RESUMO
The objectives of the study were to determine the effect of seminal plasma ß-NGF on Corpus Luteum morphology and function and level of mRNA expression of steroidogenic enzymes. Llamas were assigned (n = 12/per group) to receive an intramuscular dose of: (a) 1 ml phosphate buffered saline (PBS), (b) 5 µg gonadorelin acetate (GnRH), or (c) 1.0 mg of purified llama spß-NGF. Ovaries were examined by transrectal B-mode ultrasonography from treatment to ovulation (Day 0 = treatment). B mode/Power Doppler ultrasonography and blood samples collection were performed at Days 4, 8 and 10 (n = 3 llamas per treatment group/per time point) to determine CL diameter, vascularization and plasma progesterone concentration respectively. Plasma progesterone concentration was analyzed in all llamas at Day 0. Then females were submitted to ovariectomy at Days 4, 8 and 10 (n = 3 llamas/treatment/time), CL was removed to determine vascular area, proportion of luteal cells and CYP11A1/P450scc and STAR expression by RT-PCR. Ovulation was similar between llamas treated with GnRH or spß-NGF and CL diameter did not differ between GnRH or spß-NGF groups by Day 4, 8 or 10. Vascularization area of the CL was higher (P < 0.01) in llamas from the spß-NGF than GnRH-treated group by Day 4 and 8. Plasma progesterone concentration was higher (P < 0.05) in llamas from the spß-NGF compared to females of GnRH group by Day 4 and 8. The proportion of small and large luteal cells did not differ between GnRH or spß-NGF groups by Day 8. CYP11A1/P450scc was upregulated 3 folds at day 4 and 10 by spß-NGF compared to GnRH. STAR transcription was 3 folds higher at day 4 in females treated with spß-NGF. In conclusion, the luteotrophic effect of spß-NGF could be related to an increase of vascularization and up regulation of CYP11A1/P450scc and STAR transcripts enhancing progesterone secretion.
Assuntos
Camelídeos Americanos/fisiologia , Corpo Lúteo/irrigação sanguínea , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Sêmen/química , Animais , Corpo Lúteo/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sêmen/metabolismoRESUMO
Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment.
Assuntos
Feminino , Animais , Bovinos , Bovinos/embriologia , Bovinos/fisiologia , Hormônio Foliculoestimulante/análise , Hormônio Foliculoestimulante/síntese químicaRESUMO
Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles<5 mm decreased during superstimulation suggesting that there was no continuous recruitment of small follicles, and the number of follicles>1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment. (AU)
Assuntos
Animais , Feminino , Bovinos , Bovinos/embriologia , Bovinos/fisiologia , Hormônio Foliculoestimulante/análise , Hormônio Foliculoestimulante/síntese químicaRESUMO
The objective of the study was to compare the pituitary and ovarian responses after intramuscular, intravenous, or intrauterine administration of ß-nerve growth factor (ß-NGF) of seminal plasma origin (SP-NGF) in llamas. In experiment 1, mature female llamas with a growing follicle of 7 mm or greater were assigned randomly to four groups (n = 7/group) and given 2 mg of purified SP-NGF in a volume of 2 mL by (1) intramuscular administration, (2) intravenous administration, and (3) intrauterine infusion, or (4) intrauterine infusion of 2 mL of PBS (negative control). Because ovulations were not detected after intrauterine infusion in experiment 1, a second experiment was done to determine if a higher dose of SP-NGF given by intrauterine infusion, similar to a natural dose during copulation, will elicit an ovulatory response. In experiment 2, llamas with a growing follicle of 7 mm or greater were assigned randomly to three groups (n = 6/per group) given an intrauterine infusion of (1) 4 mL of raw seminal plasma, (2) 4 mL of PBS containing 20 mg of purified llama SP-NGF, or 3) 4 mL of PBS (negative control). In both experiments, the ovaries were examined daily by transrectal ultrasonography using a B-mode scanner and power Doppler mode to detect ovulation and to monitor CL growth, regression, and vascularization. Blood samples were collected to determine plasma LH and progesterone concentrations. In experiment 1, only llamas treated by intramuscular or intravenous administration of SP-NGF ovulated (7 of 7 and 6 of 7, respectively). Plasma LH concentration did not differ between the intramuscular and intravenous SP-NGF-treated groups, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. In experiment 2, the ovulation rate was 100% for llamas treated by intrauterine infusion of raw seminal plasma or llama SP-NFG, whereas no ovulations were detected in females treated with PBS. Plasma LH concentrations did not differ between groups that ovulated, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. We conclude that ß-NGF from llama seminal plasma origin elicits a preovulatory LH surge, followed by ovulation and the development of a functional CL, regardless of the route of administration. However, the dose required to elicit pituitary and ovarian responses is higher when administered by intrauterine infusion than by intramuscular or intravenous routes.
