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ABSTRACT: The interovulatory intervals of mares contain various combinations of minor follicular waves (largest follicle does not become dominant) and major waves (largest follicle becomes dominant). After emergence of an ovulatory follicular wave, the follicles grow in a common-growth phase until the beginning of deviation. At deviation, the dominant follicle continues to grow and the subordinate follicles begin to regress. Devia-tion begins when the future dominant follicle is about 22.5 mm. The capacity for follicle dominance is similar among the four largest follicles at the beginning of deviation, as indicated by a study involving ablation of one, two, or three largest follicles. The ovulatory waves, as well as major anovulatory waves and minor waves, originate from the stimulation of an FSH surge, which reaches a peak when the largest follicle is about 13 mm. The initial decline in the FSH surge appears to be a function of inhibin; circulating estradiol does not begin to increase until about 2 days after the FSH peak or about 1 day before the beginning of deviation. Con-centrations of LH of the ovulatory LH surge reach a transient plateau encompassing deviation of the ovula-tory wave. The intrafollicular concentrations of estra-diol, IGF-1, inhibin-A, and activin-A increase differen-tially in the future dominant follicle versus the future subordinate follicles about 1 day before the beginning of diameter deviation. These factors may be enablers for differentially enhancing the FSH and LH respon-siveness of the future dominant follicle, based on the results of in vitro studies in nonequine species. Injec-tion of a physiologic dose of IGF-1 into the second-largest follicle of mares at the expected beginning of deviation increased the concentrations of inhibin-A and activin-A, but not estradiol, within 24 hours. Injection of an IGF binding protein into the largest follicle at the expected beginning of deviation resulted in decreases within 24 hours in several follicular-fluid factors in the largest follicle and ovulation from the second-largest follicle. Ablation of the largest follicle at the expected beginning of deviation resulted in experimental devia-tion between the two largest remaining follicles begin-ning 24 hours after ablation; concentrations of IGF-1 increased differentially in the converting future domi-nant follicle 12 hours before the beginning of experi-mental deviation, whereas inhibin-A, activin-A, and estradiol did not begin to increase differentially until 24–48 hours after the beginning of deviation. Results of these three experimental approaches indicated that the IGF-1 system is critical for the initiation of devia-tion in mares.
; 11:45-63.
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ABSTRACT: Recent innovations in ultrasonographic tech-niques have advanced our knowledge on the effects of seasonality on the changing populations of antral folli-cles in mares. The seasonal patterns in the monthly in-cidence of ovulations is at least partly a function of daylength, even at latitudes as low as 10º where the longest day may be less than 13 hours. Follicle dynam-ics involves a mid-anovulatory phase in winter when the follicles may not exceed 16 or 17 mm. A minor follicular wave (no dominant follicle) ends the mid-anovulatory phase and begins a resurging phase, raising the diameter of the largest follicle to at least 21 mm. The largest follicle of subsequent minor waves reaches a similar diameter forming an approximate plateau. However, in some mares, the later portion of the re-surging phase or the entire phase may consist of a pe-riod of major anovulatory waves in which the largest follicle approaches or is similar to the diameter of a dominant ovulatory follicle. In one study, the length of the resurging phase was a mean of 52 days and con-sisted of minor waves, unless a period of major anovu-latory waves occurred. Each minor or major wave is stimulated by an FSH surge, and the surges are similar regardless of the diameter reached by the largest folli-cle. Results of a Doppler study has led to the hypothe-sis that the future dominant-sized (≥ 30 mm) follicles of the period of major anovulatory waves develop dur-ing an LH deficient environment beginning when the follicles are as small as 15 mm. These future dominant-sized anovulatory follicles are already dysfunctional at about 20 mm as indicated by an increase in circulating FSH. Despite an aberrant vasculature and low concen-trations of estradiol, free IGF-1, inhibin-A, and VEGF, the follicles continue to expand at a rate comparable to the growth of an ovulatory follicle. The effects of daylength on follicle dynamics continue through the ovulatory season. For example, follicular activity of the estrous cycle is greater during the first half of the sea-son as indicated by more follicles ≥ 20 mm, greater diameter of largest follicle, and greater incidence of minor and major anovulatory waves. After the last ovu-lation of the year, most mares, during a receding phase, have an increase in follicle activity at the time ovula-tion would have occurred. A major anovulatory wave or a minor wave develops during the increased follicle activity. Concentrations of LH are greater in associa-tion with major waves than with minor waves, but the wave-stimulating FSH surges are similar. A persistent corpus luteum forms from the site of the last ovulation of the year in about 25% of mares. In these mares, a period of major anovulatory waves may ensue until the corpus luteum regresses.
