Morphological characterization of follicle deviation in Nelore (Bos indicus) heifers and cows

Follicle diameter deviation is defined as the beginning of the differential change in growth rates between the largest and next largest follicles subsequent to wave emergence and is considered a key component of follicle selection. Follicle selection has been extensively studied in European breeds of cattle (Bos taurus) but has not been critically studied in Zebu breeds (Bos indicus). The objectives of the present study were to determine and compare the morphological characteristics of deviation associated with the first post-ovulatory wave (Wave 1) of the estrous cycle in Nelore heifers (n=8) and nonlactating cows (n=11). Beginning on the day of ovulation (day 0), the three largest follicles (F1-F3, respectively) were individually tracked every 12 h for 6d using transrectal ultrasonography. In individual animals, deviation was determined graphically using visual inspection of the diameter profiles of F1, F2 and sometimes F3 (observed deviation) and mathematically using segmented regression analysis of the diameter differences between F1 and F2 or sometimes F3 (calculated deviation). Mean day of emergence of Wave 1 when F1 reached >3 mm (approximately 1 d after ovulation) and growth rate of F1 during deviation (approximately 1.4 mm/d) were not significantly different between heifers and cows. The results of determining the beginning of deviation within heifers and cows using the observed and calculated methods were not significantly different. Averaged over both methods, diameter deviation occurred 2.8 d after ovulation when F1 reached 5.7 mm in heifers, and 2.4 d after ovulation when F1 reached 6.1 mm in cows. In conclusion, the emergence of Wave 1 and growth rates and diameters of the future dominant follicles at the beginning of deviation were similar in Nelore heifers and nonlactating cows, regardless of the methods used to determine deviation. Relative to Holstein cattle, emergence of Wave 1 appeared to occur about 1 d later and diameter of the future dominant follicle at the beginning of deviation was about 2 mm smaller in Nelore.     

Follicle deviation and ovulatory capacity in Bos indicus heifers

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.     

Ultrasonographic and endocrine aspects of follicle deviation, and acquisition of ovulatory capacity in buffalo (Bubalus bubalis) heifers

The objectives of this study were to determine the interval from ovulation to deviation and the diameter of the dominant (DF) and largest subordinate (SF) follicles at deviation in buffalo (Bubalus bubalis) heifers. Two methods of evaluation (observed vs. calculated) were used. FSH and LH profiles encompassing follicle deviation (Experiment 1), and the follicular diameter when the DF acquired ovulatory capacity (Experiment 2) were also determined. The time of deviation and the diameter of the DF and the largest SF at deviation did not differ between observed and calculated methods. Overall, follicle deviation occurred 2.6 ± 0.2d (mean ± SEM) after ovulation, and the diameters of the DF and SF at deviation were 7.2 ± 0.2 and 6.4 ± 0.2mm, respectively. No changes in plasma levels of FSH or LH were observed (P=0.32 and P=0.96, respectively). Experiment 2 was conducted in two phases according to the diameter of the DF during the first wave of follicular development at the time of LH challenge (25mg of pLH). In the first phase, follicles ranging from 5.0 to 6.0mm (n=7), 6.1 to 7.0mm (n=11), or 7.1 to 8.0mm (n=9) were used, and in the second phase, follicles ranging from 7.0 to 8.4mm (n=10), 8.5 to 10.0mm (n=10), or 10.1 to 12.0mm (n=9) of diameter were used. After the pLH treatment, the DF was monitored by ultrasonography every 12h for 48h. No ovulations occurred in heifers in the first phase. However, in the second phase, an effect of follicular diameter was observed on ovulation rate [7.0-8.4mm (0.0%, 0/10), 8.5-10.0mm (50.0%, 5/10), and 10.0-12.0mm (55.6%, 5/9)]. In summary, follicle deviation occurred 2.6d after ovulation in buffalo (B. bubalis) heifers, when the diameters of the DF and SF were 7.2 and 6.4mm, respectively. No significant changes in plasma concentrations of FSH or LH were detected. Finally, the acquisition of ovulatory capacity occurred when the DF reached 8.5mm in diameter.     

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.     

