M.O. Gastal

Comparative Study of the Dynamics of Follicular Waves in Mares and Women

Abstract 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.

Regulation of Circulating Gonadotropins by the Negative Effects of Ovarian Hormones in Mares

Abstract 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 ≥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.

Relationship of Vascular Perfusion of the Wall of the Preovulatory Follicle to In Vitro Fertilisation and Embryo Development in Heifers

The effect of the extent of vascular perfusion of the wall of the preovulatory follicle on in vitro cleavage rate of the recovered oocyte and embryo development to >8 cells was studied in 52 heifers. Heifers received a luteolytic dose of prostaglandin F2alpha (PGF2alpha) when the largest follicle was > or =11 mm. An ovulation-inducing injection of GnRH was given 36 h later (hour 0), and collection of follicular fluid and the oocyte was done at hour 26. Vascular perfusion of the follicular wall was assessed by colour Doppler ultrasonography at hours 0 and 26. Each of the recovered oocytes (41/52; 79%) was mature (extruded polar body). Cleavage and embryo development were assessed at 48 h and 120 h respectively, after in vitro fertilisation (IVF). The percentage of cleaved oocytes and >8 cell embryos was 80% (31/39) and 55% (17/31) respectively. Vascular perfusion of the follicular wall was greater (lower pulsatility index; P<0.001) for follicles that produced cleaved versus non-cleaved oocytes and greater (P<0.04) for follicles that produced >8 cell versus < or =8 cell embryos. Percentage of follicular wall with Doppler signals of blood flow was greater (P<0.001) for >8 cell versus < or =8 cell embryos. Follicular-fluid concentration of free IGF1 was lower for cleaved oocytes (P<0.001) and >8 cell embryos (P<0.05), and oestradiol was lower (P<0.05) for >8 cell embryos. Results supported the hypothesis that greater vascular perfusion of the wall of the preovulatory follicle was positively associated with IVF and embryo development.

The suitability of echotexture characteristics of the follicular wall for identifying the optimal breeding day in mares

Ultrasonically detected changes in the equine preovulatory follicle were characterized for the 3 d preceding ovulation early (n = 47) and late (n = 14) in the ovulatory season. Values for the following follicle end points increased progressively over the 3 d: diameter, incidence of nonspherical shape, echogenicity of the apparent granulosa layer, and prominence of an anechoic layer beneath the granulosa. The latter 2 echotexture end points were scored from 1 to 3 (minimal to maximal). Follicle diameter and the 2 echotexture characteristics were more prominent early than late in the ovulatory season. Early in the season, both echotexture characteristics were at the maximal score of 3 in 33/47 (70%) follicles on Day -1 (Day 0 = ovulation). None of the follicles ovulated before both characteristics reached a score of > or = 2. Use of follicle diameter alone to predict impending ovulation seemed ineffective because of a wide range in diameters on Day -1 (31 to 49 mm). The efficiency of a score of > or = 2 for both granulosa echogenicity and prominence of the anechoic layer as an echotexture indicator for the initiation of breeding early in the ovulatory season was compared to diameter indicators of > or = 30 mm, > or = 35 mm, or > or = 40 mm. Data were evaluated as though mares had been bred every other day beginning when an indicator was attained. If the echotexture and > or = 30 mm indicators had been used, none of 34 mares would have ovulated before breeding. However, the mean number of breedings per bred mare would have been greater (P < 0.05) for the > or = 30 mm indicator (2.1 +/- 0.1) than for the echotexture indicator (1.6 +/- 0.1 breedings). The number of breedings per mare would have been equivalent for the echotexture indicator and the diameter indicators of > or = 35 mm (1.5 +/- 0.1) and > or = 40 mm (1.4 +/- 0.2). However, 21 and 74% of the mares would have ovulated before breeding for the > or = 35 mm and > or = 40 mm indicators, respectively. Results suggested that the echotexture indicator would have been more efficient for initiation of breeding than any of the diameter indicators.

Temporal interrelationships among luteolysis, FSH and LH concentrations and follicle deviation in mares

