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.     

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.     

In vivo effects of an intrafollicular injection of insulin-like growth factor 1 on the mechanism of follicle deviation in heifers and mares

In cattle and mares, free insulin-like growth factor 1 (IGF-1) is higher in the future dominant follicle (F1) than in the future largest subordinate follicle (F2) before deviation in diameter or selection is manifested between the two follicles. The effect of IGF-1 on other follicular-fluid factors and on the destiny of F2 were studied in two experiments in each species, using a total of 40 heifers and 42 mares. An injection of IGF-1 was made into F2 at the expected beginning of deviation (heifers, F1 >or= 8.5 mm; mares, F1 >or= 20.0 mm; Hour 0). In heifers, follicular fluid was taken from F2 at Hours 3, 6, 12, or 24; each heifer was sampled only once. In mares, sequential F2 samples were taken from each mare at Hours 0, 6, and 24 or at Hours 12 and 24. Transvaginal ultrasound guidance was used for treatment and sample collection. In heifers, IGF-1 treatment of F2 stimulated the secretion of estradiol (P < 0.05) between Hours 3 and 6 and androstenedione (P < 0.05) between Hours 3 and 12. In F2 of control heifers, estradiol decreased (P < 0.05) and androstenedione did not change significantly. In mares, IGF-1 treatment of F2 did not affect the concentrations of estradiol during the 24-h posttreatment period; androstenedione decreased (P < 0.04) in the IGF-1 group and increased (P < 0.006) in the controls. Compared with control mares, the IGF-1 group had higher (P < 0.04) activin-A at Hours 12 and 24 and higher (P < 0.0006) inhibin-A at Hour 24. After ablating F1 at Hour 24 in mares, F2 became dominant and ovulated in more mares (P < 0.0002) in the IGF-1 group (12/14) than in the control group (2/14). These results are consistent with reported temporal relationships among follicular factors during deviation in both species and indicate that IGF-1 plays a key role in controlling the temporal relationships; however, no indication was found that IGF-1 stimulated estradiol production in mares during the 24 h after treatment.     

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.     

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.     

Differential blood flow changes between the future dominant and subordinate follicles precede diameter changes during follicle selection in mares

Diameter deviation during a follicular wave is characterized by the continued growth of the developing dominant follicle and reduced growth and regression of the subordinate follicles. This study considered the hypothesis that reduced blood flow in the future largest subordinate follicle precedes the beginning of diameter deviation. The hypothesis was tested by quantifying the daily changes in blood-flow velocities and blood-flow area within the wall of follicles before and during diameter deviation in mares (n = 7). The blood-flow end points were quantified daily by transrectal color Doppler ultrasonography. Follicles were identified retrospectively by rank as F1 (largest) and F2 according to the maximum attained diameter. Follicles were grouped into nine F1 diameter ranges of 3.0 mm each (equivalent to 1 day's growth) centered on 6.5, 9.5, 12.5, 15.5, 18.5, 21.5, 24.5, 27.5, and 30.5 mm. Diameter deviation began in the 24.5-mm group, as indicated by a smaller (P < 0.05) difference between F1 and F2 in the 24.5-mm group than in the 27.5-mm group. Based on a similar approach, peak systolic velocity and time-averaged maximum velocity of blood flow began to deviate between F1 and F2 in the 18.5-mm group (P < 0.04) and blood flow area began to deviate in the 21.5-mm group (P < 0.009). Thus, differential blood flow area between F1 and F2 began an average of 3.0 mm (equivalent to 1 day) and differential blood-flow velocities began an average of 6.0 mm before the beginning of diameter deviation. The results demonstrated that deviation between F1 and F2 in the blood flow of the follicle walls occurred 1 or 2 days before deviation in follicle diameter during follicle selection in mares.     

