Follicle and endocrine dynamics during experimental follicle deviation in mares

Deviation during a follicular wave in mares begins when the largest follicle (F1) reaches a mean diameter of 22.5 mm and is characterized by continued growth of F1 to become the dominant follicle and regression of F2 to become the largest subordinate follicle. In the present study, F1 was ablated at the expected beginning of deviation (Hour 0) to provide a reference point for characterizing the intrafollicular changes preceding experimental deviation between F2 and F3. Diameters and concentrations of follicular fluid factors in F2 and F3 were determined in F1-ablated mares at Hours 0, 12, 24, 48, or 72 (n = 8 mares/group). Circulating FSH concentrations were greater (P < 0.05) in the Hour 72 ablation group than in controls 12 h after ablation and then progressively decreased. The diameters of F2 and F3 increased (P < 0.05) during Hours 0 to 24. Thereafter, F2 continued to increase but F3 did not, indicating that experimental deviation began at Hour 24. The diameter of F2 and circulating FSH concentration at Hour 24 were similar (P > 0.1) to the diameter of F1 and FSH concentration at Hour 0, respectively. A differential change between F2 and F3 was not detected in follicular fluid concentrations of estradiol, inhibin-A, and activin-A by the beginning of experimental deviation. However, estradiol was higher in F2 at Hours 0 and 12 and inhibin-A was higher in F2 throughout the experiment, and both factors could have been involved in experimental deviation. Free insulin-like growth factor-1 (IGF-1) increased (P < 0.05) in F2 beginning at Hour 12 and was higher (P < 0.05) in F2 than in F3 by the beginning of experimental deviation. Temporally, this result indicated that intrafollicular IGF-1 was involved in conversion of F2 from a destined subordinate follicle to a dominant follicle.     

Activin A,estradiol, and free insulin-like growth factor I in follicular fluid preceding the experimental assumption of follicle dominance in cattle

In cattle, the two largest follicles of a wave (F1, F2) begin to deviate into a dominant follicle and a subordinate follicle when F1 is a mean of 8.5 mm in diameter. After the beginning of deviation, F1 and F2 are diameter-defined dominant and subordinate follicles. Changes associated with the conversion of F2 into a future dominant follicle were studied by ablating F1 at the expected beginning of deviation (F1, 8.5 mm; Hour 0) and assessing the follicular-fluid factors in F2. Follicles were designated F1C and F2C in controls and F2A in F1-ablated heifers. Follicular-fluid collections were made at Hours 0, 4, 8, or 12 (n = 7 heifers per hour; fluid from F1C, F2C, and F2A; experiment 1) or at Hours 4, 6, 8, 10, or 12 (n = 9 heifers per hour; fluid from F2A; experiment 2). Postablation concentrations of circulating FSH increased (P < 0.05) between Hours 2 and 6. Diameter of F2A increased (P < 0.05) after Hour 8 in both experiments so that the diameter of F2A at Hours 10 or 12 was not different (P > 0.1) from the diameter of F1 at Hour 0. A transient elevation (P < 0.05) in follicular-fluid activin A occurred in F2A at Hour 8 in both experiments. Concentrations of estradiol (P < 0.05) and insulin-like growth factor I (IGF-I; P < 0.1) decreased in F2C by Hour 8. In F2A, the concentrations of both factors began to increase (P < 0.05) after Hours 4 or 8 so that there was no difference (P > 0.1) between F1C and F2A at Hour 12. Concentrations of IGF-I and IGF binding protein 2 (IGFBP-2) in F2A changed in opposite directions at the same hours. No differences between follicles were found for concentrations of progesterone, androstenedione, inhibin A, and inhibin B. The order of events in the conversion of a future subordinate follicle to a future dominant follicle was an increase in systemic FSH, a transient elevation in follicular-fluid activin A, and a simultaneous increase in follicular-fluid estradiol and restoration of an apparent growth-compatible balance of free IGF-I and IGFBP-2.     

