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E L Gastal, M O Gastal, and O J Ginther

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.

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E L Gastal, M O Gastal, M A Beg, and O J Ginther

The changing diameter interrelationships among follicles during the interval from emergence to deviation (common-growth phase) were studied in 59 mares. All follicles of ≥6.0 mm were ablated 10 days after ovulation. The four largest follicles of the postablation wave were ranked D1, D2, D3 and D4 at the expected beginning of deviation (D1 ≥ 20.0 mm), according to descending diameter. The four follicles were also ranked independently, according to order of emergence at 6.0–6.9 mm as E1 (first to emerge), E2, E3 and E4. The follicles emerged during 1.3 ± 0.1 to 3.1 ± 0.1 days, and expected deviation began 6.5 ± 0.1 days after ablation. The frequency of emerging follicles becoming the largest follicle at the beginning of deviation was different (P < 0.0001; chi-square test) among follicles E1 (61%), E2 (25%), E3 (9%) and E4 (5%). There were no differences in growth rates among the four follicles throughout the common-growth phase (overall, 2.8 ± 0.04 mm/day). The differences in diameters between follicles E1 and E2 were similar between 3 days (2.7 ± 0.2 mm) and 6 days (2.9 ± 0.4 mm) after ablation. In controls and after ablation of D1; D1 and D2; or D1, D2 and D3 at the expected beginning of deviation, the largest remaining follicle became dominant in 26 of 34 mares (76%). In 10 of 15 mares (67%), the second-largest follicle became dominant when the largest follicle was ablated 1 or 2 days after the expected beginning of deviation. Results indicated the following: 1) the first follicle to emerge maintained its diameter advantage in most mares and average diameter growth rates were similar among the four follicles throughout the common-growth phase; 2) the hypothesis was supported that the capacity for dominance is similar among the four largest follicles at the beginning of deviation, but dominance by a smaller follicle is blocked when a larger follicle is present; and 3) the second-largest follicle retained the capacity for dominance in most mares for as long as 2 days after the beginning of deviation.

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O J Ginther, E L Gastal, M O Gastal, and M A Beg

During a follicular wave in mares, the two largest follicles (F1 and F2) begin to deviate in diameter when F1 is a mean of 22.5 mm. The intrafollicular effects of pregnancy-associated plasma protein-A (PAPP-A), IGF-I, activin-A and vascular endothelial growth factor (VEGF) on other follicular-fluid factors during deviation were studied. In four treated groups (n = 7/group), a single dose of one of the four factors was injected into F2 when F1 was ≥20.0 mm (expected beginning of deviation). In a control group (n = 7), F2 was injected with vehicle. One day after treatment, a sample of follicular fluid was taken from F1 and F2 of the control group and from F2 of the treated groups and was assayed for free IGF-I, oestradiol, androstenedione, activin-A, inhibin-A, follistatin and VEGF. In the control group, the means for all end points were significantly greater in F1 than in F2, except that concentrations of androstenedione were lower in F1 than in F2. The treatment effects for F2 were significant as follows: PAPP-A increased the concentrations of free IGF-I, inhibin-A, follistatin and VEGF and decreased the concentrations of androstenedione; IGF-I increased the concentration of inhibin-A and decreased the concentration of androstenedione; activin-A decreased the concentrations of follistatin and androstenedione and increased the diameter of F2; and VEGF increased the concentration of IGF-I and decreased the concentration of androstenedione. These results support the hypotheses that during deviation in mares PAPP-A increases the follicular-fluid concentrations of free IGF-I, follistatin responds to changes in follicular-fluid concentrations of activin-A, and VEGF affects the concentrations of other follicular-fluid factors.

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J C Ferreira, E L Gastal, and O J Ginther

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.

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M A R Siddiqui, E L Gastal, M O Gastal, M Almamun, M A Beg, and O J Ginther

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 F2α (PGF2α) when the largest follicle was ≥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 ≤8 cell embryos. Percentage of follicular wall with Doppler signals of blood flow was greater (P<0.001) for >8 cell versus ≤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.