; 11:31-44.
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ABSTRACT: The objective was to conduct a preliminary evaluation of the efficacy of two media for in vitro culture of equine preantral follicles. Ovarian cortical strips were obtained from mares (N = 10) via the Biopsy Pick-Up method during the breeding season. Ovarian tissue was immediately submitted to histological analysis (noncultured control; D0) or cultured in situ for 1 day (D1) or 7 days (D7) in either α-MEM or TCM-199 and submitted to histological analysis, generating five treatment groups: noncultured control, α-MEM:D1, TCM-199:D1, α-MEM:D7, and TCM-199:D7. Preantral follicles were evaluated for follicle class (primordial, transitional, primary, and secondary) and morphology (normal vs. abnormal). A total of 142 preantral follicles were analyzed in five replicates. No follicles were observed in the TCM-199:D7 treatment group. The proportion of primordial follicles was higher (P < 0.03) in the control compared to the α-MEM:D7 treatment group. The proportion of primary follicles was higher (P < 0.04) in the α-MEM:D7 treatment group compared to the control. The proportion of developing follicles (transitional, primary, and secondary) was higher (P < 0.03) in the α-MEM:D7 treatment group compared to the control group. There was a greater (P < 0.004) percentage of morphologically normal developing follicles in the α-MEM:D1 treatment group compared to the TCM-199:D1 treatment group. Overall, the percentage of morphologically normal follicles was higher in the control group (72%; P < 0.02) and α-MEM:D1 group (84%; P < 0.0001) compared to the α-MEM:D7 (27%) treatment group. Mean follicle diameter was greater (P < 0.04) in the α-MEM:D7 treatment group (40.6 ± 1.1 μm) compared to the control group (37.3 ± 0.7 μm). Mean oocyte diameter was greater in the α-MEM:D1 (31.0 ± 0.7 μm; P < 0.006), TCM-199:D1 (30.7 ± 1.8 μm; P < 0.006), and α-MEM:D7 (33.2 ± 1.8 μm; P < 0.006) treatment groups compared to the control group (27.4 ± 0.9 μm). In conclusion, based on these preliminary data, in vitro culture of equine ovarian fragments obtained in vivo via the Biopsy Pick-Up method promoted preantral follicle development and follicle and oocyte growth in α-MEM for 7 days, with some follicles remaining morphologically normal throughout the culture period.
Theriogenology 02/2013; · 1.96 Impact Factor
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ABSTRACT: The aims of this study in mares were to: (1) compare preantral follicle parameters between in vitro Biopsy Pick-Up (BPU) and scalpel blade collection methods and between histological and mechanical isolation processing (experiment 1); (2) histologically evaluate preantral follicles (experiment 2); and (3) compare histological analysis with a previously established mechanical isolation technique using a tissue chopper (experiment 3) for ovarian cortical fragments obtained in vivo using a BPU instrument. In experiment 1, preantral follicles were analyzed (N = 220; 90% primordial and 10% primary). Proportions of primordial and primary follicles did not differ (P > 0.05) between tissue collection (BPU vs. scalpel blade dissection) or processing (mechanical isolation vs. histology) methods. Follicle viability and morphology rates were similar (P > 0.05) between tissue collection methods, but mechanical isolation produced more (P < 0.05) morphologically normal follicles than histology. For experiment 2, preantral follicles (N = 332) were analyzed and primordial and transitional (combined) follicles and oocytes were 36.3 ± 0.3 and 26.1 ± 0.3 μm in diameter, respectively, and primary follicles and oocytes averaged 42.9 ± 1.8 and 31.8 ± 2.1 μm. For experiment 3 (188 preantral follicles), within the same animals, the proportion of primordial versus primary follicles was higher (P < 0.03) for histological analysis (98%) compared to tissue chopper analysis (94%), and number of follicles per mg of tissue was not affected (P > 0.05) by processing methods. In conclusion, most parameters evaluated for preantral follicles were similar between histological and tissue chopper processing techniques; hence, mechanical isolation efficiently dissociated equine preantral follicles from the ovarian cortex. Therefore, the tissue chopper could be used to isolate large numbers of morphologically normal equine preantral follicles for cryopreservation and/or in vitro culture.