Follicular-fluid factors and granulosa-cell gene expression associated with follicle deviation in cattle

Intrafollicular changes in the largest follicle (F1) and second-largest (F2) follicle were examined in relation to follicle diameter deviation. Deviation is characterized by continued growth of the largest follicle and the cessation of growth of the smaller follicles. Granulosa cells and follicular fluid were obtained from slaughterhouse ovaries (n = 95 pairs, experiment 1), and follicular fluid was collected in vivo (n = 28 heifers, experiment 2). Several ranges in the diameter of F1 were used to represent the progressive growth of the follicle. The diameter range with the first significant increase in the difference between F1 and F2 was determined for each end point and was used as an indicator of the sequence of events associated with diameter deviation. An increased difference for diameter and for estradiol concentration occurred (P: < 0.05) simultaneously at the 8.5- to 8.9-mm range in both experiments. In experiment 1, the increased difference between F1 and F2 in LH receptor (LHr) mRNA expression occurred (P: < 0.05) at the 8.0- and 8.4-mm range. In F2 of experiment 2, there was a progressive decrease (P: < 0.05) in free insulin-like growth factor (IGF)-1 and a progressive increase (P: < 0.05) in IGF binding protein (BP)-2 across the follicle-diameter ranges (7.5-11.2 mm). No differences were detected between F1 and F2 for 3beta-hydroxysteroid dehydrogenase mRNA expression in experiment 1 and testosterone, total inhibin, and dimeric inhibin-A concentrations in experiment 2. The results indicated that the acquisition of granulosa cell LHrs by F1, as indicated by increased LHr mRNA expression, occurred one diameter range before an increased difference between F1 and F2 for diameter or estradiol concentrations. On a temporal basis, it is concluded that LHr acquisition plays a role in the establishment of diameter deviation. In addition, the reduced growth of F2 may have involved the reduced bioavailability of IGF-1 in association with elevated IGFBPs.     

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.     

Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle

Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 +/- 1, 33 +/- 2, 53 +/- 1, and 84 +/- 2 h after the periovulatory peak in FSH concentrations. Follicles > or = 2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r > or = 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (> or = 48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.     

Follicle deviation and intrafollicular and systemic estradiol concentrations in mares.

By definition, follicle deviation begins on the day the two largest follicles of a wave begin to differ in growth rates. The relationships between follicle deviation and intrafollicular and systemic estradiol concentrations were studied in ponies, using a two-follicle model in which all but the two largest follicles were ablated. A 20-microliter sample of follicular fluid was obtained from each of the two follicles by transvaginal ultrasonography. In experiment 1, the two follicles were sampled when the larger follicle reached 15 mm. No differences (p > 0.05) in post-sampling follicle characteristics were found between control (n = 6) and sampled (n = 8) groups except that the growth rate was slower (p < 0.01) in the larger follicle between the day of sampling and the next day (0.7 +/- 0.7 mm per day) than in the controls (3.3 +/- 0.3 mm per day). The growth rates between 2 and 5 days after sampling were not different between groups. Follicular fluid estradiol-17beta concentrations were higher (p < 0.007) in the larger follicle (460 +/- 67 ng/ml; diameter, 16.4 +/- 0.4 mm) than in the smaller follicle (322 +/- 50 ng/ml; diameter, 14.6 +/- 0.6 mm). In experiment 2, the pair of follicles was sampled when the larger follicle reached 15 mm, 20 mm, or 25 mm (n = 5 per group). There were no significant differences among the three groups for day of deviation and diameters of larger and smaller follicles at deviation. The difference in diameter between the larger and smaller follicles was similar for the 15-mm (2.2 +/- 0.9 mm) and 20-mm (3.1 +/- 1.0 mm) groups, but the difference between follicles for the 25-mm group (7.9 +/- 1.2 mm) was greater (p < 0.004) than for the other two groups. In contrast, the differences in estradiol concentrations between the larger and smaller follicles increased (p < 0.0001) progressively for the 15-mm (13.0 +/- 86.8 ng/ml), 20-mm (722.0 +/- 173.8 ng/ml), and 25-mm (1873.5 +/- 310.3 ng/ml) groups. The first significant (p < 0.007) increase in systemic estradiol occurred between the day before and the day of the beginning of deviation. Detection of an increased difference in estradiol concentrations between the two follicles before the detection of a change in differences in diameter suggests, on a temporal basis, that estradiol is a candidate for involvement in the mechanism that leads to follicle-diameter deviation in mares.     

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.     

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.     

Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers (n = 35) were ovariectomized at 36 (n = 7), 66 (n = 8), 84 (n = 12) and 108 (n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment (n = 6) or were treated with a progesterone-releasing intravaginal device (n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1-F4). Mean diameter of the F1 follicle increased (P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest (P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower (P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108h, but amounts of follicular fluid IGFBP-4 were lower (P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher (P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.     

Aberrant blood flow area and plasma gonadotropin concentrations during the development of dominant-sized transitional anovulatory follicles in mares

Color Doppler transrectal ultrasound was used to evaluate blood flow area in the wall of dominant anovulatory follicles versus ovulatory follicles in mares during the transition between anovulatory and ovulatory seasons. Daily examinations were done in 11 control mares toward the end of the anovulatory season. In 13 separate mares, follicular fluid was collected from 30-mm follicles, and blood flow areas from control mares were used as a basis for designating the sampled follicle as either anovulatory or ovulatory. Blood flow area in the controls ranged from 0.18 to 0.35 cm(2) in six mares on the day of a 30-mm anovulatory follicle and from 0.25 to 0.86 cm(2) in 11 mares on the day of a 30-mm ovulatory follicle; the ranges did not overlap except for one follicle. In the controls, mean blood flow area was lower (P < 0.05) in the anovulatory group than in the ovulatory group for each day beginning with the first Doppler examination at 25 mm. For plasma LH in controls, an effect of follicle group (P < 0.0001) and an interaction (P < 0.0001) of group by day reflected lower (P < 0.05) concentrations in the anovulatory group on Days -6, -2, and 5-8 (Day 0 = 30-mm follicle). For plasma FSH, an interaction (P < 0.0001) reflected higher (P < 0.05) concentrations in the anovulatory group on Days -3 and 1-4. More (P < 0.05) statistically identified FSH surges occurred in the anovulatory group during Days -7 to 8. In the sampled mares, follicular-fluid concentrations of estradiol, free insulin-like growth factor-1, inhibin-A, and vascular endothelial growth factor were lower (P < 0.05) in 30-mm designated anovulatory follicles than in 30-mm designated ovulatory follicles. Results were interpreted as follows: 1) The future anovulatory dominant-sized follicle developed under an LH deficiency, 2) the LH deficiency led to reductions in blood flow area and in concentrations of follicular-fluid factors, and 3) the reduction in follicle production of FSH suppressors resulted in higher plasma FSH concentrations.     

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: role of luteinizing hormone

The circulating concentrations of LH were reduced by administration of 50 mg of progesterone every 8 h for 72 h, beginning when the largest follicle was 6.0 mm (experiment 1; n = 10). Progesterone treatment prevented the transient increase in LH that accompanied deviation (partitioning into dominant and subordinate categories) in control heifers (n = 10). The reduced LH concentrations were associated with reduced growth of the largest follicle, beginning a mean of 31 h after deviation, but did not alter the time of deviation or the growth and regression of the second-largest follicle. In experiment 2, 0 mg (controls) or 50 mg of progesterone was given every 8 h for three injections, beginning when the largest follicle was 7.0 mm (predeviation group) or 9.0 mm (postdeviation group; n = 8 for each of the four groups). Blood samples from the jugular vein and follicular-fluid samples from the two largest follicles were taken 8 h after the last treatment when the largest follicle was a mean of 8.7 mm in the predeviation group and 10.8 mm in the postdeviation group. In the controls, follicular-fluid concentrations of estradiol and free insulin-like growth factor (IGF)-1 in the largest follicle and IGF binding protein (IGFBP)-2 in the second-largest follicle were higher (P: < 0.05) in the postdeviation group than in the predeviation group. Progesterone treatment lowered (P: < 0.006) the circulating LH concentrations to a similar extent in both groups. In the predeviation group, progesterone treatment did not have a significant effect on any of the characteristics of the largest follicle. In the postdeviation group, the largest follicle of the progesterone-treated heifers had significant reductions in diameter and in follicular-fluid concentrations of estradiol and free IGF-1. Follicular-fluid concentrations of immunoreactive inhibin were not different for any of the comparisons. The results supported the hypothesis that LH has a positive effect on diameter of the largest follicle but not until after the beginning of diameter deviation. In addition, the results indicated that LH is involved in the production of estradiol by the largest follicle and that free IGF-1 concentrations increase in the largest follicle during deviation.     

