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Summary. Individual follicles were monitored by ultrasonography in 15 mares during the transitional period preceding the first ovulation of the year and in 9 mares during the first interovulatory interval. During the transitional period, 7 mares developed 1–3 anovulatory follicular waves characterized by a dominant follicle (maximum diameter ≥ 38 mm) that had growing, static, and regressing phases. The emergence of a subsequent wave (anovulatory or ovulatory) did not occur until the dominant follicle of the previous wave was in the static phase. After the emergence of the subsequent wave, the previous dominant follicle regressed. The mean (± s.d.) length of the interval between successive waves was 10·8 ± 2·2 days. Before the emergence of waves (identified by a dominant follicle), follicular activity seemed erratic and follicles did not reach > 35 mm. During the interovulatory interval, 6 mares developed 2 waves (an anovulatory wave and a subsequent ovulatory wave) and 3 mares developed only 1 detected wave (the ovulatory wave). The ovulatory follicle at the end of the transitional period reached 20 mm earlier (Day − 15), grew slower (2·6 ± 0·1 mm/day; mean ± s.e.m.) but reached a larger diameter on Day − 1 (50·5 ± 1·1 mm) than for the ovulatory follicle at the end of the interovulatory interval (Day − 10, 3·6 ± 0·2 mm/day, 44·4 ± 1·0 mm, respectively; P < 0·05 for each end point). The interval from cessation of growth of the largest subordinate follicle to the occurrence of ovulation was longer (P < 0·05) for end of the transitional period (9·5 ± 0·7 days) than for the end of the interovulatory interval (6·8 ± 0·6 days). Results demonstrated the occurrence of rhythmic follicular waves during some transitional periods and the occurrence of 2 waves during some of the first oestrous cycles of the year.

Keywords: follicles; follicular waves; mares; reproductive seasonality

The characteristics of follicle evacuation during ovulation and the development of the corpus luteum until day 5 (day 0 = ovulation) were studied in seven nulliparous Holstein heifers using real-time ultrasonography. Ovulation was induced and synchronized with a single injection of PGF_2α followed in 36 h by GnRH. Continuous scanning and videotaping was performed from apparent stigma formation until antral fluid was no longer detected. The beginning of follicular evacuation (second 0) was defined, retrospectively, after the antral area decreased 10% or more in 1 s. The completion of evacuation was defined as the inability to detect the antrum (the beginning of luteal development, 0 h). Corpora lutea development was monitored at 0, 4 and 20 h, and every 24 h thereafter until day 5. Changes in the maximal cross-sectional area of the antrum, luteal tissue, and central luteal cavities and in the pixel intensity of luteal tissue were determined using a computerized image program. The initial antral fluid evacuation occurred in two patterns that could be readily separated: (1) rapid, means of 58 and 89% evacuation in 1 and 4 s, respectively (four heifers); and (2) slow, means of 17 and 35% in 1 and 4 s, respectively (three heifers). The initial loss that distinguished the two patterns involved about 4 and 20 s for rapid and slow evacuation, respectively. Thereafter, the loss patterns were similar for the two types. The time from the beginning to the completion of evacuation ranged from 6 s to 14.5 min. Mean luteal tissue area increased (P < 0.05) between completion of evacuation (91.2 ± 6.5 mm²) and day 3 (164.4 ± 13.7 mm²) and between day 3 and day 4 (263.4 ± 26.6 mm²). The growth rate of the luteal tissue area between day 3 and day 4 (103.2 ± 16.0 mm² day⁻¹) was greater (P < 0.05) than that between day 2 and day 3 (41.9 ± 12.4 mm² day⁻¹) and between day 4 and day 5 (49.7 ± 22.0 mm² day⁻¹). In contrast to increasing luteal tissue area, mean pixel intensity decreased (P < 0.05) progressively between the completion of evacuation (78.4 ± 6.3) and day 2 (60.4 ± 2.5) and did not change significantly thereafter. In conclusion, initial follicular fluid loss during ovulation occurred in two patterns, involving about 4 and 20 s, respectively. The most intensive luteal tissue growth occurred between day 3 and day 4, and the echogenicity of the luteal tissue decreased between day 0 and day 2.