Theriogenology 01/2013; · 1.96 Impact Factor
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ABSTRACT: A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphologic normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production.
Theriogenology 12/2012; · 1.96 Impact Factor
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ABSTRACT: Long-term in vitro culture (16 days) of caprine ovarian cortical tissue was performed to test the effect of FSH and IGF-I on the viability and development of preantral follicles and mRNA expression for FSH and IGF-I receptors. Fragments were cultured in α-MEM(+) alone or supplemented with different combinations of FSH and IGF-I (sequential medium). The culture period was divided into two parts. Follicles were isolated and classified as normal or abnormal and primordial, primary or secondary. Viability of isolated follicles was determined by staining with Trypan Blue dye. Expression of FSHR and IGFR-1 mRNA was evaluated by qPCR. At day 8 of culture, more (P < 0.05) follicles in treatments containing IGF-I alone or associated with FSH were normal and viable (overall mean, 81 % and 79 % respectively) than the treatments cultured with FSH or α-MEM(+) alone (68 % and 63 %). At day 16 of culture, treatments with FSH and/or IGF-I had more (P < 0.05) viable follicles (69 %) than α-MEM(+) (38 %). The percentages of follicular development observed in the IGF-I/FSH, FSH+IGF-I/FSH+IGF-I and FSH/IGF-I treatments were similar but higher (P < 0.05) than the other treatments. FSH and IGF-I during the entire culture period maximized (P < 0.05) follicular and oocyte diameters and the percentage of secondary follicles (28 %). FSHR mRNA expression in the non-cultured control was similar to the treatment supplemented with FSH and IGF-I but higher (P < 0.05) than α-MEM(+). IGFR-1 expression did not differ among treatments. Association of FSH and IGF-I in long-term in vitro culture promoted follicular development, maintaining FSHR mRNA expression.
Cell and Tissue Research 10/2012; · 3.11 Impact Factor
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D M Magalhães-Padilha,
P M Andrade,
E T Sales,
V R Araujo,
I M T Lima,
S V Castro,
L R Faustino,
C A P Lopes,
C C Campello,
S N Báo,
M O Gastal, E L Gastal,
J R Figueiredo
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ABSTRACT: A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.
Animal reproduction science 06/2012; 132(3-4):159-68. · 1.56 Impact Factor
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ABSTRACT: In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to -6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), -1 (68%), -2 (63%), -3 (66%), -4 (76%), -5 (61%), and -6 (27%). The -6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to -5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.
Theriogenology 12/2011; 77(6):1159-66. · 1.96 Impact Factor
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ABSTRACT: To study early follicular development in the equine species, a biopsy pickup (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. The hypotheses tested in this study were that (1) preantral follicle quantity, viability and morphology do not differ according to stage of the oestrous cycle; (2) rate of atresia increases with follicle class; and (3) the BPU method provides sufficient material for studies on the early stages of folliculogenesis in mares. The transvaginal, ultrasound-guided BPU device was a 48-cm-long automated, spring-loaded device with an inner trocar point plunger containing a 15-mm specimen notch surrounded by an outer 16G cutting needle (US Biopsy, Franklin, IN, USA). A total of 33 ovarian biopsy procedures were performed on 19 mares during the breeding season. Mares were 5 to 21 years of age and biopsies were performed during oestrus, diestrus, or both as confirmed by transrectal ultrasonography. Follicles were isolated mechanically using a tissue chopper and were analysed for either viability (live vs dead using trypan blue dye to analyse cell membrane integrity) or morphological normality (intact oocyte surrounded by granulosa cells that are well organised in 1 or more layers). Statistical analyses were performed using Fisher's exact test, paired t-test and one-way ANOVA. A total of 256 biopsy attempts were made, resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25mg. An average of 18.8±1.9 (mean±s.e.m.) follicles were isolated per biopsy procedure. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). Primordial and primary follicles had an average diameter of 31.3±6.2μm and 41.1±6.6μm, respectively. Viability rate was higher (P<0.001) for primordial follicles (91%) when compared with primary follicles (50%), with an overall follicle viability rate of 88%. Primordial follicles tended (P<0.06) to have a higher rate of morphological normality (96%) when compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested and number of follicles per milligram of tissue did not differ (P>0.05) according to reproductive status. Younger mares (ages 5 to 7) had more (P<0.05) follicles isolated per procedure than older mares (ages 14 to 21). The length of the interovulatory interval was not affected (P>0.05) by the biopsy procedure. No adverse effects were observed in cyclicity or general health following any procedure. The results supported the hypotheses tested. In conclusion, the BPU method provides material for the study of early follicular development in the equine species. In the future, the BPU method might be a feasible technique to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary to preserve genetic material or for large-scale embryo production.