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O J Ginther, M A Beg, E L Gastal, M O Gastal, A R Baerwald, and R A Pierson

Changes in systemic concentrations of FSH, LH, oestradiol and progesterone during the ovulatory follicular wave were compared between 30 mares and 30 women. Based on a previous study, the emergence of the future ovulatory follicle was defined as occurring at 13.0 mm in mares and 6.0 mm in women, and deviation in diameter between the two largest follicles was expected to begin at 22.7 mm in mares and 10.3 mm in women. Mean FSH concentrations were high in mares during the luteal phase, resulting from statistically identified FSH surges occurring in individuals on different days and in different numbers (mean, 1.5 ± 0.2 surges/mare); the internadir interval was 3.9 ± 0.3 days. In contrast, mean FSH in women was low during the luteal phase and increased to a prolonged elevation during the follicular phase. The prolonged elevation was apparent in each individual (internadir interval, 15.2 ± 0.4 days). Changes in LH or oestradiol concentrations encompassing deviation were not detected in mares, but both hormones increased slightly but significantly between emergence and deviation in women. The hypothesis that a greater number of growing follicles causes a greater predeviation decrease in FSH was supported for mares (r, −0.39; P< 0.04), but a similar negative correlation (r, −0.36) was not significant in women. The hypothesis that the increase in oestradiol during the luteal phase in women was at least partly attributable to luteal-phase anovulatory follicular waves was not supported. Normalization of FSH concentrations to the day of emergence showed maximum value on the day of emergence with a significant increase and decrease on each side of emergence in both species. The day of expected deviation occurred 3 days after emergence during the decline in FSH in both species. These results indicated that the previously reported striking similarities in emergence and deviation between mares and women during the ovulatory follicular wave are associated with species similarities in the temporal relationships between follicle events and FSH concentration changes. Thus, mares may be useful research models for studying the role and mechanism of the action of FSH in emergence and deviation during the ovulatory follicular wave in women.

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S T Bashir, M O Gastal, S P Tazawa, S G S Tarso, D B Hales, J Cuervo-Arango, A R Baerwald, and E L Gastal

Luteinized unruptured follicle (LUF) syndrome is a recurrent anovulatory dysfunction that affects up to 23% of women with normal menstrual cycles and up to 73% with endometriosis. Mechanisms underlying the development of LUF syndrome in mares were studied to provide a potential model for human anovulation. The effect of extended increase in circulating LH achieved by administration of recombinant equine LH (reLH) or a short surge of LH and decrease in progesterone induced by prostaglandin F2α (PGF2α) on LUF formation (Experiment 1), identification of an optimal dose of COX-2 inhibitor (flunixin meglumine, FM; to block the effect of prostaglandins) for inducing LUFs (Experiment 2), and evaluation of intrafollicular endocrine milieu in LUFs (Experiment 3) were investigated. In Experiment 1, mares were treated with reLH from Day 7 to Day 15 (Day 0=ovulation), PGF2α on Day 7, or in combination. In Experiment 2, FM at doses of 2.0 or 3.0 mg/kg every 12 h and human chorionic gonadotropin (hCG) (1500 IU) were administered after a follicle ≥32 mm was detected. In Experiment 3, FM at a dose of 2.0 mg/kg every 12 h plus hCG was used to induce LUFs and investigate the intrafollicular endocrine milieu. No LUFs were induced by reLH or PGF2α treatment; however, LUFs were induced in 100% of mares using FM. Intrafollicular PGF2α metabolite, PGF2α, and PGE2 were lower and the ratio of PGE2:PGF2α was higher in the induced LUF group. Higher levels of intrafollicular E2 and total primary sex steroids were observed in the induced LUF group along with a tendency for higher levels of GH, cortisol, and T; however, LH, PRL, VEGF-A, and NO did not differ between groups. In conclusion, this study reveals part of the intrafollicular endocrine milieu and the association of prostaglandins in LUF formation, and indicates that the mare might be an appropriate model for studying the poorly understood LUF syndrome.

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G D A Gastal, B G Alves, K A Alves, M E M Souza, A D Vieira, A S Varela Jr, J R Figueiredo, J M Feugang, T Lucia Jr, and E L Gastal

The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) or kept intact (whole ovary group) and stored at 4°C for up to 24 h in α-MEM-enriched solution. The effect of the size of the ovarian tissue was observed on the morphology of preantral follicles, stromal cell density, DNA fragmentation and mitochondrial membrane potential. The results showed that (i) biopsy size fragments had more morphologically normal preantral follicles after 24 h of storage at 4°C; (ii) mitochondrial membrane potential was the lowest during each storage time when the whole ovary was used; (iii) DNA fragmentation rate in the ovarian cells of all sizes of fragments increased as storage was prolonged and (iv) transplant size fragments had increased stromal cell density during storage at cool temperature. In conclusion, the biopsy size fragment was the best to preserve follicle morphology for long storage (24 h); however, transportation/storage should be prior determined according to the distance (time of transportation) between patient and reproduction centers/clinics.