Colour-Doppler ultrasonography was used to study the spatial relationship between vascular perfusion in the middle of each uterine horn and the reported location of the embryo proper and expanding conceptus using endometrial vascularity scores 1–4 (nil–maximal). Vascularity increased in both uterine horns between days 14 and 18 (day 0=ovulation) in nonpregnant heifers (n=6) but not in pregnant heifers (n=11). The increase was temporally associated with decreasing plasma progesterone and increasing oestradiol. In pregnant heifers, a transient increase in endometrial vascularity in the ipsilateral horn (horn with embryo) was not detected before day 18, despite a reported transient increase in blood flow in the ipsilateral uterine artery between days 13 and 17. Endometrial vascularity in the ipsilateral horn first increased (P<0.05) between days 18 and 20. Day 20 is the reported day of adhesiveness between chorion and uterus. An increase (P<0.05) in the contralateral horn between days 18 and 22 was slight, but a greater increase occurred after day 32. Day 32 is the reported day of entry of the allantochorion into the contralateral horn. By day 42, scores were similar between the two horns, and the allantochorion reportedly fills both horns. On days 42–60, at a time when placentomes apparently are limited to the ipsilateral horn, vascularity remained elevated in the ipsilateral horn but decreased in the contralateral horn. Results support the hypothesis that vascular perfusion in each uterine horn during early pregnancy is mediated by direct contact between conceptus and uterus.

Summary. Seasonally anovulatory mares were injected, i.m., twice daily with a GnRH analogue (GnRH-A), and hCG was given when the largest follicle reached 35 mm in diameter. In Exp. 1, treatment was initiated on 23 December when the largest follicle per mare was ⩽ 17 mm. An ovulatory response (ovulation within 21 days) occurred in 17 of 30 (57%) GnRH-A-treated mares on a mean of 15·8 days. The shortest interval to ovulation in control mares (N = 10) was 57 days. The diameter of the largest follicle first increased significantly 6 days after start of treatment. In Exp. 2, treatment was begun on 15 January and mares were categorized according to the largest follicle at start of treatment. The proportion of mares ovulating within 21 days increased significantly according to initial diameter of largest follicle (⩽ 15 mm, 9/25 mares ovulated; 15–19 mm, 13/21; 20–24 mm, 20/24; ≥ 25 mm, 3/3). The multiple ovulation rate was greater (P < 0·01) for treated mares (27/86 mares had multiple ovulations) than for control mares (2/35). Treated mares in which the largest follicle at start of treatment was ≥ 25 mm had a higher (P < 0·01) multiple ovulation rate (9/14) than did mares in which the largest follicle was <25 mm (18/72). The pregnancy rate for single ovulators was not different between control mares (26/30 pregnant mares) and treated mares (43/54). There was a significant interaction for diameter of largest follicle during pregnancy between day and group (control group and 3 treated groups in which the largest follicle on first day of treatment was < 15, 15–19 or 20–24 mm). On all days from Days 17 to 38, the largest follicle was larger (P < 0·05) in the control group (largest mean, 27·9 mm) than in the groups first treated when the largest follicle was < 15 mm (largest mean, 15·3 mm), 15–19 mm (largest mean, 17·9 mm), or 20–24 mm (largest mean 20·2 mm).

Keywords: GnRH; anovulatory season; ovulation; follicles; horse

The temporal associations between increases in FSH and growth of small follicles (2–20 mm) were studied during one oestrous cycle (ovulation to ovulation) in 15 horse mares. Follicular diameters were obtained ultrasonically. For each day, follicles were combined for both ovaries, grouped from largest to smallest (excluding dominant follicles), and divided into three to five tiers of six follicles for each mare (for example: tier 1, six largest follicles; tier 2, next six largest follicles). A significant increase in mean diameters followed by a significant decrease was used to define a follicular wave within each of the tiers for each mare. Day of wave emergence was defined by the lowest mean preceding the increase. Follicular waves were detected in all tiers in each mare. The number of detected waves per interovulatory interval was greater (P < 0.05) for tier 5 (2.8 ± 0.4) than for tier 1 (1.1 ± 0.1) and tier 2 (1.9 ± 0.3). A primary follicular wave (wave giving rise to the dominant follicle that ovulated during the subsequent oestrus was identified in tier 1 in all mares. Composite profiles were constructed by normalizing each tier for each mare to the mean day of emergence of the primary wave for all mares, as detected in tier 1. The composite follicular profiles for the five tiers were approximately parallel; the follicles for tiers 2–5 emerged on the mean day of emergence of the primary wave (6 days after ovulation). The range of follicular means on the common day of wave emergence was 3 mm (tier 5) to 12 mm (tier 1). The follicles of tier 1 on the day of emergence of the primary wave would have been 3 mm before ovulation, if the rate of growth before emergence was similar to the rate after emergence. The common day of emergence for each of the five composite tiers was temporally associated with the highest (P < 0.05) mean FSH concentrations of the oestrous cycle. In another analysis, which did not use the tiering procedure, a temporal association between increased exposure to FSH and growth of 2–3 mm follicles was suggested by a higher (P < 0.05) frequency of increased FSH concentrations than of decreased concentrations on the days of increased numbers of 2–3 mm follicles.