Reproduction Fertility and Development 12/2011; 24(1):174. · 2.11 Impact Factor
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ABSTRACT: Colour- and power-Doppler ultrasonography have recently been used as potential new technologies to assess the degree of vascular perfusion of the ovary and follicles for research and clinical studies of ovarian and follicle hemodynamics and to predict fertility in horses, cattle and humans. In the present study, the following hypotheses were tested: (1) preovulatory follicle (POF) diameter (≥30mm), but not blood flow, is repeatable between cycles within the same mare; (2) POF diameter and blood flow are good indicators of follicle status; (3) double POF have similar blood flow; and (4) highly vascularized POF produce corpora lutea (CL) with greater blood flow. Non-lactating mares (n=13; 5 to 21 years old) of mixed breeds were used from March to May in the Northern Hemisphere. Follicle diameter and vascularity of the follicle wall before the first and second ovulations of the season and vascularity of the first CL were measured daily using transrectal colour-Doppler ultrasonography. The vascularity of the follicle wall and CL was based on the display of the blood-flow signals visualised in a slow, continuous-motion evaluation. Statistical analyses were performed by the SAS MIXED procedure, ANOVA and Student's t-tests and Spearman's correlation. A total of 26 periovulatory periods were evaluated. Unexpectedly, there were 84.6% (11/13) and 61.5% (8/13) double dominant POF and 30.8% (4/13) and 46.2% (6/13) double ovulations in the first and second periovulatory periods, respectively. The POF diameters were highly correlated (r=0.68; P<0.0001) between the first and second periovulatory periods. The diameter of the POF 5 days before the first ovulation was larger (P<0.004) than before the second ovulation of the year. However, the POF vascularity did not differ between those periods. For 4 days before ovulation (Day 0), the diameter and blood flow of the POF were greater (P<0.05) than for those follicles that underwent atresia in single- and double-ovulatory mares. The POF diameter and blood flow were positively correlated in ovulatory (r=0.51; P<0.0001) and in atretic (r=0.32; P<0.02) follicles. In double-ovulatory mares, POF diameter and blood flow increased (P<0.0006) for 5 days before ovulation, with no difference between the 2 follicles in the same cycle for each parameter. The POF blood flow was positively correlated (r=0.32; P<0.0009) with CL vascularity during the first periovulatory period (Day -7 to +6) of the season. Furthermore, a positive correlation (r=0.58; P<0.01) was observed between the maximum vascularity of the POF and its subsequent CL. In conclusion, although preliminary, our results demonstrated that (a) POF vascularity is not repeatable within individuals; (b) potential atretic POF have low blood flow; (c) double POF have similar vascularity; and (d) greater blood flow to the POF is associated with higher CL vascularity.