Comparative study of the dynamics of follicular waves in mares and women

Deviation in growth rates of the follicles of the ovulatory wave begins at the end of a common growth phase and is characterized by continued growth of the developing dominant follicle (F1) and regression of the largest subordinate follicle (F2). Follicle diameters during an interovulatory interval were compared between 30 mares and 30 women, using similar methods for collecting and analyzing data. Follicles were tracked and measured daily by ultrasonography. Diameter at follicle emergence (mares, 13 mm; women, 6 mm) and the required minimal attained diameter for assessment of follicles (mares, 17 mm; women, 8 mm) were chosen to simulate the reported ratio between the two species in mean diameter of F1 at the beginning of deviation (mares, 22.5 mm; women, 10.5 mm). F1 emerged before F2 (P < 0.02) in each species, and the interval between emergence of the two follicles was similar (not significantly different) between species. Growth rate for F1 and F2 during the common growth phase was similar within species, and the percentage of diameter increase was similar between species. Proportionality between species in diameter of F1 at deviation (2.2 times larger for mares than for women) and at maximum preovulatory diameter (2.1 times larger) indicated that relative growth of F1 after deviation was similar between species. A predeviation follicle was identified in 33% of mares and 40% of women and was characterized by growth to a diameter similar to F1 at deviation but with regression beginning an average of 1 day before the beginning of deviation. The incidence of a major anovulatory wave preceding the ovulatory wave was not different between species (combined, 25%). Results indicated that mares and women have comparable follicle interrelationships during the ovulatory wave, including 1) emergence of F1 before F2, 2) similar length of intervals between sequential emergence of follicles within a wave, 3) similar percentage growth of follicles during the common growth phase, and 4) similar relative diameter of F1 from the beginning of deviation to ovulation. Similar follicle dynamics between mares and women indicate the mare may be a useful experimental model for study of folliculogenesis in women, with the advantage of larger follicle size.     

Stimulation of the largest subordinate follicle by intrafollicular treatment with insulin-like growth factor 1 is associated with inhibition of the dominant follicle in heifers

The effect of intrafollicular treatment of the second-largest follicle (F2) with insulin-like growth factor (IGF) 1 on the largest follicle (F1) and F2 was studied in heifers. Treatment of F2 was done when F1 reached ≥8.2 mm (expected beginning of follicle deviation; Day 0 or Hour 0). In each of two experiments, three groups (n = 6 or 7 heifers/group) were used: controls, F2 treated with vehicle and F2 treated with IGF1. The IGF1 treatment consisted of 200 μg of recombinant human IGF1 (pharmacological dose) in 20 μL of vehicle. In Experiment 1, the hypothesis that treatment of F2 with IGF1 has a stimulatory effect on F2 was supported by a greater (P < 0.05) incidence of F2 dominance (≥10 mm) in the IGF1 group (71%) than in the other two groups (8%), and a greater (P < 0.02) growth rate of F2 on Days 0-2. Unexpectedly, treatment of F2 with IGF1 had an inhibitory effect on F1, as indicated by a reduced (P < 0.03) growth rate of F1 during Days 0-1 and Days 0-4 and a lesser (P < 0.05) maximum diameter of F1 in the IGF1 group. In Experiment 2, the hypothesis of an inhibitory effect on F1 when F2 was treated with IGF1 was supported by a lesser (P < 0.04) increase in diameter of F1 and a lesser (P < 0.04) percentage of follicle wall with power-Doppler signals of blood flow between Hours 0 and 14 in the IGF1 group. Circulating concentrations of FSH and LH were not altered significantly in either experiment. In conclusion, treatment of F2 with IGF1 at the expected beginning of deviation had a stimulatory effect on F2, but an inhibitory effect on F1.     

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.     

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.     

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 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.     

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.     

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.     

Response of estradiol and inhibin to experimentally reduced luteinizing hormone during follicle deviation in mares

The increase in LH concentrations at the time of the decrease in FSH concentrations during follicle deviation in mares was studied to determine the role of LH in the production of estradiol and immunoreactive inhibin (ir-inhibin). Ten days after ovulation, all follicles > or =6 mm were ablated, prostaglandin F(2 alpha) was given, and either 0 mg (control group, n = 15) or 100 mg of progesterone in safflower oil (treated group, n = 16) was given daily for 14 days, encompassing the day of diameter deviation. The follicular and hormonal data were normalized to the expected day of the beginning of diameter deviation when the largest follicle first reached > or =20 mm (Day 0). The experimentally induced decrease in LH concentrations during follicle deviation beginning on Day -4 delayed and stunted the increase in circulating concentrations of ir-inhibin and estradiol beginning on Days -3 and -1, respectively, but did not alter the predeviation FSH surge and the initiation of diameter deviation between the two largest follicles. Combined for both groups, the interval to the expected day of deviation was 16.6 days after ovulation when the largest follicle was a mean of 21.6 mm. After deviation, the largest follicle started to regress in the treated group beginning on Day 1 and was associated with decreased concentrations of ir-inhibin and estradiol, and increased concentrations of FSH. The negative influence of the dominant follicle on the postdeviation decrease in FSH observed in the control group was alleviated and concentrations resurged in the treated group. Apparently this is the first in vivo evidence that the increase in LH that precedes follicle deviation has a positive effect in supporting the production of inhibin during diameter deviation. It was concluded that the increase in LH concentrations before diameter deviation played a role in the production of estradiol and inhibin by the largest follicle during deviation.     

