored every 24 h by transrectal ultrasonography using a 7.5-MHz linear-array transducer (Aloka SDD-900; Instruments for Science and Medicine, Vancouver, BC). According to company specifications, the scanner provided a lateral resolution of 2 mm and an axial resolution of 1 mm. The integrated electronic calipers are designed to move in 0.1-mm increments; however, follicles <1 mm could not be detected consistently or monitored reliably. Ultrasound examinations of heifers commenced irrespective of the day of the estrous cycle and continued until two successive ovulations were recorded, so as to encompass one complete interovulatory interval. Based on the previous day's record of the topographic location and diameter of follicles and corpora lutea [7], all follicles ≥4 mm were recorded, and attempts were made to monitor individually identified follicles 1–3 mm in size. In addition, the number of follicles in the 1- to 3-mm and ≥4-mm categories was recorded during each examination. Ultrasound examinations were performed by one person (R.J.) to minimize variation in data acquisition.

Experiment 2: Developmental Pattern of 1- to 3-mm Follicles at the Time of Wave Emergence

In experiment 1, follicles ≥4 mm could be individually identified on a daily basis; however, identification of individual 1- to 3-mm follicles was difficult due to 1) their comparatively large numbers, 2) similarity in shape, and 3) insufficiently frequent ultrasound examinations. Experiment 2 was, therefore, designed to overcome difficulties encountered during experiment 1 by incorporating specific animal-selection criteria, the use of alternative methods of identifying individual follicles, and more frequent ultrasound examinations.

Animals Hereford-cross cows (n = 24), 3–4 yr of age and weighing 600–650 kg were selected during the fall (September) from a group of 37 using the same criteria as in experiment 1. Results of earlier studies indicate that the total number of follicles within the ovaries varies widely among cows [21], but that, within cows, the number of follicles ≥4 mm recruited into a follicular wave remains consistent from one wave to the next [22, 23]. To minimize the complexity of monitoring small follicles and to reduce interanimal variation, cows at the upper and lower extremes of follicle numbers were excluded; i.e., only cows near the median for follicle numbers were selected. To this end, the cows were given two luteolytic doses of cloprostenol 12 h apart (500 µg Estrumate, i.m.; Schering-Plough Animal Health, Canada) and the number of ovarian follicles ≥1 mm in diameter was recorded during daily ultrasound examinations from the day of prostaglandin treatment until 1 day after ovulation. Ovulation was detected in 21 cows within 5 days of cloprostenol treatment. Cows (n = 21) were ranked on the basis of the cumulative number of follicles ≥1 mm detected in both ovaries –1, 0 and +1 days from ovulation. The median value of the cumulative number of follicles was 119, and cows (n = 9) nearest the median rank (range 77–154 follicles) were selected for detailed ultrasound examinations.

Ovarian ultrasound examinations The ovaries of each cow were examined by transrectal ultrasonography at 6-h intervals from 5 to 13 days after ovulation so as to encompass the emergence of the second follicular wave of the estrous cycle. Ultrasound examinations were done with the equipment described in experiment 1 using a technique similar to that previously validated for assessing follicles ≥2 mm [36]. Examinations were done by one operator (R.J.), and a technical routine was established to optimize follicle enumeration and minimize errors. The right ovary was examined first, followed by the left ovary, and the ultrasound transducer was moved from the lateral to medial aspect of the ovary and back again. The transducer was moved slowly and kept steady for a few seconds when a follicle was resolved at its full diameter. The ovaries were then scanned a second time in a similar fashion except that follicular images were frozen on the screen and measured using the integrated electronic calipers. Two values, measured at right angles to each other, were recorded and averaged to obtain an estimate of follicle diameter [37].