Dose-response study of intrafollicular injection of insulin-like growth factor-I on follicular fluid factors and follicle dominance in mares

The effect of insulin-like growth factor-I (IGF-I) on the concentrations of follicular fluid factors during follicle deviation and the development of dominance was studied in mares in two experiments. Transvaginal ultrasound guidance was used for intrafollicular injection and subsequent sequential sampling of follicular fluid. Treatment involved a single injection of IGF-I into the second-largest follicle (F2) at the expected beginning of deviation (Hour 0) based on diameter (> or =20 mm) of the largest follicle (F1). Mares in IGF-I groups were given a dose of 500 microg (experiment 1) or 250, 25, or 2.5 microg (experiment 2). Ablation of F1 at Hour 24 was done in experiment 1, but not in experiment 2. The 500- and 250-microg doses stimulated growth, leading to ovulation of F2 in 10 of 10 and 4 of 5 mares in the two experiments, respectively, compared to 4 of 12 and 0 of 5 in saline-injected controls. These doses prevented (P < 0.05) the increase in IGF binding protein-2 and androstenedione that occurred in F2 of controls and increased (P < 0.05) the concentrations of activin-A, inhibin-A, and vascular endothelial growth factor (VEGF). The 500-microg dose stimulated higher (P < 0.05) concentrations of estradiol, but not until Hour 48, whereas the lower doses were ineffective. In experiment 2, free IGF-I concentrations in F2 at Hour 24 decreased progressively as the dose decreased so that concentrations for the 2.5-microg dose were higher (P < 0.05) than in F2 of controls and similar (not significantly different) to endogenous concentrations in F1. Correspondingly, concentrations of androstenedione in F2 at Hour 24 were lower (P < 0.05) and concentrations of activin-A, inhibin-A, and VEGF were higher (P < 0.05) after treatment of F2 with the 2.5-microg dose than in F2 of controls and were similar to concentrations in F1. Hence, a physiologic intrafollicular dose of IGF-I did not stimulate estradiol production but reduced the production of androstenedione and stimulated the production of activin-A, inhibin-A, and VEGF during follicle selection in mares.     

Selection of the dominant follicle in cattle: role of estradiol

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.     

Concentrations of circulating hormones during the interval between pulses of a PGF2α metabolite in mares and heifers

The temporal relationship of several hormones to a metabolite of prostaglandin F2α (PGFM) was studied in mares and heifers from the beginning of the first PGFM pulse during luteolysis to the end of the second pulse. Mares (n=7) were selected with a 9-h interval between the peaks of the two pulses. In mares, estradiol-17β (estradiol) increased (P<0.05) within each PGFM pulse and plateaued for a mean of 6h between the pulses, resulting in a stepwise estradiol increase. Progesterone decreased linearly (P<0.0001) throughout the intra-pulse and inter-pulse intervals of PGFM. In heifers (n=6), inter-pulse intervals were variable, and therefore Hours 1-4 of the first pulse (Hour 0=PGFM peak) and Hours -4 to -1 of the second pulse were used to represent the mean 8-h interval between peaks of the two pulses. Estradiol increased (P<0.05) during the ascending portion of each PGFM pulse and then decreased (P<0.05) beginning at Hour -1 of the first PGFM pulse and Hour 0 of the second pulse. The 1-h delay during the second pulse was accompanied by an apparent increase in PRL. A transient decrease in estradiol occurred in individuals between PGFM pulses at a mean of 5h after the first PGFM peak, concomitant with a transient LH increase (P<0.05). Results indicated that estradiol plateaued in mares and fluctuated in heifers during the interval between PGFM pulses. Heifers also showed temporal relationships between estradiol and LH and apparently between estradiol and PRL.     

Follicle selection in cattle: dynamics of follicular fluid factors during development of follicle dominance.

Follicle diameter deviation during follicular waves in cattle begins with a reduction in growth rates of developing subordinate follicles, in contrast to the maintenance of a constant growth rate by a developing dominant follicle. In experiment 1, the temporal changes encompassing deviation in concentrations of follicular fluid factors relative to one another in the three largest follicles (F1, F2, and F3) were studied. Follicular fluid samples were collected when F1 reached diameter ranges of 7.0-7.9, 8.0-8.9, 9.0-9.9, and 10.0-10.9 mm (n = 12 per range). The first increase (P < 0.05) in the difference between F1 and F2 for estradiol occurred at the 8.0- to 8.9-mm range, which was one range earlier than for diameter (P < 0.05). Free insulin-like growth factor (IGF)-1 concentrations in F1 were similar among diameter ranges, but concentrations in F1 were higher (P < 0.05) than in F2 for each range except 7.0-7.9 mm. Concentrations of free IGF-1 in F2 decreased (P < 0.05). No significant differences were detected in concentrations of progesterone, androstenedione, total inhibin, and inhibin-A. Averaged over follicles, inhibin-B decreased (P < 0.05) between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges, and activin-A increased (P < 0.05) between the 7.0- to 7.9- and 9.0- to 9.9-mm ranges. However, no differences were found among follicles. In experiment 2, changes associated with the development of dominance by F2 were studied using ablation of F1 at the beginning of expected deviation (F1, 8.5 mm; Hour 0) as the reference point. Follicular fluid factors were compared at Hour 12 between F2 of a control group (F1 intact; n = 10) and an ablated group (F1 ablated; n = 10). Diameter (P < 0.02), estradiol (P < 0.001), free IGF-1 (P < 0.002), and progesterone (P < 0.003) were greater and IGF-binding protein-2 was lower (P < 0.01) in F2 of the ablated group at Hour 12. No differences were detected in concentrations of androstenedione, total inhibin, and inhibin-A. The results of the two experiments indicated, on a temporal basis, that intrafollicular changes in estradiol and the IGF system, but not in the inhibin/activin system, could account for a reported greater FSH responsiveness by the future dominant follicle than by the future subordinate follicles by the beginning of diameter deviation in cattle.     