Role of increased estradiol on altering the follicle diameters and gonadotropin concentrations that have been reported for double-ovulating heifers

During the preovulatory period in heifers that ovulate from two compared to one follicle, circulating concentrations of estradiol-17β (E2) are greater, diameter of follicles and concentration of FSH are reduced, and the LH surge occurs sooner. The effect of increased E2 on the reported characteristics of double ovulation was studied by treating heifers with 0.07 mg E2, 0.09 mg E2, or vehicle in four treatments at 6-h intervals (n=6 heifers/group), beginning at the time of expected follicle deviation (largest follicle, 8.5mm). There were no significant differences on follicle diameters or hormone concentrations between the 0.07 and 0.09 mg E2 groups, and heifers were combined into one E2 group (n=12). The E2 treatments induced concomitant preovulatory surges in LH and FSH at 34.0 ± 2.6h after first treatment, compared to 57.6 ± 4.5h in the vehicle group (P<0.0002). The E2 treatments did not affect FSH concentrations during the preovulatory gonadotropin surge. The diameter of the preovulatory follicle at the LH peak was smaller (P<0.0001) in the E2-treated group (10.2 ± 0.2mm) than in the vehicle group (13.1 ± 0.6mm). The hypothesis was not supported that the previously reported increase in circulating E2 in heifers with double preovulatory follicles accounts for the reported lesser concentrations in the preovulatory FSH surge in heifers with double ovulations. Hypotheses were supported that the reported earlier occurrence of the preovulatory LH surge and smaller preovulatory follicles in heifers with double ovulations are attributable to the reported increase in E2 from the double preovulatory follicles.     

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.     

Emergence and deviation of follicles during the development of follicular waves in cattle

The nature of emergence and deviation of follicles during follicular waves in cattle was studied in 3 experiments by re-examining data from previous projects. Wave emergence was defined as the day or examination (when more than 1 examination per day) the future dominant follicle was 4 mm (Day 0 or Examination 0). Deviation was defined as the beginning of the greatest difference in growth rates between the 2 largest follicles and between 2 consecutive examinations. The search for deviation in an individual wave was done retrospectively from the examination with the maximum diameter of the second largest follicle. In Experiment 1, follicles were assessed ultrasonically for 28 waves every 8 h. The number of examinations that encompassed the emergence of all growing 3-mm follicles was 10.0 +/-0.5 (mean +/-SEM; equivalent to 3.3 d) and extended from mean Examination -3.1 +/-0.3 to mean Examination 6.0 +/-0.6. A mean of 24 growing 3-mm follicles was found, and the maximal attained diameters were 4 mm (46%), 5 mm (25%), and >/=6 mm (29%). More (P<0.05) 3-mm follicles at Examinations -2 and -1 grew to >/=6 mm than to 4 or 5 mm, whereas more 3-mm follicles at Examinations 2 to 6 grew to only 4 mm. On average, the future dominant follicle appeared as a 3-mm follicle (Examination -2.1 +/-0.2) 6 and 10 h earlier (P<0.03) than for the largest (Examination -1.4 +/-0.3) and second-largest (Examination -0.8 +/-0.4) future subordinates, respectively. This result supported the hypothesis that the future dominant follicle has, on the average, an early developmental advantage. In Experiment 2 (n=33 waves), data were normalized to the day at the beginning of deviation (Day 2.8 +/-0.2) when the mean diameters of the dominant and largest subordinate follicle were 8.5 +/-0.2 mm and 7.2 +/-0.2 mm, respectively. This result suggests that the follicle selected to become dominant, as manifested by deviation, is the first follicle to develop to a decisive stage. In Experiment 3 (n=19 waves), FSH concentrations were lower (P<0.05) on the day at the beginning of deviation (8.5 +/-0.5 ng/ml) than on the day before (10.1 +/-0.8 ng/ml), with no continuing decrease after deviation. This temporal result suggests that the attainment of approximate basal levels of FSH is a component of the deviation mechanism.     