Methods of follicle data recording The conventional method of profiling daily changes in individual follicles by ultrasonography involves retrospective evaluation of serial ovarian sketches that provide topographical and dimensional information of follicles ≥4 mm. Such sketches are usually rendered as a three-dimensional impression of the amalgamated series of two-dimensional images (Fig. 1). In experiment 1, we used this conventional amalgamation method of sketching daily changes in the small follicles; however, retrospective tracking of individual small follicles was difficult for reasons previously mentioned. Importantly, we noticed that, as small follicles grow, the plane of view changed relative to other follicles, which made it difficult to follow them retrospectively. To circumvent the problem of tracking individual small follicles, we employed a sectional method of sketching follicles in which multiple ovarian maps (Fig. 1a) were used to record images of follicles in sequential sections of each ovary while moving the transducer from the lateral to the medial aspect of the ovary. However, we found that this sectional method of sketching follicles was labor intensive. To simply it, we followed the procedure of sectional sketching (Fig. 1a) only for the first ultrasound examination. On subsequent ultrasound examinations, changes in individual follicle diameter were recorded against the respective first sectional sketch (Fig. 1b), without the necessity of redrawing ovarian structures. To minimize the error in monitoring daily changes in the follicular diameter, we recorded each ultrasound examination on S-VHS videotapes (Video Cassette Recorder model PV-VS4821-K, Panasonic; PT Matsushita Kotobuki Electronic Industries, Indonesia). A separate videotape cassette was used for each animal. Individual small follicles were identified by retrospective analysis of ovarian sketches and recorded ultrasound images.



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  		FIG. 1. Illustration of follicular changes in the right ovary of cow 29 over a 12-h period as determined by a sectional method of data recording (a). A portion, or section, of the ovary was sketched when a new follicle(s) was imaged at its full diameter while moving the ultrasound transducer from lateral to medial (sections 1–4 for this example). Hence, the number and thickness of sectional images varied from ovary to ovary and from time to time, depending on the size and number of ovarian structures present. To minimize time and labor, the sectional method was modified so that changes in follicular diameter were recorded against a single ovarian sketch for a given 24-h period (b). Aggregate sketches at the bottom represent the conventional method of follicle monitoring (c) used in experiment 1, wherein structures overlap each other

Plasma sampling and radioimmunoassay for FSH Jugular blood samples were collected in heparinized tubes (10 ml; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) during day-time examinations (i.e., 0800, 1400, 2000 h) 5–13 days after ovulation and were centrifuged for 15 min at 1500 x g within 30–60 min of collection. Plasma was aspirated and stored at –20°C. Plasma concentration of FSH was measured using a double antibody radioimmunoassay [38]. The primary antibody was NIDDK-anti-ovine FSH, and the concentrations were expressed using standards prepared from USDA-bovine FSH-I-l. The minimum detectable limit of the assay was 0.13 ng/ml. The range of the standard curve was 0.13–16 ng/ml. The intra- and interassay coefficients of variation were 8% and 8% for the low-reference sample (mean 0.89 ng/ml) and 11% and 9% for the high-reference sample (mean 2.15 ng/ml), respectively.

Data Analysis

In experiment 1, ovarian follicular data of heifers were grouped into two categories based on the number of follicular waves displayed during the interovulatory interval (IOI) i.e., two-wave IOI and three-wave IOI. The day of wave emergence (Day 0) was determined by retrospective analysis of follicular data and defined as the day on which the dominant follicle of a wave was first detected at a diameter of 4–5 mm [4, 5, 39]. The dominant follicle was defined as the largest follicle of a wave and subordinate follicles were defined as those that appeared to originate from the same pool of follicles [4, 7]. For statistical analysis and preparation of figures, individual heifer's follicle data for each wave were centralized to the day of wave emergence [5, 18]. The numbers of 1- to 3-mm and ≥4-mm follicles were analyzed from Day –3 to Day 5 to determine a day effect [10, 11]. Data were analyzed by analysis of variance for repeated measures using the mixed procedure [40] in the Statistical Analysis System software package (SAS version 8.2 for MS Windows; SAS Institute Inc., Cary, NC). Five covariance structures (compound symmetry; autoregressive, order 1; unstructured; unstructured 1; and Huynh-Feldt) were fitted to the data and the best model was selected based on the smallest Akaike information criterion values. Data were analyzed for the effect of day (Day –3 to Day 5), follicle type (1–3 mm and ≥4 mm), IOI type (two-wave versus three-wave IOI), and wave type (wave 1 versus wave 2 versus wave 3; anovulatory versus ovulatory wave). If main effects or their interaction were statistically significant (P ≤ 0.05), multiple comparisons were made using Tukey post hoc test. Correlation between the change in the number of 1- to 3-mm and ≥4-mm follicles over time was estimated using Pearson correlation analysis.