The effect of altered LH concentrations on the deviation in growth rates between the 2 largest follicles was studied in pony mares. The progestational phase was shortened by administration of PGF2alpha on Day 10 (Day 0=ovulation; n=9) or lengthened by daily administration of 100 mg of progesterone on Days 10 to 30 (n=11; controls, n=10). All follicles > or = 5 mm were ablated on Day 10 in all groups to initiate a new follicular wave. The interovulatory interval was not altered by the PGF2alpha treatment despite a 4-day earlier decrease in progesterone concentrations. Time required for growth of the follicles of the new wave apparently delayed the interval to ovulation after luteolysis. The FSH concentrations of the first post-ablation FSH surge were not different among groups. A second FSH surge with an associated follicular wave began by Day 22 in 7 of 11 mares in the progesterone group and in 0 of 19 mares in the other groups, indicating reduced functional competence of the largest follicle. A prolonged elevation in LH concentrations began on the mean day of wave emergence (Day 11) in the prostaglandin group (19.2 +/- 2.2 vs 9.0 +/- 0.7 ng/mL in controls; P<0.05), an average of 4 d before an increase in the controls. Concentrations of LH in the progesterone group initially increased until Day 14 and then decreased so that by Day 18 the concentrations were lower (P<0.05) than in the control group (12.9 +/- 1.6 vs 20.2 +/- 2.6 ng/mL). Neither the early and prolonged increase nor the early decrease in LH concentrations altered the growth profile of the second-largest follicle, suggesting that LH was not involved in the initiation of deviation. However, the early decrease in LH concentrations in the progesterone group was followed by a smaller (P<0.05) diameter of the largest follicle by Day 20 (26.9 +/- 1.7 mm) than the controls (30.3 +/- 1.7 mm), suggesting that LH was necessary for continued growth of the largest follicle after deviation.

Differential Blood Flow Changes Between the Future Dominant and Subordinate Follicles Precede Diameter Changes During Follicle Selection in Mares

Abstract 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 days 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.

Temporal Relationships and Repeatability of Follicle Diameters and Hormone Concentrations within Individuals in Mares

Data were collected daily from 23 mares during two consecutive interovulatory intervals (IOIs). Several significant (p < 0.05) new observations on temporal relationships were made. The FSH increase that begins before ovulation temporarily plateaued on the day of discharge of follicular fluid into the peritoneal cavity in association with ovulation. During the declining portion of the pre-ovulatory oestradiol surge, an abrupt reduction in the rate of decrease occurred in synchrony with the peak of the LH surge and is consistent with a negative effect of LH on oestradiol. Repeatability within mares was based on the following positive and significant correlations between the two IOIs: (i) length of the interval between ovulations and between ovulation and the beginning of follicle deviation; (ii) diameter of the pre-ovulatory follicle on days -3 to -1; (iii) number of follicles in diameter classes of 2-5 mm (correlation for 22/23 days of the IOI), 5.1-10 mm (18/23 days), 10.1-15 mm (12/23 days) and 15.1-20 mm (12/23 days) and (iv) concentrations of FSH (18/23 days) and LH (22/23 days). The greatest repeatability for the follicle-diameter classes occurred in the 2-5 mm class, and thereafter the repeatability progressively decreased as the diameters for the classes increased. Results demonstrated measurable repeatability within mares for several end points between consecutive IOIs.

Negative Effect of Estradiol on Luteinizing Hormone Throughout the Ovulatory Luteinizing Hormone Surge in Mares

Abstract The negative effect of estradiol-17β (E2) on LH, based on exogenous E2 treatments, and the reciprocal effect of LH on endogenous E2, based on hCG treatments, were studied throughout the ovulatory follicular wave during a total of 103 equine estrous cycles in seven experiments. An initial study developed E2 treatment protocols that approximated physiologic E2 concentrations during the estrous cycle. On Day 13 (ovulation = Day 0), when basal concentrations of E2 and LH precede the ovulatory surges, exogenous E2 significantly depressed LH concentrations to below basal levels. Ablation of all follicles ≥10 mm when the largest was ≥20 mm resulted in an increase in percentage change in LH concentration within 8 h that was greater (P < 0.03) than for controls or E2-treated/follicle-ablated mares. Significant decreases in LH occurred when E2 was given when the largest follicle was either ≥25 mm, ≥28 mm, ≥35 mm, or near ovulation. Treatment with 200 or 2000 IU of hCG did not affect E2 concentrations during the initial portion of the LH surge (largest follicle, ≥25 mm), but 2000 IU significantly depressed E2 concentrations before ovulation (largest follicle, ≥35 mm). Results indicated a continuous negative effect of E2 on LH throughout the ovulatory follicular wave and may be related to the long LH surge and the long follicular phase in mares. Results also indicated that a reciprocal negative effect of LH on E2 does not develop until the E2 surge reaches a peak.

Critical Role of Insulin-Like Growth Factor System in Follicle Selection and Dominance in Mares

Abstract 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 ⩾ 20 mm; Day 0). The experimental groups were control (no injection, n = 8), vehicle (injection of vehicle; n = 6), and BP (injection of 250 μg 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.

Dose-Response Study of Intrafollicular Injection of Insulin-Like Growth Factor-I on Follicular Fluid Factors and Follicle Dominance in Mares

Abstract 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 (≥20 mm) of the largest follicle (F1). Mares in IGF-I groups were given a dose of 500 μg (experiment 1) or 250, 25, or 2.5 μg (experiment 2). Ablation of F1 at Hour 24 was done in experiment 1, but not in experiment 2. The 500- and 250-μg 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-μg 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-μg 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-μg 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.