Follicular and hormonal response to experimental suppression of FSH during follicle deviation in cattle

A near steroid-free fraction of bovine follicular fluid was used to suppress FSH concentrations at the expected time of follicle deviation or when the largest follicle of Wave 1 reached > or = 8.0 mm (actual mean diameter, 8.4 mm; Hour 0). It was hypothesized that the low concentrations of FSH associated with deviation are inadequate for the smaller follicles but are needed for continued growth of the largest follicle. Control heifers (n=8) received 10 mL of saline, and treated heifers (n=16) received either 8.8 mL or 13.3 mL of the follicular-fluid fraction at Hours 0, 12, and 24. Between Hours -48 and 0, FSH concentrations decreased (P<0.05) and diameters of the 4 largest follicles increased (Hour effect, P<0.0001) similarly between groups. Concentrations of LH in the controls increased (P<0.05) between Hours -24 and -12 and decreased (P<0.05) between Hours 8 and 36, demonstrating a transient LH surge encompassing the expected beginning of deviation. In the treated group, a comparable increase in LH occurred before deviation but a decrease did not occur until after Hour 48. By Hour 4.5, the FSH concentrations in the treated group decreased (P<0.05) to below the concentrations in the controls. Suppressed diameter (P<0.001) of the largest follicle was detected at the first post-treatment examination (Hour 12; 7.5 h after FSH suppression) and was accompanied by reduced (P<0.04) systemic estradiol concentrations. The mean growth rates of the 3 smaller follicles in both the treated and control groups began to decrease at Hours -12 to 24 and were not different between groups during Hours 0 to 36. Concentrations of FSH in the treated group returned to control concentrations by Hour 24 (hour of last treatment). A rebound (P<0.05) in concentrations of FSH to >100% above control concentrations occurred by Hour 48 and was accompanied by resumed growth of the largest follicle in 75% of the heifers between Hours 48 and 72. The results demonstrated that the low concentrations of FSH associated with deviation can be further reduced by treatment with a nonsteroidal factor of follicular origin. Transient reduction of FSH concentrations to below the already low control concentrations inhibited the largest follicle but did not further inhibit the smaller follicles. These results support the hypothesis that the low FSH concentrations associated with follicle deviation are below the minimal requirements of the smaller or subordinate follicles but are needed for continued growth of the largest or dominant follicle in cattle.     

Follicle Selection in Cattle: Relationships among Growth Rate, Diameter Ranking, and Capacity for Dominance

Follicles of wave 1 were designated F1, F2, and so forth, according to descending diameter at the expected (F1, > or =8.2 mm) or observed beginning of deviation (Hour 0), as indicated by a reduction in growth rate of F2. During Hours -24 to 0 (experiment 1; n = 34 waves) and Hours -16 to 0 (experiment 2; n = 21), F1 and F2 grew in parallel (no significant differences). During Hours -16 to 0, growth rate was greater (P < 0.05) for F1 (1.4 +/- 0.1 mm/16 h) and F2 (1.0 +/- 0.1) than for F3 (0.6 +/- 0.1) and F4 (0.5 +/- 0.1). During Hours 0 to 16, growth rate was greater (P < 0.05) for F1 (1.4 +/- 0.2 mm/16 h) than for F2 (0.1 +/- 0.1), F3 (0.1 +/- 0.1), and F4 (0.1 +/- 0.2). In experiment 1, zero, one, two, or three largest follicles were ablated by aspiration of contents at Hour 0 (n = 7/group). For heifers with a single dominant follicle, the dominant follicle formed from the largest retained follicle more often when it was >7.0 mm (14 of 15) than when it was <7.0 mm (0 of 10). When the retained follicles were <7.0 mm, the first follicle to reach 7.0 mm became dominant in seven of eight heifers. Mean hour of observed deviation (occurring after Hour 0 in the ablation groups) increased progressively in groups with increasing number of ablated follicles. Plasma concentrations of FSH for groups with one, two, or three ablated follicles increased to a similar extent between Hours 0 and 12. Results supported the following: 1) during the 24 h before the beginning of deviation, small follicles grew more slowly than large follicles and the largest follicles grew in parallel; 2) after ablation of large follicles, the small retained follicles did not deviate until one reached a diameter characteristic of the beginning of deviation; 3) the potential for dominance at the expected beginning of deviation was greatest for the largest follicle and decreased progressively for the smaller follicles but only when the retained follicles were >7.0 mm; and 4) the three largest subordinate follicles began to deviate simultaneously.     