Growth hormone priming as an adjunct treatment in superovulatory protocols in the ewe alters follicle development but has no effect on ovulation rate

This study investigated the effect of FSH alone and rGH priming followed by FSH treatment on follicle populations, follicular fluid concentrations of components of the IGF system and steroids, and the ovulation rate in sheep. Estrus was synchronized with progestagen sponges. Ewes (n = 10/group) in Group 1 served as untreated controls, while those in Groups 2 to 5 received a standard superovulatory treatment of 1.1 mg i.m. oFSH twice daily for 4 d. In addition, ewes in Groups 3 and 5 were administered rGH (15 mg/d, i.m.) for the 7 d prior to FSH treatment. Groups 1, 2 and 3 were sacrificed just prior to the LH surge; Groups 4 and 5 were allowed to ovulate. Daily plasma samples were collected to monitor GH, IGF-1 and insulin levels. All follicles > or = 1.0 mm from Groups 1, 2 and 3 were counted, and follicular fluid from follicles > or = 2.5 mm was assayed for estradiol, testosterone, IGF-1 and IGFBPs. Compared with the control, treatment with rGH + FSH but not FSH alone increased (P < 0.001) plasma concentrations of GH, IGF-1 and insulin. The mean number of large-(> or = 4.5 mm) and medium-sized (2.5 to 4.0 mm) follicles was increased (P < 0.01), and the mean number of small (< or = 2.0 mm) follicles was decreased (P < 0.001) by FSH treatment. The mean number of medium-sized (2.5 to 4.0 mm) follicles was further increased (P < 0.05) by rGH priming. Estradiol concentration in medium but not in large estrogenic follicles was increased (P < 0.05) by rGH priming, whereas testosterone concentration in estrogenic follicles was not altered. Components of the IGF system in medium-sized estrogenic follicles were similar in all treatment groups; however, in large estrogenic follicles rGH increased IGF-1 concentrations (P < 0.05) and intensity of the 44-42 kDa IGFBP band (P < 0.01). Priming with rGH did not alter superovulatory responses. These results show that rGH priming, when used as an adjunct to FSH treatment in ewes, alters components of the IGF system in large estrogenic follicles and increases the number and physiological maturity of medium-sized follicles in the ovary; it does not however alter ovulation rate responses.     

Regulation of circulating gonadotropins by the negative effects of ovarian hormones in mares

The functional and temporal relationships between circulating gonadotropins and ovarian hormones in mares during Days 7-27 (ovulation = Day 0) was studied using control, follicle ablation, and ovariectomy groups (n = 6 mares/group). In the follicle-ablation group, all follicles > or = 6 mm were ablated on Day 7, and every 2 days thereafter, newly emerging follicles were also ablated. Estradiol concentrations decreased (P < 0.01) similarly in the controls and the follicle-ablation group between Days 7 and 11 and by Day 15 began to increase in the controls and continued to decrease in the follicle-ablation group. Concentrations of progesterone were not affected by follicle ablation, but diameter of the corpus luteum was greater (P < 0.05) by Day 21 in the follicle-ablation group; these results indicated that the follicles were involved in morphologic luteolysis, but not in functional luteolysis. Concentrations of LH were higher (P < 0.05) on Days 15 and 16 in the follicle-ablation group than in the controls, indicating an initial negative effect of follicles on LH. Immunoreactive inhibin and estradiol decreased (P < 0.0001) and FSH and LH increased (P < 0.05) within 1 or 2 days after ovariectomy; these changes occurred more slowly in the follicle-ablation group. The maximum value for an FSH surge in each control mare was below the lower 95% confidence limit in the ovariectomy group. Maximum concentration for the periovulatory LH surge in the controls was not different from the mean maximum LH concentrations in the ovariectomy group. Our interpretation is that the gonadotropin surges resulted from changes in the magnitude of the negative effects of ovarian hormones on the positive effects of extraovarian control. There was no indication of a positive ovarian effect on either FSH or LH.     

Developmental pattern of small antral follicles in the bovine ovary

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).     

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.