In experiment 2, data (follicle diameter and growth rates) were analyzed for the effects of day (Day 5 to Day 13 after ovulation) and follicle type (dominant, first and second subordinates) by analysis of variance for repeated measures as described in experiment 1. Data were analyzed in three ways: 1) by centralization to the day of wave emergence (i.e., when dominant follicle was first detected at 4–5 mm) to determine the time of emergence of the dominant and subordinate follicles relative to the conventional definition of wave emergence, 2) by centralization to the peak in circulating concentrations of FSH, to determine the association between changes in FSH and the emergence of follicles at 1 mm, and 3) by centralization to the day of detection of the dominant follicle at 1 mm, to compare growth rates. If main effects or their interaction were significant (P ≤ 0.05), multiple comparisons were made using Tukey post hoc test. The association between FSH and emergence of dominant and subordinate follicles was estimated using Pearson correlation analysis after centralizing the follicular data to the FSH peak (0 h).

All procedures described within were reviewed and approved by the University of Saskatchewan Protocol Review Committee, under the umbrella of the University Committee on Animal Care and Supply, and were performed in accordance with the principles outlined by the Canadian Council on Animal Care.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Data from one heifer were missing from 2 to 5 days after the first ovulation, resulting in a loss of follicle identification and ambiguity in follicle data from Day 6 onward; hence, this heifer was excluded from statistical analyses. Data from the remaining 17 heifers were divided into two groups based on the number of follicular waves observed during the IOI; nine heifers displayed two follicular waves and eight heifers displayed three follicular waves. Wave emergence (i.e., when the prospective dominant follicle was first detected at 4–5 mm in diameter) was detected, on average, 0 and 9 days after ovulation for two-wave IOI and 0, 9, and 17 days after ovulation for three-wave IOI. It was feasible to detect and count small follicles (1–3 mm), but individual identity of small follicles could not be traced on the basis of daily examinations. Therefore, only data pertaining to follicle numbers were used to investigate the developmental pattern of small follicles in two-wave and three-wave IOI.

In two-wave IOI (Fig. 2), the number of small (1- to 3-mm) and large (≥4-mm) follicles changed over days (P ≤ 0.05). A maximum in the small follicle population occurred on Day –1 of wave emergence (defined conventionally as the day on which the dominant follicle of a wave is 4–5 mm in diameter) for both wave 1 (anovulatory wave) and wave 2 (ovulatory wave), whereas a maximum in large follicles occurred between Day 1 and Day 2 after wave emergence. There was an inverse relationship between the number of small and large follicles during wave 1 (r = –0.66; P = 0.05) and wave 2 (r = –0.62; P = 0.04). The pattern of maxima and minima in the number of small and large follicles in three-wave IOI (Fig. 3) were similar to those in two-wave IOI except that the day effect was statistically nonsignificant for large follicles (P = 0.18) during wave 1 and for small follicles (P = 0.49) during wave 3. An inverse relationship between the number of small and large follicles existed for wave 1 (r = –0.79; P = 0.01) and wave 3 (r = –0.90; P = 0.001), but not for wave 2 (r = –0.57; P = 0.14).



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  		FIG. 2. Comparative changes (mean ± SEM) in the number of small (1- to 3-mm) and large (≥4-mm) follicles and the diameter of the dominant follicles during two-wave interovulatory intervals in cattle. For statistical and illustrative purposes, follicle number data from each wave were centralized to the day of wave emergence (defined as the day on which the dominant follicle was detected at 4–5 mm in diameter). Arrows indicate emergence of successive waves. The first wave includes data from –3 to 5 days from ovulation, and the second wave includes data from 6 to 16 days from ovulation. Data from the last 4 days of the interovulatory interval are provided for completeness. *, Within the 1- to 3-mm category, values differed (P ≤ 0.05). •, Within the ≥4-mm category, values differed (P ≤ 0.05)