Assuntos
Camelídeos Americanos/fisiologia , Hormônio Luteinizante/sangue , Fator de Crescimento Neural/administração & dosagem , Ovulação/efeitos dos fármacos , Sêmen/química , Administração Intravenosa/veterinária , Animais , Corpo Lúteo/anatomia & histologia , Corpo Lúteo/irrigação sanguínea , Relação Dose-Resposta a Droga , Feminino , Masculino , Músculos/efeitos dos fármacos , Ovário/diagnóstico por imagem , Progesterona/sangue , Ultrassonografia , Útero/efeitos dos fármacosRESUMO
The objective of the study was to test the hypothesis that repeated administrations of OIF/NGF during the peri-ovulatory period (pre-ovulatory, ovulatory, early post-ovulatory), will enhance the luteotrophic effect in llamas. Female llamas were examined daily by transrectal ultrasonography in B- and Doppler-mode using a scanner equipped with a 7.5-MHz linear-array transducer to monitor ovarian follicle and luteal dynamics. When a growing follicle ≥7mm was detected, llamas were assigned randomly to one of the three groups and given 1mg of purified OIF/NGF im (intramuscular) (a) pre-ovulation (single dose; n=12), (b) pre-ovulation and at the time of ovulation (2 doses, n=10), or (c) pre-ovulation, at the time of ovulation, and 24h after ovulation (3 doses, n=10). The pre-ovulatory follicle diameter at the time of treatment, ovulation rate and the first day of CL detection did not differ (P=0.3) among groups. However, maximum CL diameter was greatest (P=0.003) in llamas in the 2-dose group, and smallest in the 3-dose group. Accordingly, the 2 dose-group had the largest day-to-day profile for CL diameter (P<0.01), area of CL vascularization (<0.01), and plasma progesterone concentration (P=0.01) compared to the other groups. Interestingly, the luteal response to 3-doses of OIF/NGF during the peri-ovulatory period was not different from a single dose. In conclusion, OIF/NGF isolated from llama seminal plasma is luteotrophic and the effect on CL size and function is affected by the number and timing of treatments during the peri-ovulatory period.
Assuntos
Camelídeos Americanos/fisiologia , Corpo Lúteo/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Ovulação/efeitos dos fármacos , Ovulação/fisiologia , Animais , Esquema de Medicação , Feminino , Fator de Crescimento Neural/administração & dosagemRESUMO
Ovulation-inducing factor (OIF) is a protein present in llama seminal plasma that has recently been identified as ß-Nerve Growth Factor (NGF) and it induces not only a high rate of ovulation but also appears to have luteotrophic properties in this species. A 2-by-2 experimental design was used to determine the effect of treatments (OIF/NGF vs GnRH) and categories of preovulatory follicle diameter (7-10 vs >10mm) on ovulation rate, CL diameter and function in llamas. Llamas (n=32 llamas per group) were randomly assigned to receive an intramuscular dose of: (a) 1mg purified OIF/NGF in the presence of a follicle of 7-10mm in diameter; (b) 50 µg of GnRH in the presence of a follicle of 7-10mm in diameter; (c) 1mg purified OIF/NGF in the presence of a follicle >10mm in diameter; (d) 50 µg of GnRH in the presence of a follicle >10mm in diameter. Llamas were examined by ultrasonography every 12h from treatment to Day 2 (Day 0=treatment) to detect ovulation, and again on Day 8 to determine CL diameter. Ovulation rates did not differ among groups. There was an effect of preovulatory follicle size on Corpus Luteum diameter at Day 8 (P<0.001), however plasma progesterone concentration (n=15/per group) was higher (P<0.05) in the OIF/NGF - than that of the GnRH - treated group by the same day. We conclude that OIF/NGF treatment enhances CL function regardless preovulatory follicle size at the time of treatment.
Assuntos
Camelídeos Americanos/fisiologia , Corpo Lúteo/efeitos dos fármacos , Fator de Crescimento Neural/isolamento & purificação , Fator de Crescimento Neural/farmacologia , Folículo Ovariano/citologia , Sêmen/química , Animais , Tamanho Celular , Corpo Lúteo/diagnóstico por imagem , Corpo Lúteo/fisiologia , Feminino , Fase Folicular , Hormônio Liberador de Gonadotropina/farmacologia , Masculino , Folículo Ovariano/diagnóstico por imagem , Ovulação/efeitos dos fármacos , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Ultrassonografia , Regulação para Cima/efeitos dos fármacosRESUMO
The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 µg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.