Reproduction Fertility and Development 12/2011; 24(1):174. · 2.11 Impact Factor
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D M Magalhães-Padilha,
A B G Duarte,
V R Araújo,
M V A Saraiva,
A P Almeida,
G Q Rodrigues,
M H T Matos,
C C Campello,
J R Silva,
M O Gastal, E L Gastal,
J R Figueiredo
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ABSTRACT: The objectives were to quantify insulin-like growth factor receptor-1 (IGFR-1) mRNA in preantral follicles on Days 0 and 18 of in vitro culture in the presence or absence of FSH, and to evaluate the effects of IGF-I on the rate of normal follicles, antral cavity formation, and in vitro growth and maturation of caprine oocytes on Days 0, 6, 12, and 18 of culture. The expression of IGFR-1 was analyzed using real-time RT-PCR before and after follicle culture. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the presence or absence of bovine IGF-I (50 or 100 ng/mL). At the end of the culture period, the oocytes were submitted to IVM. The expression of IGFR-1 mRNA in preantral follicles cultured in vitro only approached being significantly higher in follicles supplemented with FSH when compared to follicles immediately after recovery (P<0.06) and cultured without FSH (P<0.1). There was a higher (P<0.05) percentage of normal follicles on Days 6, 12, and 18 of culture in IGF-I 50 (97, 92, 67%, respectively) and IGF-I 100 (100, 90, 80%) groups versus the control (90, 64, 36%). In addition, the rate of antrum formation at 6 and 12 d of culture was higher (P<0.05) in IGF-I groups (IGF-I 50: 72 and 90% and IGF-I 100: 69 and 85%) than the control group (41 and 59%). After 18 d of culture, the percentages of grown oocytes acceptable for IVM were higher (P<0.05) in follicles cultured in the presence of IGF-I (82 vs 49%). Furthermore, follicles cultured in the presence of IGF-I 50 and IGF-I 100 had higher (P<0.05) meiotic resumption rates (63 and 66%, respectively) than the control group (11%). In conclusion, treatment with FSH tended to increase IGFR-1 mRNA expression during the in vitro culture of preantral follicles and the addition of IGF-I to the culture medium clearly improved the in vitro development of caprine preantral follicles.
Theriogenology 09/2011; 77(1):206-13. · 1.96 Impact Factor
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ABSTRACT: The objective of this study was to evaluate the effect of short-term feed restriction on characteristics of the preovulatory follicle and on concentrations of systemic hormones (leptin, follicle-stimulating hormone [FSH], luteinizing hormone [LH]) and follicular fluid hormones and growth factors (leptin, estradiol, inhibin-A, activin-A, free insulin-like growth factor-1 [IGF1], insulin-like growth factor binding protein 2 [IGFBP2], vascular endothelial growth factor [VEGF]). Mares were submitted to a short-term (48 h) feed restriction when the expected ovulatory follicle was > or =27 mm (Hour 0) or served as controls (n=8/group). No effect of short-term feed restriction was detected for systemic concentrations of FSH and LH and for intrafollicular concentrations of estradiol, activin-A, free IGF1, and IGFBP2. Restricted mares had decreased systemic concentrations of leptin at Hour 24 (approached significance) and at Hours 36 and 48 (P<0.04). Follicular fluid of restricted mares at Hour 48 had lower (P<0.02) concentration of leptin and a tendency (P<0.1) for greater concentrations of inhibin-A and VEGF. The percentage of wall of the preovulatory follicle with color-Doppler signals of blood flow at Hour 48 was greater (P<0.04) in the restricted group. Intrafollicular concentration of leptin (combined groups) was positively correlated with score for body condition (r=+0.60; P<0.002) and negatively correlated with the percentage of the follicle wall with blood-flow signals (r=-0.60; P<0.02). Our favored interpretation is that the preovulatory follicle seems to compensate for a nutritional deficiency by increasing the blood flow in the follicle wall.
Theriogenology 03/2010; 73(9):1202-9. · 1.96 Impact Factor
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ABSTRACT: Changes in echotexture and blood flow in the wall of preovulatory follicles in heifers were studied in relation to the LH surge and ovulation in gonadotrophin-releasing hormone-induced (n = 7; Experiment 1) and spontaneous (n = 8; Experiment 2) ovulators. Ultrasonographic examinations and blood sampling were performed either every hour (Experiment 1) or every 6 h (Experiment 2). The interval from LH peak to ovulation in induced and spontaneous ovulators was 27.1 +/- 0.3 and 34.5 +/- 1.5 h, respectively. Follicle diameter did not increase between the LH peak and ovulation. In the induced ovulators, serration of the stratum granulosum was detected in one (14%), two (29%), three (43%) and four (57%) heifers at 4, 3, 2 and 1 h before ovulation, respectively. An initial increase in blood flow (P < 0.001) encompassed the LH peak in both experiments. In the induced ovulators, blood flow increased (P < 0.02) to maximum 3 h after the LH peak, maintained a plateau for 5 h, decreased (P < 0.05) between 9 and 14 h, increased (P < 0.05) again between 19 and 21 h and then decreased (P < 0.01) between 25 and 26 h (1 h before ovulation). The biphasic increase and decrease in blood flow and serration of the granulosum in the wall of the preovulatory follicle in cattle are novel findings.