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  		FIG. 3. Comparative changes (mean ± SEM) in the number of small (1- to 3-mm) and large (≥4-mm) follicles and the diameter of the dominant follicles during three-wave interovulatory intervals in cattle. For statistical and illustrative purposes, follicle number data from each wave were centralized to the day of wave emergence (defined as the day on which the dominant follicle was detected at 4–5 mm in diameter). Arrows indicate emergence of successive waves. The first wave includes data from –3 to 5 days from ovulation, the second wave includes data from 6 to 13 days from ovulation, and the third wave includes data from 14 to 22 days from ovulation. *, Within the 1- to 3-mm category, values differed (P ≤ 0.05). •, Within the ≥4-mm category, values differed (P ≤ 0.05)

Changes in the number of 1- to 3-mm follicles during wave emergence were similar (P = 0.63) between waves in two-wave IOI (Fig. 4a), but differed (P < 0.01) among waves in three-wave IOI as a result of a greater number of follicles in the ovulatory wave (P < 0.04; Fig. 4b). The follicle number profile during emergence of wave 1 was similar between two- and three-wave IOI (P = 0.81); therefore, data were combined to compare with the second anovulatory wave in three-wave IOI. The number of follicles in the second anovulatory wave (three-wave IOI) tended to be greater (P < 0.07) than in the first anovulatory wave (two- and three-wave IOI combined; Fig. 5a). The number of follicles in the ovulatory wave tended to be greater (P < 0.06) in three-wave IOI than in two-wave IOI (Fig. 5b).



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  		FIG. 4. Number of small (1- to 3-mm) follicles (mean ± SEM) at the time of emergence of each wave (defined conventionally as the day on which the dominant follicle was detected at 4–5 mm in diameter) of two- and three-wave IOIs in cattle



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  		FIG. 5. Number of small (1- to 3-mm) follicles (mean ± SEM) in cattle at the time of emergence of the anovulatory (wave 1 of two- and three-wave IOI, and wave 2 of three-wave IOI) and ovulatory (last wave of two- and three-wave IOI) waves (defined conventionally as the day on which the dominant follicle was detected at 4–5 mm in diameter) in two-wave and three-wave IOI in cattle

Data for all but the ovulatory wave of three-wave IOI, which was significantly different from other waves, were combined to characterize the relationship between the number of small (1- to 3-mm) and large (≥4-mm) follicles during wave emergence (Fig. 6). The number of small and large follicles changed over days (P < 0.01). A significant follicle category-by-day interaction (P < 0.01) and Pearson correlation coefficient (r = –0.79; P = 0.01) documented an inverse relationship between the number of small and large follicles.



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  		FIG. 6. Relationship between changes in the number (mean ± SEM) of small (1- to 3-mm) and large (≥4-mm) follicles during a follicular wave in cattle. Data for all waves for two-wave (n = 9 heifers) and three-wave (n = 8 heifers) interovulatory intervals were combined (n = 34 waves) with the exception of the ovulatory wave of three-wave IOI. Within follicle categories, values denoted with an asterisk (*) or dot (•) are different (i.e., maxima and minima; P ≤0.05)

Experiment 2

Sketching of follicles using the sectional method (Fig. 1a) provided information about the location and number of small follicles in the ovary, but was labor and time intensive. Modification of the sectional method (Fig. 1b) was helpful but not entirely effective because the plane in which small follicles were detected within the ovary changed as they grew and regressed. Hence, data tabulated using the sectional sketching method were systematically compared with video recordings of each examination to enable individual identification of follicles as small as 1 mm. The follicle destined to become dominant was first detected at a diameter of 1 mm 66 h earlier, before it reached 4 mm (i.e., conventional definition of wave emergence; Fig. 7a). The largest (first) subordinate follicle was first detected at 1 mm 48–54 h earlier (i.e., 6–12 h later than the future dominant follicle).



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  		FIG. 7. Growth (mean ± SEM) of dominant and subordinate follicles in cattle (n = 9) relative to (a) wave emergence (defined as the day on which the dominant follicle was detected at 4–5 mm in diameter) and (b) the peak in plasma FSH concentrations (mean ± SEM). FSH concentrations changed over time (P < 0.01). The diurnal pattern of plasma FSH concentration is illustrated in the inset (mean ± SEM). Values (mean ± SEM) in the inset were taken from samples drawn at 0800, 1400 and 2000 h from –60 to 90 h (0 h = FSH peak). ab, Values with no common superscript are different (P≤0.05)