A duplex grey-scale and colour-Doppler ultrasound instrument was used to study the changes in the wall of the preovulatory follicle in mares. When the follicle reached ≥35 mm (hour 0), mares were randomized into control (n = 16) and human chorionic gonadotropin (hCG)-treated (n = 16) groups. The hCG treatment was given at hour 0. Scanning was done every 12 h until hour 36, every hour between hours 36 and 48, and every 12 h thereafter until ovulation. Blood was sampled every 12 h for oestradiol assay. During the period 0–24 h post-treatment, oestradiol concentrations decreased in the hCG group and increased in the controls (significant interaction). During the period 0–36 h post-treatment, thickness and echogenicity of the granulosa increased in the hCG group but not in the controls. During the period 36 to 12 h before ovulation, granulosa and colour-Doppler end-points increased in the control and hCG groups (hour effects), while oestradiol was decreasing in both groups. The prominence and percentage of follicle circumference with an anechoic band peripheral to the granulosa and colour-Doppler signals in the follicle wall, indicating arterial blood flow, decreased during the period 4 to 1 h before ovulation (hour effects). Results indicated that the ultrasonographic changes of the wall of the preovulatory follicle were not associated temporally with changes in oestradiol concentrations and prominence of an anechoic band, and colour-Doppler signals decreased during the few hours before ovulation. The hypothesis that the latter portion of the ovulatory LH surge has a negative effect on systemic oestradiol was supported by the immediate decrease in oestradiol concentrations when hCG was injected.

Summary. The effects of lactational status and reproductive status on patterns of follicle growth and regression were studied in 41 llamas. Animals were examined daily by transrectal ultrasonography for at least 30 days. The presence or absence of a corpus luteum and the diameter of the largest and second largest follicle in each ovary were recorded. Llamas were categorized as lactating (N = 16) or non-lactating (N = 25) and randomly allotted to the following groups (reproductive status): (1) unmated (anovulatory group, N = 14), (2) mated by a vasectomized male (ovulatory non-pregnant group, N = 12), (3) mated by an intact male and confirmed pregnant (pregnant group, N = 15). Ovulation occurred on the 2nd day after mating with a vasectomized or intact male in 26/27 (96%) ovulating llamas. Interval from mating to ovulation (2·0 ± 0·1 days) and growth rate of the preovulatory follicle (0·8 ± 0·2 mm/day) were not affected by lactational status or the type of mating (vasectomized vs intact male). Waves of follicular activity were indicated by periodic increases in the number of follicles detected and an associated emergence of a dominant follicle that grew to ≥7 mm. There was an inverse relationship (r = −0·2; P = 0·002) between the number of follicles detected and the diameter of the largest follicle. Successive dominant follicles emerged at intervals of 19·8 ± 0·7 days in unmated and vasectomy–mated llamas and 14·8 ± 0·6 days in pregnant llamas (P = 0·001). Lactation was associated with an interwave interval that was shortened by 2·5 ± 0·05 days averaged over all groups (P = 0·03). Maximum diameter of anovulatory dominant follicles ranged from 9 to 16 mm and was greater (P < 0·05) for non-pregnant llamas (anovulatory group, 12·1 ± 0·4 mm; ovulatory group, 11·5 ± 0·2 mm) than for pregnant llamas (9·7 ± 0·2 mm). In addition, lactation was associated with smaller (P < 0·05) maximum diameter of dominant follicles averaged over all reproductive statuses (10·4 ± 0·2 vs 11·7 ± 0·3 mm). The corpus luteum was maintained for a mean of 10 days after ovulation in non-pregnant llamas and to the end of the observational period in pregnant llamas. The presence (ovulatory non-pregnant group) and persistence (pregnant group) of a corpus luteum was associated with a depression in the number of follicles detected and reduced prominence of dominant follicles (anovulatory group > ovulatory non-pregnant group > pregnant group). Lactation was also associated with reduced prominence of dominant follicles. The results demonstrate that follicular activity occurred in waves for llamas of all types of reproductive status and that lactation and the presence of a corpus luteum were associated with depressed follicular development.