Follicle selection in cattle: follicle deviation and codominance within sequential waves

Follicle deviation during bovine follicular waves is characterized by continued growth of a developing dominant follicle and reduction or cessation of growth of subordinate follicles. Characteristics of follicle deviation for waves with a single dominant follicle were compared between wave 1 (begins near ovulation; n = 15) and wave 2 (n = 15). Follicles were defined as F1 (largest), F2, and F3, according to maximum diameter. No mean differences were found between waves for follicle diameters at expected deviation (F1, > or =8.5 mm; Hour 0) or observed deviation or in the interval from follicle emergence at 4.0 mm to deviation. For both waves, circulating FSH continued to decrease (P < 0.05) after Hour 0, estradiol began to increase (P < 0.05) at Hour 0, and immunoreactive inhibin began to decrease (P < 0.05) before Hour 0. A transient elevation in circulating LH reached maximum concentration at Hour 0 (P < 0.01) in both waves and was more prominent (P < 0.0001) for wave 1. Waves with codominant follicles (both follicles >10 mm) were more common (P < 0.02) for wave 1 (35%) than for wave 2 (4%). Codominants (n = 6) were associated with more (P < 0.05) follicles > or=4 mm and a greater concentration (P < 0.04) of circulating estradiol at Hours -48 to -8 than were single dominant follicles (n = 15). A mean transient increase in FSH and LH occurred in the codominant group at Hour -24 and may have interfered with deviation of F2. In codominant waves, deviation of F3 occurred near Hour 0 (F1, approximately 8.5 mm). A second deviation involving F2 occurred in four of six waves a mean of 50 h after the F3 deviation and may have resulted from a greater suppression (P < 0.05) of FSH in the codominant group after Hour 0. In conclusion, follicle or hormone differences were similar for waves 1 and 2, indicating that the deviation mechanisms were the same for both waves. Waves that developed codominant follicles differed in hormone as well as follicle dynamics.     

Unilateral ablation of follicles ≥ 4 mm leads to compensatory follicle response from the contralateral ovary in heifers

In each of two experiments, heifers were assigned to a control group and a unilaterally ablated (UA) group (n = 6/group). In the UA group, follicles ≥ 4 mm in the left ovary were ablated by transvaginal ultrasound-guided technique at Hour 0 (8:00 AM) on the day of ovulation. Follicles in the CL-bearing right ovary remained intact. In Experiment 1, ablations continued until the next ovulation, and new follicles emerged in the right ovary in 9 of 14 (64%) waves. The number of follicles/wave (combined, 6.4 ± 0.4) did not differ between groups. In Experiment 2, follicles were counted at Hours 0, 4, 8, 12, and 24; the resistance index (RI) for blood flow in the ovarian pedicle was determined at Hours 0 and 12; and blood samples were collected every hour from Hours 0 to 12 and at Hour 24. An increase (P < 0.05) in the number of follicles in the follicle-intact ovary began at Hour 4 with complete compensation by Hour 24. Concentrations of FSH did not change between Hours 0 and 24 in the UA group but decreased (P < 0.05) in the controls by Hour 7. At Hour 12, RI to the right ovary approached being lower (P < 0.06) in the UA group than in the control group. Results indicated that unilateral ablation of follicles ≥ 4 mm led to compensatory follicle response in the follicle-intact ovary, and initially circulatory FSH concentrations were maintained and blood flow to the follicle-intact ovary increased.     