Ovarian follicular dynamics in heifers during early pregnancy

Daily ultrasonic monitoring of individual follicles was used to compare follicular wave characteristics of nonbred (n = 6) and pregnant heifers (n = 6). The dominant follicle of the first wave (Wave 1) did not differ significantly between reproductive statuses for any endpoint. The dominant follicle of Wave 2 was the ovulatory follicle in all nonbred heifers. The maximum diameter of the dominant follicle of Wave 2 was greater (p less than 0.05) for the nonbred heifers (14.8 mm) than for the pregnant heifers (13.0 mm). The dominant follicle of Wave 3 was detected later (p less than 0.003; Day 19.7 vs. Day 17.3) and reached a greater diameter (p less than 0.05; 16.6 mm vs. 12.0 mm) in the nonbred than in the pregnant heifers. On the mean day of onset of luteolysis (Day 15.2) in the nonbred heifers, the dominant follicle was similar in diameter for the two groups. Within a few days, the follicle began to regress in the pregnant heifers but maintained or increased in diameter in the nonbred heifers so that a greater maximum diameter was attained. During Days 0 70 of pregnancy, the interval from emergence of a wave to the emergence of the next wave was constant (not significantly different; mean intervals, 8.5 9.8 days). The mean maximum diameter attained by the dominant follicles differed significantly among the first 6 follicular waves; diameter was greatest for Wave 1 (15.7 mm), smallest for Waves 2 (13.1 mm) and 3 (12.6 mm), and intermediate for Waves 4 (14.0 mm), 5 (13.7 mm), and 6 (14.5 mm).(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular and hormonal dynamics during the first follicular wave in heifers

A few days after the first follicular wave emerges as 4-mm follicles, follicular deviation occurs wherein 1 follicle of the wave continues to grow (dominant follicle) while the others regress. The objectives of this study were to characterize follicle growth and associated changes in systemic concentrations of gonadotropins and estradiol at 8-h intervals encompassing the time of follicle deviation. Blood samples from heifers (n = 11) were collected and the ovaries scanned by ultrasound every 8 h from 48 h before to 112 h after the maximal value for the preovulatory LH surge. The follicular wave emerged at 5.8 +/- 5.5 h (mean +/- SEM) after the LH surge, and at this time the future dominant follicle (4.2 +/- 0.8 mm) was larger (P < 0.001) than the future largest subordinate follicle (3.6 +/- 0.1 mm). There was no difference in growth rates between the 2 follicles from emergence to the beginning of the deviation (0.5 mm/8 h for each follicle), indicating that, on average, the future dominant follicle maintained a size advantage over the future subordinate follicle. Deviation occurred when the 2 largest follicles were 8.3 +/- 0.2 and 7.8 +/- 0.2 mm in diameter, at 61.0 +/- 3.7 h after wave emergence. Diameter deviation was manifested between 2 adjacent examinations at 8-h intervals. Mean concentrations of FSH decreased, while mean concentrations of LH increased 24 and 32 h before deviation, respectively, and remained constant (no significant differences) for several 8-h intervals encompassing deviation. In addition to the increase and decrease in circulating estradiol concentrations associated with the preovulatory LH surge, an increase (P < 0.05) occurred between the beginning of deviation and 32 h after deviation. The results supported the hypotheses that deviation occurs rapidly (within 8 h), that elevated systemic LH concentrations are present during deviation, and that deviation is not preceded by an increase in systemic estradiol.     

Ovarian follicular populations during early pregnancy in heifers

Ovarian follicles >/=2mm were studied in 14 pregnant and 14 nonpregnant Holstein heifers by daily ultrasound examinations. There were significant differences among days, from Day 0 (day of ovulation) to Day 21, in the diameter of the largest follicle and the diameter of the second largest follicle in pregnant and nonpregnant heifers. There was an interaction of day and reproductive status (P < 0.001) for the diameter of the largest follicle. Significant differences among days were also observed in the numbers of follicles 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm, and the total number of follicles >/=2 mm. There was a significant main effect of reproductive status for the number of follicles 11 to 13 mm. An interaction of day and reproductive status was observed for the number of follicles >13 mm, but not for any of the other diameter categories. The effect of reproductive status for number of follicles 11 to 13 mm and the interactions for the number of follicles >13 mm and the diameter of the largest follicle seemed due to the selective growth and ovulation of the follicle destined to ovulate in nonpregnant heifers. The differences in ovarian follicular populations between pregnant and nonpregnant heifers were attributed solely to the presence of a physiological mechanism for the selection of an ovulatory follicle in nonpregnant heifers. There were no significant differences among days for any follicular endpoint during Days 22 to 60 in the pregnant heifers.     

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.