FSH and Small Follicle Emergence

Data centralized to the peak in FSH (Fig. 7b) revealed a change (P < 0.01) in circulating concentrations of FSH over time. Plasma FSH concentrations were positively correlated (P = 0.01) with follicle diameter (dominant follicle, r = 0.71; first subordinate follicle, r = 0.73; second subordinate follicle, r = 0.76) from the time of follicle detection at 1 mm to the time at which FSH concentrations peaked. A negative correlation (P < 0.01) was detected thereafter (dominant follicle, r = –0.90; first subordinate follicle, r = –0.68; second subordinate follicle, r = –0.78). Growth of the three largest follicles began well before the peak in circulating concentration of FSH (Fig. 7, a and b). Although, the experiment was not designed specifically to examine the acute pattern of FSH secretion, the wave-eliciting surge in FSH (spanning 4–5 days) appeared to be composed of subsurges that occurred every 24 h (Fig. 7b). Retrospective analysis based on this observation revealed that each subsurge in FSH was followed by an increase in follicle diameter, on average, 5.6 ± 0.33 h later. Subsurge increases in FSH followed a diurnal pattern (time effect, P < 0.01) with maxima during the evening hours (Fig. 7b, inset).

Growth Rates of Dominant and First and Second Subordinate Follicles

When diameter and growth rate profiles of dominant and subordinate follicles were centralized to the time of detection of the future dominant follicle at 1 mm (0 h), the diameter of the dominant follicle differed (Fig. 8a) from that of the first and second subordinate follicles at 24 h (P = 0.04) and 12 h (P = 0.01), respectively, when the dominant follicle was 2.4 ± 0.17 mm and 1.7 ± 0.14 mm. However, a divergence in growth rates (Fig. 8b) was not detected until 108 h (dominant versus second subordinate; P = 0.02) and 120 h (dominant versus first subordinate; P = 0.03), when the dominant follicle was 8.8 ± 0.49 mm and 9.5 ± 0.30 mm, respectively.



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  		FIG. 8. Diameter (a) and growth rate (b) changes (mean ± SEM) in the dominant and first two subordinate follicles during wave emergence in cattle (n = 9). Data were centralized to the hour of detection of the dominant follicle at 1 mm. Asterisks (*) indicate the first significant differences between the dominant follicle and its subordinates


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanism controlling recruitment of primordial follicles into the growing pool and the stage at which growing follicles join follicular waves are unknown. However, based on the well-documented developmental pattern of large follicles (≥4 mm), we hypothesized that small follicles (1–3 mm) develop in a wave-like manner. Consistency in the number of follicles ≥4 mm recruited into a follicular wave from one wave to the next [22, 23] suggests that follicular development is entrained to waves before follicles become ultrasonographically detectable (i.e., <4 mm). The impetus to test the hypothesis was derived from observations that 1) FSH receptors are present in small follicles shortly after entering the growing pool [31, 32]; 2) FSH binds to granulosa cells of very small follicles, i.e., follicles with only a single layer of granulosa cells [41]; and 3) the development of primary follicles to secondary follicles in the developing fetus at Day 120 of gestation is associated with an increase in the serum concentration of FSH [42]. These observations, plus the knowledge that circulating concentrations of FSH surge in a rhythmic and periodic manner during the estrous cycle [9], provide rationale for the hypothesis that small follicles (1–3 mm) develop in a wave-like manner.