Surges of FSH during the follicular and early luteal phases of the estrous cycle in heifers

Surges of FSH were characterized in each of 12 Holstein heifers using a computerized cycle detector program, and as mean changes averaged over all heifers. Blood samples were collected 6 times a day at 4-h intervals beginning at late diestrus. Concentrations of FSH were adjusted relative to the preovulatory LH peak (Hour 0) and profiled beginning 48 h before and ending 120 h after the LH peak. Peak concentrations of FSH and LH occurred synchronously in 11 of 12 (92%) heifers, and only a 4-h interval separated peak concentrations in the remaining heifer. The FSH surge that was synchronous with the LH surge was designated FSH Surge 1 and was used as a reference to designate other FSH surges. Surge -1 of FSH was detected in 58% of the heifers at mean Hour -21.2, and Surges 2, 3 and 4 were detected in 92%, 92% and 75% of the heifers, respectively, at mean Hours 25.1, 57.8 and 78.7. Mean peak levels and duration of FSH Surges-1, 2, 3 and 4 were significantly lower than for FSH Surge 1. Mean concentrations of FSH significantly increased and decreased before and after the LH peak, resulting from the synchrony between FSH Surge 1 and the LH surge in individual heifers. Additionally, there was a tendency (P < 0.08) for a second and third increase in mean FSH concentrations at Hours 24 and 60, which was attributed to FSH Surges 2 and 3 that occurred in individuals. Peak FSH concentrations of Surge 2 occurred (mean, Hour 25.1) within 8 h of maximal mean concentrations at Hour 24 in 91% of the heifers. Correspondingly, peak FSH concentrations of Surge 3 occurred (mean, Hour 57.8) within 8 h of maximal mean concentrations at Hour 60 in 64% of the heifers. Surges -1 and 4 of FSH occurred less frequently and at various times within and among heifers compared with Surges 1 to 3; therefore, they were not detected as mean increases in FSH concentrations but were masked as a result of concentrations being averaged over all heifers. In summary, FSH surges were detected in individual heifers before and after the combined FSH/LH surge. The interpeak intervals for FSH Surges 1 to 2 (25 h), 2 to 3 (33 h) and 3 to 4 (21 h) suggests a rhythmic nature to the surges.     

Disruption of the periovulatory LH surge by a transient increase in circulating 17β-estradiol at the time of ovulation in mares

The mechanism for a reported temporal association between ovulation and a transient disruption in the periovulatory increase in LH concentrations was studied in nine mares treated with human chorionic gonadotropin when the preovulatory follicle was ≥32 mm. Examinations for ovulation detection and blood collection were done at 2-h intervals and the results were retrospectively centralized to ovulation (Hour 0). Concentrations of LH began to increase (P < 0.03) rapidly at Hour ?18, decreased (P < 0.04) between Hours 0 and 6, and again increased (P < 0.0001) after Hour 12. A progressive decrease (P < 0.0001) in estradiol between Hours ?30 and 24 was interrupted temporarily by an increase (P < 0.01) between Hours ?2 and 0 and a decrease (P < 0.007) between Hours 0 and 2. Results indicated that a disruption and depression in the periovulatory LH surge occurred at the time of detected ovulation in mares and was temporally associated with a transient increase in estradiol during an overall progressive decline. The disruption in LH concentrations is attributable to the estradiol increase, based on a reported negative effect of estradiol on LH. The source of the circulating estradiol was likely from the discharge of estradiol-laden follicular fluid into the abdomen during ovulation and rapid absorption of estradiol into the circulation.     

Disruption of periovulatory FSH and LH surges during induced anovulation by an inhibitor of prostaglandin synthesis in mares

The role of passage of follicular fluid into the peritoneal cavity during ovulation in the transient disruption in the periovulatory FSH and LH surges was studied in ovulatory mares (n=7) and in mares with blockage of ovulation by treatment with an inhibitor of prostaglandin synthesis (n=8). Mares were pretreated with hCG when the largest follicle was ≥32 mm (Hour 0). Ultrasonic scanning was done at Hours 24 and 30 and every 2h thereafter until ovulation or ultrasonic signs of anovulation. Blood samples were collected at Hours 24, 30, 32, 34, 36, 38, 48, and 60. Ovulation in the ovulatory group occurred at Hours 38 (five mares), 40, and 44. Until Hour 36, diameter of the follicle and concentrations of FSH, LH, and estradiol-17β (estradiol) were similar between groups. Between Hours 34 and 36, a novel transient increase in estradiol occurred in each group, and color-Doppler signals of blood flow in the follicular wall decreased in the ovulatory group and increased in the anovulatory group. In each group, FSH and LH periovulatory surges were disrupted by a decrease or plateau between Hours 38 and 48 and an increase between Hours 48 and 60. The discharge of hormone-laden follicular fluid into the peritoneal cavity at ovulation was not an adequate sole explanation for the temporally associated transient depression in FSH and LH. Other routes from follicle to circulation for gonadotropin inhibitors played a role, based on similar depression in the ovulatory and anovulatory groups.     