Keywords: ovaries; follicles; follicular waves; lactation; llamas; camelids

Summary. For 18 two-wave interovulatory intervals in heifers, the follicular waves were first detected on Days −0·2 ± 0·1 and 9·6 ± 0·2, and for 4 three-wave intervals on Days −0·5 ± 0·3, 9·0 ± 0·0 and 16·0 ± 1·1 (ovulation is Day 0). The day-to-day mean diameter profile of the dominant follicle of the 1st wave and the day of emergence of the 2nd wave were not significantly different between 2-wave and 3-wave intervals. There were no indications, therefore, that events occurring during the first half of the interovulatory interval were associated with the later emergence of a 3rd wave. The dominant ovulatory follicle differed significantly (P < 0·05 at least) between 2-wave and 3-wave intervals in day of emergence (Day 9·6 ± 0·2 and 16·0 ± 1·1), length of interval from emergence of follicle to ovulation (10·9 ± 0·4 and 6·8 ± 0·6 days), and diameter on day before ovulation (16·5 ± 0·4 and 13·9 ± 0·4 mm). The mean length of 2-wave interovulatory intervals (20·4 ± 0·3 days) was shorter (P < 0·01) than for 3-wave intervals (22·8 ± 0·6 days). The mean day of luteal regression for 2-wave and 3-wave intervals was 16·5 ± 0·4 and 19·2 ± 0·5 (P < 0·01). For all intervals, luteal regression occurred after emergence of the ovulatory wave, and the next wave did not emerge until near the day of ovulation at the onset of the subsequent interovulatory interval. In conclusion, the emergence of a 3rd wave was associated with a longer luteal phase, and the viable dominant follicle present at the time of luteolysis became the ovulatory follicle.

Keywords: follicles; corpus luteum; cattle; follicular waves

Summary. Characteristics of spontaneous embryonic loss in 21 mares were compared with those of 52 contemporary mares that maintained pregnancy. Embryonic losses were, in retrospect, grouped according to day of loss and length of the interovulatory interval, respectively, as follows: group 1, ⩽day 20 and ⩽30 days (n = 10); group 2, ⩽day 20 and >30 days (n = 3); and group 3, >day 20 and >30 days (n = 8); ovulation was day 0. Mean diameter of the embryonic vesicle in group 1 was smaller (P < 0·05) on days 12-18 than in the pregnancy-maintained group, but among the pregnancy-maintained group and the embryonic-loss groups, the mean individual growth rates of vesicles was similar (no significant difference). A more frequent (P < 0·05) location of the vesicles in the uterine body on day 13 in group 1 was due to a greater proportion of small vesicles and for day 18 was due to a greater incidence of fixation failure. Luteal regression occurred at the expected time in 77% of the mares with loss sooner than day 20. Low concentration of progesterone on days 12, 15 and 18, a detected decrease in diameter of the corpus luteum on days 15 and 18, and an interovulatory interval of ⩽ 30 30 days indicated that luteolysis was not prevented by the embryonic vesicle in group 1. However, in mares in group 2, the corpus luteum was maintained as indicated by luteal diameters, progesterone concentrations, and prolonged interovulatory intervals. Thus, luteal maintenance occurred in 23% of mares with embryonic loss at ⩽day 20. In group 3 (loss >day 20), circulating concentrations of progesterone were either maintained (four mares) after embryonic death (cessation of embryonic heartbeat) or appeared to decrease before death (two mares) or on the day of death (one mare). The decrease in progesterone on the day of death seemed to be associated with acute endometritis. In another mare, embryonic loss was associated with continuing low concentrations of progesterone, a flaccid uterus, an oversized embryonic vesicle, failure of development of an embryo proper and collapse of the large vesicle on day 42. The apparent drop in progesterone before embryonic death in two of eight mares with loss after day 20 could have been associated with primary luteal insufficiency.

Keywords: mare; embryonic loss; embryonic vesicle; progesterone; interovulatory interval

Transrectal color and power Doppler ultrasonography was used to study uterine blood flow and perfusion in mares with and without uterine cysts. Vascular perfusion of the uterus and blood flow velocities, vascular perfusion, diameter, circumference, and area of a cross section of the mesometrial attachment were evaluated. To study the effect of internal cysts, two matched groups (cystic and control, n=21 mares/group) were used. Uterine vascular perfusion in mares with cysts was less (P<0.0001) in the cystic than the noncystic region and less (P<0.0009) than that for controls. Mares with cysts had lower (P<0.04) pulsatility index (PI) and greater end diastolic velocity (EDV; P<0.03) and time-averaged maximum velocity (TAMV; P<0.05) of the mesometrial vessels than the controls. To study the effect of the size of internal uterine cystic area, paired mares were arranged in four groups (n=8–11/group): small uterine cystic area (≤275 mm²) versus controls and large uterine cystic area (>410 mm²) versus controls. A small uterine cystic area did not affect uterine hemodynamics. Mares with large uterine cystic area had lower PI (P<0.05) and greater peak systolic velocity (P≤0.05), EDV (P<0.009), and TAMV (P<0.005). To study the effect of age, old versus young mares without cysts were compared (n=11/group). Old mares had greater EDV (P<0.02) and TAMV (P<0.01) than young mares. Results demonstrated, for the first time in any species, reduced uterine vascular perfusion in mares with uterine cysts and a positive association between size of the cystic area and disturbed uterine hemodynamics.