Reproduction Fertility and Development 01/2010; 22(7):1110-7. · 2.11 Impact Factor
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ABSTRACT: The effect of the ovarian follicles on plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) before versus after the expected emergence of the ovulatory follicular wave was studied on Days 0 to 18 (Day 0=ovulation) in four groups of mares (n=6/group). In addition to a control group, all follicles >/=6mm in diameter were ablated on Days 0.5, 6.5, or 12.5 in a herd of mares with reported emergence at 6mm of the future ovulatory follicle on mean Day 10.5. Concentrations of FSH were not different between the Day-0.5 or Day-6.5 ablation groups and the corresponding controls. However, ablation on Day 12.5 resulted in an immediate FSH increase (group-by-day interaction, P<0.003). For LH, ablation on Day 0.5 resulted in an interaction (P<0.02), partially from lower (P<0.05) concentrations on each of Days 15.5 to 18.0 than that in the controls, whereas ablation on Days 6.5 or 12.5 did not result in a significant group effect or interaction. Testosterone concentration, but not progesterone or estradiol concentration, was lower (P<0.04) on Day 2 in the Day-0.5 ablation group than that in the controls. We inferred that follicles did not contain adequate FSH suppressors on Days 0.5 and 6.5 and that they were present only in the Day-12.5 ablation group or after the expected emergence of the ovulatory wave. The hypothesis of an association between low postovulatory concentrations of an ovarian steroid and low concentrations of LH after Day 15 was supported.
Theriogenology 07/2009; 72(4):445-52. · 1.96 Impact Factor
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ABSTRACT: The follicle and hormone aspects of diameter deviation and development of one dominant (>/=28 mm) follicle (1DF) vs two dominant follicles (2DF) were studied in 32 ovulatory follicular waves in mares. Follicles were ranked each day as F1 (largest) to F3. The beginning of deviation was designated day 0 and preceded the first increase in the differences in diameter between F1 and F2 in the 1DF group and between a combination of F1 and F2 vs F3 in the 2DF group. One dominant follicle and 2DF developed in 21 (66%) and 11 (34%) waves, respectively. Double ovulations occurred in only one of the waves with 2DF. In 8/11 waves with 2DF, a second deviation occurred between F1 and F2 on 2.5 +/- 0.4 days after the first deviation. On day 0, 1DF and 2DF waves were similar in number of days after ovulation, number of follicles, difference in diameter between F1 and F2, and plasma concentrations of LH, estradiol and immunoreactive inhibin. The interval from maximum FSH concentration to day 0 was longer (p < 0.05) and FSH concentration was lower (p < 0.05) on days -1 to 4 in the 2DF group. The similarities on day 0 in the characteristics of 1DF and 2DF waves despite the differences in the declining portions of the FSH profile indicated that a specific day of the FSH decline or a specific concentration were not factors in initiating deviation. Unlike reported results in heifers, the results in mares did not indicate a hormonal basis for the development of 2DF or two deviations.
Reproduction in Domestic Animals 11/2008; 44(2):248-54. · 1.36 Impact Factor
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ABSTRACT: Horse oocytes (n = 37) were recovered in vivo from pre-ovulatory follicles 30 h after an ovulation-inducing hCG injection and were examined by fluorescent staining and confocal microscopy. Percentages of metaphase-I (MI), metaphase-II (MII) and atypical oocytes were 11%, 78% and 11% respectively. Microtubules were concentrated in the meiotic spindle in both MI and MII oocytes. Chromosomes in the metaphase plate were anchored at the equatorial region of the spindle. Spindle orientation was perpendicular to the oolema in all MI oocytes, whereas in MII oocytes, 66% were parallel and 34% were perpendicular. In MII oocytes, the nuclear material in the polar body had no specific organization and was intertwined with microtubules. Discrete foci of microfilaments at the sub-cortical region of the ooplasm formed an F-actin band, as seen in the inner confocal sections. The percentage area of oocyte image with discrete foci and/or the thickness of F-actin band was used to indicate microfilament content. Microfilament content was greater (p < 0.006) in MII oocytes than in MI oocytes and greater (p < 0.03) in MII oocytes with a perpendicular spindle than with a parallel spindle. The perpendicular spindle orientation in MII oocytes may have represented a later stage of maturation. Atypical oocytes were based on microtubules that were detached from the kinetochores and spread in the ooplasm or by microtubules that accumulated as an amorphous mass near the condensed chromatin. This is the first description of the nuclear configuration, spindle morphology and cytoskeletal organization of in vivo maturing horse oocytes.