Stimulation of pulses of 13,14-dihydro-15-keto-PGF2α (PGFM) with estradiol-17β and changes in circulating progesterone concentrations within a PGFM pulse in heifers

A single physiologic dose (0.1 mg) of estradiol-17β in sesame-oil vehicle or vehicle alone (n = 8) was given to heifers on day 14 after ovulation to study the effect on circulating 13-14-dihydro-15-keto-PGF2α (PGFM), PGFM pulses, and changes in progesterone concentrations within a PGFM pulse. Blood samples were collected hourly for 16 h after treatment. The estradiol group had a greater mean concentration of PGFM, greater number of heifers with PGFM pulses and number of pulses/heifer, and greater prominence of the PGFM pulses. Changes in progesterone concentrations were not detected during the 16 h sampling session in the vehicle group, indicating that the heifers were in preluteolysis. Progesterone decreased after 12 h in the estradiol group, indicating a luteolytic effect of the estradiol-induced PGF secretion as represented by PGFM concentrations. Intrapulse changes in progesterone were detected during a PGFM pulse in the estradiol group (P < 0.006), but not in the vehicle group. Progesterone increased (P < 0.01) between Hours −2 and −1 of an estradiol-induced PGFM pulse (Hour 0 = peak of pulse), decreased (P < 0.004) between Hours −1 and 0, and increased (P < 0.01) or rebounded between Hours 0 and 1. Results were compatible with previous reports of a role for estradiol in the induction of PGFM pulses in cattle and demonstrated intrapulse changes in progesterone concentrations during an induced PGFM pulse.     

Suppression of circulating concentrations of FSH and LH by inhibin and estradiol during the initiation of follicle deviation in mares

The role of estradiol and inhibin in suppression of FSH and LH during the initiation of follicle deviation was examined in mares. In Experiment 1, the two largest follicles (F1, F2) were retained during a wave and the rest were ablated as they reached > or =10 mm. The largest follicle was left intact (control, n=12) or was ablated when it reached > or =20.0 mm (Day 0; expected beginning of deviation). The second largest follicle continued growing (n=9) or regressed (n=4) after F1 ablation. Circulating estradiol and total inhibin decreased after Day 0 in the F2-regressing group, whereas estradiol increased after Day 0.5 and inhibin was unaltered in the control and F2-growing groups. Circulating FSH decreased in the control group and increased in the F2-regressing group after Day 0. In the F2-growing group, FSH increased between Days 0 and 0.5 and then decreased. Circulating LH increased between Days 0 and 2 in the F2-regressing group and between Days 0 and 0.5 in the F2-growing group. In Experiment 2, 0 or 1 follicle was retained in a wave followed by administration of 0 or 1 mg of estradiol at the expected beginning of deviation (Hour 0; 2 x 2 factorial design, n=4-6/group). Circulating total inhibin was higher and FSH was lower at Hour 0 in the 1-follicle than in the 0-follicle groups. Follicle-stimulating hormone decreased after Hour 0 in the 1-mg but not in the 0-mg groups, and the decrease in the 0-follicle/1-mg group was not to the level of that in the 1-follicle/1-mg group. Circulating LH was not affected by follicle number but was reduced by estradiol. Results supported the hypotheses that F1 near the beginning of deviation produces inhibin and estradiol and that the increase in circulating estradiol at the beginning of deviation induces FSH suppression in combination with other follicle substances (presumably inhibin). Results also indicated that the increase in estradiol induces suppression of LH.     

Critical role of insulin-like growth factor system in follicle selection and dominance in mares