Results of experiment 1 supported our first hypothesis that small antral follicles develop in a wave-like pattern. A significant inverse relationship was detected in the profiles of the number of small follicles (1–3 mm) and large follicles (≥4 mm), consistent with a wave-like developmental pattern [43]. The periodic shifts from a maximum number of small follicles to a maximum number of large follicles resulted when small follicles grew as a cohort to a larger diameter and were not immediately replaced by another set of small follicles. Although changes in the number of 1- to 3-mm follicles during the third wave of three-wave IOI did not reach significance, the pattern of change appeared similar to that of other waves in two- and three-wave IOI; the lack of statistical significance was attributed to the small number of observations. Alternatively, the increase in variability in follicle numbers associated with the final wave of three-wave IOI may reflect a different mechanism than that controlling other waves, and the difference warrants more critical study. Results of the present study are consistent with those of a previous study [43] in which an inverse relationship was found between the number of 2- to 3-mm and ≥4-mm follicles. The statistical rigor of this inverse relationship is exemplified by its detection in the previous study, despite that data were tabulated and analyzed irrespective of wave emergence [43]. In an early study [44], no cyclic changes in the number of vesicular follicles up to 5 mm in diameter were detected; however, statistical inference was not possible because only one cow was used for each day of the estrous cycle. In a later study, wherein the ovaries of cows were examined by laparotomy on Days 3, 8, 13, and 18 [45], an increase in the number of small follicles was noted on Day 3. The timing of the maximum in the number of small follicles observed in the present study (i.e., 1 day before ovulation, or 1 day before detection of the dominant follicle at 4 mm in diameter) was earlier than that reported in the laparotomy study; however, in the latter, the point of reference (i.e., estrus or ovulation) is not clear and follicle enumeration was done by examining only the superficial surface of the ovary. In addition, follicular measurements in the laparotomy study were made using vernier calipers from the ovarian surface, in contrast with that of the present study in which electronic calipers were used to measure follicles throughout the depth of the ovary from images frozen on the ultrasound scanner.

The pattern of ovarian follicular development remained uncertain until a tool became available that enabled repeated, serial observation of the dynamic process, i.e., ultrasonography [8]. The elusiveness of the dynamics of follicles too small to be monitored by ultrasonography persists for the same reason. The difficulty experienced in experiment 1 in serial identification of individual follicles <3 mm was perhaps not surprising because the small diameter was near the limit of image resolution, and smaller follicles grew more slowly and were greater in number than larger follicles. In addition, daily changes were more difficult to track because the tendency of small follicles to change plane within the ovarian tissue during growth confounded the use of topographic landmarks. To address these issues, the design of experiment 2 incorporated special criteria for animal selection to minimize variation, more frequent ultrasonography to detect subtle changes among small follicles, and modifications to data recording and tabulation. Critical comparison of methodically recorded videotape images with previous section-by-section sketches of serial ultrasound images permitted individual profiling of follicles as small as 1 mm. With this approach, the dominant follicle was initially identified at a diameter of 1 mm; i.e., 66 h before it reached the previously stated time of wave emergence at a diameter of 4 to 5 mm [8].

The temporal relationship between the surge in circulating concentrations of FSH and the growth of small follicles in the present study is consistent with the results of an earlier study [9]. In the earlier study, the surge in FSH began 2–4 days (~48–96 h) before ultrasonographic detection of a dominant follicle at 4–5 mm (conventionally defined as wave emergence). The ultrasonographic detection of a dominant follicle at 1 mm in the present study, 66 h earlier than previously detected, was coincident with the beginning of the surge in FSH. In addition, the peak in the number of 1- to 3-mm follicles in the present study is coincident with the peak in circulating concentrations of FSH reported previously; i.e., 1–2 days before detection of a dominant follicle at 4–5 mm [9]. Exquisite sensitivity of small antral follicles to changes in circulating FSH was reflected in their ability to respond in less than 6 h to slight rises in FSH; transient increases in FSH were followed by transient increases in the growth rate of the three largest follicles from their earliest detection at 1 mm. Although detailed examination of the pattern of FSH secretion was not part of the original design, retrospective analysis of data revealed a diurnal pattern of circulating concentrations of FSH. The acrophases (clock time for maximal value) of FSH were observed during evening hours, which is consistent with diurnal patterns recently reported in humans [46, 47]. In association with nocturnal elevations in FSH, wave emergence (i.e., dominant follicle detected first at 4–5 mm) was detected more commonly during the morning than during the afternoon (75% versus 25%, respectively). Results of the present study indicated that emergence of the dominant follicle at 1 mm occurred 6–12 h earlier than that of subordinate follicles in the same wave. In an earlier study [12, 19], the future dominant follicle was identified at a diameter of 3 mm 6 h earlier than the future first subordinate follicle. Hence, the observation that the selected dominant follicle often has a size advantage at the time of its earliest detection [19] is in agreement with results of the present study.