Reproduction in Domestic Animals 11/2008; 44(3):435-40. · 1.36 Impact Factor
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ABSTRACT: An ovulatory follicular wave was induced by ablation of follicles > or =6mm and treatment with prostaglandin F2alpha (PGF) on Day 10 (ovulation=Day 0). Follicle and hormone dynamics of the induced waves were compared among three age groups: young (5-6 y, n=14 waves), intermediate (10-14 y, n=16), and old (> or =18, n=15). During the common-growth phase of the induced wave (Days 12-17), diameter of the future ovulatory follicle was not different among ages, but the young group had more (P<0.05) follicles that reached > or =10mm. The number was correlated (r=+0.7; P<0.0001) within mares between consecutive interovulatory intervals, indicating repeatability. Concentrations of LH increased in all age groups during Days 12-17, but were greatest (P<0.002) in the young group and continued to be greater (P<0.0001) throughout the ovulatory LH surge. During several days before Day-1, there were no age-related effects on systemic estradiol concentrations, diameter of the preovulatory follicle, or B-mode echotexture or color-Doppler signals of blood flow in the follicle wall. Interpretations were: (1) greater number of follicles in the young group reflected a greater follicle reserve, (2) greater LH concentrations throughout the ovulatory surge in the young group reflected a more positive response to an extraovarian/environmental influence after removal of the negative effect of progesterone, and (3) lower LH concentrations in the older groups were adequate for the preovulatory changes in the follicle.
Theriogenology 11/2008; 71(5):780-8. · 1.96 Impact Factor
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ABSTRACT: 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.
Reproduction in Domestic Animals 10/2008; 44(1):92-9. · 1.36 Impact Factor
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ABSTRACT: A GnRH antagonist (Acyline) was used to study the role of FSH in early development of a follicular wave in 61 mares. In Experiment 1, a single dose of 3 mg per mare, compared with 0 and 1 mg, suppressed both the FSH and follicle responses to exogenous GnRH. In Experiment 2, high concentrations of FSH were induced by two successive ablations of all follicles >/= 6 mm on days 10 and 13 (day 0 = ovulation). A single treatment with Acyline resulted in significantly greater suppression of plasma concentrations of FSH than a single treatment with charcoal-extracted follicular fluid (source of inhibin) or oestradiol. Suppression of FSH was not significantly different between the group treated with Acyline alone and a group treated with a combination of Acyline, inhibin and oestradiol. In Experiment 3, all follicles were ablated on day 10 to induce an FSH surge and a new follicular wave. Acyline treatment on day 10 resulted in an immediate decrease in FSH, without a significant effect on day of emergence of a new wave or growth of follicles from 7 to 11 mm on days 11-13. Treatment on day 15, a day before expected follicle deviation and after the peak of the wave-stimulating FSH surge, resulted in an immediate decrease in FSH and cessation of follicle growth. Results indicated that growth of follicles for about 2 days after wave emergence was independent of FSH. In contrast, during the decline in the wave-stimulating FSH surge and before follicle deviation, growth of follicles was dependent on FSH.
Reproduction in Domestic Animals 10/2008; 44(3):504-11. · 1.36 Impact Factor
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ABSTRACT: Follicle blood flow, follicular-fluid and plasma hormone concentrations, and oocyte quality were studied 30 h after an ovulation-inducing hCG treatment when the pre-ovulatory follicle was 32 mm. Mares were grouped as positive (n = 16) and negative (n = 44) for hCG antibodies before the experimental hCG treatment. Percentage of the follicle wall with blood flow signals was less (p < 0.05) in the antibody positive group than in the negative group. The concentrations of follicular-fluid oestradiol and free IGF1, and plasma oestradiol were greater (p < 0.001), and follicular-fluid progesterone (p < 0.001) and plasma LH (p < 0.02) were less in the antibody-positive group than in the negative group. For recovered oocytes at 30 h (n = 37), the antibody-positive group had fewer (p < 0.001) mature (MII) oocytes than the antibody-negative group. Results were attributable to highly effective neutralization of the hCG in the antibody-positive group.
Reproduction in Domestic Animals 10/2008; 44(3):474-9. · 1.36 Impact Factor