The role of the insulin-like growth factor (IGF) system in the deviation in growth rates among follicles (follicle selection) was studied in mares using an IGF binding protein (BP) to reduce the follicular-fluid concentrations of IGFs. The future dominant follicle (F1) was treated by intrafollicular injection at the expected beginning of deviation (F1 > or = 20 mm; Day 0). The experimental groups were control (no injection, n = 8), vehicle (injection of vehicle; n = 6), and BP (injection of 250 microg of recombinant human IGFBP-3; n = 6). A sample of follicular fluid was taken from F1 on Day 1 in all groups. Compared with the control group, IGFBP-3 reduced (P < 0.05) the follicular-fluid concentration of free IGF-1 by 90%; lowered (P < 0.05) the concentrations of estradiol, activin-A, inhibin-A, and vascular endothelial growth factor; and increased (P < 0.05) the concentration of androstenedione. The diameter of F1 decreased and the diameter of F2 increased after Day 0 in the BP group, compared with the control and vehicle groups. A greater (P < 0.05) increase in circulating concentrations of FSH between Days 0 and 1 occurred in the BP group than in the other groups and accounted for the increased growth of F2. Dominance and ovulation from F1 occurred from fewer (P < 0.03) mares in the BP group (1 of 6) than from the control and vehicle groups combined (11 of 14); the remaining mares in the BP group ovulated from F2. Results indicated that the IGF system has a critical intrafollicular role in the differential changes in concentrations of follicular-fluid factors between the future dominant and subordinate follicles, leading to the development of follicle dominance (selection) and ovulation in mares.     

Associated and independent comparisons between the two largest follicles preceding follicle deviation in cattle

Follicle diameters and concentrations of follicular fluid factors were studied in the two largest follicles (F1 and F2) using F1 diameters in increments of 0.2 mm (equivalent to 4 h intervals) and extending from 7.4 to 8.4 mm (12 heifers in each of 6 groups). Changes were compared between follicles using the F2 associated with each F1-diameter group. Diameter deviation began in the 8.2-mm group as indicated by a greater (P < 0.05) diameter difference between F1 and F2 in the 8.4-mm group than in the 8.2-mm group. In the 8.0-mm group, estradiol concentrations began to increase (P < 0.05) differentially in F1 versus F2, and free insulin-like growth factor-1 (IGF-1) began to decrease differentially in F2 (P < 0.06). Combined for F1 and the associated F2, activin-A concentrations increased (P < 0.05) between the 7.6- and 8.2-mm groups and then decreased (P < 0.05). Results supported the hypothesis that estradiol and free IGF-1 concentrations simultaneously become higher in F1 than in the associated F2 by the beginning of diameter deviation. Results did not support the hypothesis that a transient elevation in activin-A is present in F1 but not in the associated F2 at the beginning of the estradiol and IGF-1 changes; instead, a mean transient elevation in activin-A occurred at this time only when data for the two follicles were combined. Comparisons between F1 and F2 also were made by independently grouping F2 and using diameter groups at 0.2-mm increments for F2 as well as for F1. In the diameter groups common to F1 and F2 (7.4, 7.6, 7.8, and 8.0 mm) there was a group effect (P < 0.003) for estradiol involving an increase (P < 0.05) beginning at the 7.6-mm group averaged over F1 and F2. For free IGF-1 concentrations, a fluctuation (a significant increase followed by a significant decrease) occurred independently in F1 between the 7.4- to 7.8-mm groups and independently in F2 between the 7.0- to 7.4-mm groups.     

Selection of the dominant follicle in cattle: establishment of follicle deviation in less than 8 hours through depression of FSH concentrations

Deviation in follicle diameter in cattle is characterized by continued growth of the largest follicle of a follicular wave and a reduction or cessation of growth of the smaller follicles. Deviation begins when the largest follicle reaches about 8.5 mm. Two experiments were done to test the hypothesis that the deviation mechanism is established in < 8 h, as indicated by the temporal relationships between follicle removal and an increase in FSH concentrations (Experiment 1) and between a decrease in FSH concentrations and follicle inhibition (Experiment 2). In Experiment 1, the role of the first follicle to reach 8.5 mm was studied by follicle ablation (Hour 0). The combined mean FSH concentrations for the control group (n = 8) and ablation group before ablation (n = 7) progressively decreased (P < 0.02) over two 8-h intervals before the largest follicle reached > or = 8.5 mm (Hour-16, 1.77 +/- 0.11 ng/mL; Hour 0, 1.49 +/- 0.08 ng/mL). In controls, the concentrations continued to decrease (P < 0.02) until Hour 10 (1.21 +/- 0.09 ng/mL). Ablation of the largest follicle at > or = 8.5 mm resulted in increased (P < 0.02) circulating FSH concentrations between Hours 5 (1.34 +/- 0.04 ng/mL) and 8 (1.61 +/- 0.09 ng/mL). Growth rate of the second-largest follicle between Hours 0 and 8 was greater (P < 0.05) in the ablation group than in the controls, and the second largest follicle became dominant in 7 of 7 heifers following ablation of the largest follicle. In Experiment 2, a minimal single injection of a depressant of FSH concentrations (4.4 mL of steroid-reduced follicular fluid) was given when the largest follicle was a mean of 8.4 mm (Hour 0; controls, n = 4; treated, n = 4). An interaction of group and hour (P < 0.005) for FSH concentrations was attributable to an FSH decrease (P < 0.002) by Hour 6 and an increase (P < 0.002) between Hours 9 and 12 in the treated group. The growth rate of the largest follicle between Hours 0 and 12 was less (P < 0.05) in the treated group (0.2 +/- 0.2 mm/12 h) than in the control group (1.2 +/- 0.4 mm/12 h). The reduced diameter was recorded within 6 h after suppression of FSH concentrations, supporting the hypothesis. Our preferred interpretation is that when the largest follicle reaches a critical diameter of about > or = 8.5 mm, FSH concentrations continue to decrease and become lower than required by the smaller follicles but not the largest follicle. The results further indicate that a close temporal coupling between a change in FSH concentrations and the follicular response could establish the deviation mechanism in < 8 h or before the second largest follicle reaches a similar critical diameter.     