The occurrence of follicle selection has been inferred by differences in diameter or growth rates of the three largest follicles of a wave. The prospective dominant follicle of a wave became significantly larger than all others by 2.5 days after its emergence at 4–5 mm [10]. Based on an apparent change in the growth rate from one day to the next, the diameter profile of the dominant follicle deviated from that of its subordinates when it reached a diameter of 8.5 mm [19]. Among studies, the divergence in diameters [11] and growth rates [19] of the two largest follicles of a wave are temporally consistent, i.e., on average, 2.5 days after wave emergence at 4–5 mm. In the present study, however, the prospective dominant follicle was significantly larger than the first and the second subordinate follicles by Day –2 of conventionally defined wave emergence, i.e., 4.5 days earlier than previously reported. The difference in growth rates of the prospective dominant and subordinate follicles observed in the present study was, however, similar to that observed in previous studies [19]. The growth rate of the dominant follicle was significantly greater than that of the second subordinate by 42 h and the first subordinate by 54 h of emergence at 4–5 mm. The mean diameter of the dominant follicle at these two times was 8.8 and 9.5 mm, respectively. The ability of the dominant follicle to reach a critical diameter before others in the cohort (i.e., ≥8 mm), when it is imbued with the capacity to suppress its subordinates and the emergence of the next wave [10, 11], was attributed to a size advantage from its earliest detection at 1 mm.

Interestingly, a hierarchical pattern of follicular selection was apparent in the present study, reflected in the progressive magnitude of the diameter profiles of the three largest follicles of the wave. The profile of the dominant follicle was larger than that of the first subordinate throughout its growth and, in turn, the profile of the first subordinate follicle was larger than that of the second subordinate throughout its growth. Results provide rationale for the hypothesis that selection of the dominant follicle involves sequential suppression of progressively larger follicles of a wave, i.e., a selection hierarchy, where progressive attrition of follicles within a wave results from gonadotropin suppression of progressively fewer follicles until only one (dominant) survives. This is consistent with the notion that subordinate follicles have a lesser capability of surviving during the postsurge decline in FSH [10].

The design of the present study permitted critical comparison of follicle dynamics between waves within and among two-wave and three-wave IOI. The greater number of follicles recruited into the second and the ovulatory waves of three-wave IOI may be associated with a shorter interwave interval [48], i.e., a shorter period of FSH suppression between waves (~8 days versus ~10 days) than in other waves [9]. This is consistent with the observation that FSH suppressed apoptosis in serum-free culture of rat preantral [49] and antral [50] follicles in vitro, suggesting that a physiological role of FSH may be to prevent atresia. The smaller, shorter-lived dominant follicle of the first and second waves in three-wave IOI [4, 11, 18] may be responsible for less profound follicular and gonadotropin suppression than other dominant follicles. Perhaps less interwave suppression and an early surge in circulating concentrations of FSH preceding emergence of the second and third waves is responsible for rescuing more follicles from atresia, resulting in recruitment of more follicles into the respective waves.

In summary, small antral follicles (1–3 mm) developed in a wave-like manner in association with surges in circulating concentrations of FSH. The number of follicles 1–3 mm in diameter recruited into the second and third wave of three-wave IOI tended to be greater than that of other waves. Among cows selected to represent the median population with respect to the number of follicles present at wave emergence, ovarian follicles 1–3 mm in diameter were acutely sensitive to changes in circulating concentrations of FSH; transient nocturnal elevations in plasma FSH concentration were followed within 6 h by an increase in the growth rate of 1- to 3-mm follicles. The future dominant follicle had a size advantage over all other follicles of the wave much earlier than previously documented. Successive slowing of the growth rate of the third, second, and first largest follicles of a wave during the decline in the FSH surge suggests that the selection mechanism involves sequential suppression of progressively larger follicles over a period of 72 h (i.e., selection hierarchy).


   ACKNOWLEDGMENTS
 
We thank Bill Kerr and his helpers at the Goodale Research Farm for animal maintenance. We also thank Ms. Tammy Orban and Dr. Miguel Dominguez for assistance with data collection and Schering-Plough Animal Health, Canada, for providing Estrumate.


   FOOTNOTES
 
1 Supported by a grant from the Natural Sciences and Engineering Research Council of Canada. Back

2 Correspondence: FAX: 306 966 7405; gregg.adams{at}usask.ca Back

Received: 6 April 2004.

First decision: 26 April 2004.

Accepted: 2 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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