Assuntos
Camelídeos Americanos , Corpo Lúteo/efeitos dos fármacos , Fatores de Crescimento Neural/administração & dosagem , Folículo Ovariano/irrigação sanguínea , Ovulação/efeitos dos fármacos , Sêmen/química , Animais , Corpo Lúteo/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/administração & dosagem , Injeções Intramusculares/veterinária , Hormônio Luteinizante/sangue , Masculino , Fatores de Crescimento Neural/isolamento & purificação , Folículo Ovariano/diagnóstico por imagem , Folículo Ovariano/fisiologia , Progesterona/sangue , Sêmen/fisiologia , UltrassonografiaRESUMO
The use of ultrasonography has changed our understanding of the ovarian function in live animals. However, most of the studies that have used ultrasonography to image the ovary have provided data only of structures >1mm in diameter. The recent availability of high-resolution ultrasound technology with high-frequency transducers (25-70 MHz), offers the potential to examine the developmental dynamics of small antral follicles and the cumulus-oocyte complex (COC) in vivo. In this review we provide data from a series of studies performed by Veterinary Biomedical Sciences Laboratory describing the advantages and disadvantages, as well as image characteristics, of ultrasound biomicroscopy (UBM) to study ovarian biology in mammals. Data and images of small ovarian structures in rabbits, cattle, mice and humans are shown. The UBM technique allowed visualisation of small antral follicles ranging in size from 300 to 700 µm in all species examined, as well as COC within follicles in rabbits, cattle and humans. Furthermore, UBM permitted clear distinction of the follicular wall from the surrounding ovarian stroma in cattle and humans. At present, the limited depth of penetration of UBM restricts the use of this technique to an experimental setting. In that regard, further studies using UBM will probably result in a greater understanding of the pattern and control of early antral folliculogenesis and oogenesis.
Assuntos
Microscopia Acústica , Oócitos/diagnóstico por imagem , Folículo Ovariano/diagnóstico por imagem , Animais , Células do Cúmulo/diagnóstico por imagem , Feminino , Humanos , Oogênese , Fatores de TempoRESUMO
The objectives of the study were to determine the effects of nutritional restriction on ovarian function in llamas. Mature female llamas were assigned randomly to a Control group, fed 100% of maintenance energy requirements (MER) (n=8), or a Restricted group (n=8) fed from 70% to 40% of MER until a body condition score of 2.5 was attained. Blood samples were taken every-other-day to determine plasma concentrations of LH, estradiol, leptin and metabolic markers, and follicular dynamics were monitored daily by ultrasonography for 30 days (Experiment 1). Llamas were then treated with GnRH to compare the ovulatory response and corpus luteus (CL) development between groups (Experiment 2). Blood samples were taken to measure LH, leptin, progesterone and metabolic markers and ovarian structures were assessed as in Experiment 1. Llamas in the Restricted group had lower body mass and body condition scores than those in the Control group (P<0.001). Plasma concentrations of cholesterol, non-esterified fatty acids, triglycerides, and urea were higher in the Restricted group (P<0.05) than in the Control group. The day-to-day diameter profiles of the dominant follicles were smaller (P<0.05) in the Restricted group than in the Control group but plasma estradiol concentration did not differ. The ovulation rate and LH secretion in response to GnRH did not differ. Day-to-day profiles of CL diameter, plasma progesterone and leptin concentrations were smaller (P<0.01) in the Restricted group. In conclusion, nutritional restriction in llamas was associated with suppressed follicle and CL development, and lower plasma concentrations of progesterone and leptin.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Restrição Calórica , Camelídeos Americanos/metabolismo , Hormônios/sangue , Ovário/fisiologia , Animais , Constituição Corporal/fisiologia , Restrição Calórica/efeitos adversos , Restrição Calórica/veterinária , Camelídeos Americanos/sangue , Camelídeos Americanos/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Leptina/sangue , Hormônio Luteinizante/sangue , Ovário/metabolismo , Ovulação/sangue , Ovulação/metabolismo , Ovulação/fisiologia , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Progesterona/sangue , Redução de Peso/fisiologiaRESUMO
The objectives of this study were to: (1) evaluate the feasibility of ultrasonic biomicroscopy (UBM) for imaging ovarian follicles in vivo in cows and heifers, (2) compare the transvaginal to the transrectal approach, (3) compare the number of follicles detected by UBM to conventional ultrasonography (US), and (4) identify cumulus-oocyte-complexes (COC) within follicles by UBM. Mature cows (n=5) and peri-pubertal heifers (n=5) were examined once using conventional B-mode US (Aloka 900) with a 7.5 MHz transrectal, and a 5 MHz transvaginal transducers. A second series of examinations was performed using UBM (Visualsonics Vevo 660) equipped with a 30 MHz transrectal and a 40 MHz transvaginal transducer. A three- to four-fold increase in the number of small follicles (<3 mm) was detected using the transvaginal approach with UBM compared to conventional US in both heifers (32.4 ± 4.24 compared to 7.2 ± 1.4; P<0.0001) and cows (35.0 ± 13.8 compared to 10.7 ± 7.5; P=0.0013). However, fewer follicles (all sizes combined) were detected using the transrectal approach with UBM compared to conventional US in both heifers (8.6 ± 5.6 compared to 17.6 ± 4.9; P=0.02) and cows (5.3 ± 6.1 compared to 20.3 ± 7; P=0.04). In heifers, COC-like structures were identified in 39 of 202 (19.3%) follicles examined. In conclusion, UBM using a transvaginal approach is feasible and may be used for in vivo assessment of early antral follicles as small as 0.4 mm, and COC within follicles.