Changes in concentrations of follicular fluid factors during follicle selection in mares

The temporal relationships in the changes in concentrations of follicular fluid factors during follicle selection were characterized in mares. All follicles > or =5 mm were ablated 10 days after ovulation, followed by follicular fluid collection from the three largest follicles (F1, F2, and F3) when F1 of the new wave reached a diameter of 8.0-11.9, 12.0-15.9, 16.0-19.9, 20.0-23.9, 24.0-27.9, or 28.0-31.9 mm (n = 4-8 mares/range). Diameter deviation between F1 and F2 began during the 20.0- to 23.9-mm range, as indicated by a greater difference in diameter between the two follicles at the 24.0- to 27.9-mm range than at the 20.0- to 23.9-mm range. Androstenedione concentrations increased in F1, F2, and F3 between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges. In contrast, estradiol, free insulin-like growth factor (IGF)-1, activin-A, and inhibin-A concentrations increased only in F1 beginning at the 16.0- to 19.9-mm range. As a result, the concentrations of all four factors were higher in F1 than in F2 and F3 at all the later ranges, including the 20.0- to 23.9-mm range (beginning of diameter deviation). Concentrations of progesterone differentially increased in F1, concentrations of androstenedione and IGF-binding protein (IGFBP)-2 increased only in F2 and F3, and concentrations of inhibin-B differentially decreased in F2 and F3 simultaneous with the beginning of deviation. Concentrations of FSH, LH, pro-alphaC inhibin, and total inhibin did not change differentially among follicles. Results indicated that, on a temporal basis, estradiol, free IGF-1, activin-A, and inhibin-A may have played a role in the initiation of follicle deviation. In addition, these four factors as well as progesterone, androstenedione, IGFBP-2, and inhibin-B may have been involved in the subsequent differential development of the follicles.     

Concentrations of circulating hormones normalized to pulses of a prostaglandin F2α metabolite during spontaneous luteolysis in mares

The temporal relationships between a pulse of 13,14-dihydro-15-keto-PGF_2α (PGFM) and the concentrations of circulating hormones during the luteolytic period were studied for 11 pulses in 11 mares (Equus caballus) using samples collected hourly. Mean PGFM pulses encompassed 4 h before to 4 h after the peak, and hormone data were normalized to the PGFM peak (Hour 0). Concentration of progesterone decreased (P < 0.05) between Hours –4 and –3 and continued to decrease linearly throughout the PGFM pulse. The concentrations of cortisol and prolactin increased (P < 0.004) during Hours –4 to 0 and decreased (P < 0.002) during Hours 0 to 4. Estradiol concentration increased (P < 0.02) during Hours –4 to 0 but did not change significantly after Hour 0. Concentrations of follicle-stimulating hormone and luteinizing hormone did not change significantly during the PGFM pulse, and the oxytocin results were equivocal. Percentage of corpus luteum area with color-Doppler signals of blood flow did not change significantly between Hours –4 and 0 and first began to decrease (P < 0.004) between Hours 0 and 2. Results demonstrated that concentrations of progesterone decreased linearly during a PGFM pulse, and cortisol, prolactin, and estradiol increased during the ascending portion of the pulse. The progesterone and gonadotropin results supported the hypothesis that the initial progesterone and gonadotropin increases that have been reported to occur in response to a single bolus luteolytic treatment with prostaglandin F_2α do not occur in response to the natural secretion of prostaglandin F_2α.