RESUMO
This study investigated the effectiveness of different doses of estradiol benzoate (EB) to promote cervical relaxation and their effects on luteal function and outcomes of non-surgical embryo recovery (NSER) in sheep. Multiparous (MULT) and nulliparous (NULL) crossbred Lacaune X Santa Inês ewes were superovulated and naturally bred. Seven days after progesterone withdrawal, females were randomly assigned to one of three distinct cervical relaxation protocols, consisting of i.m. treatment with 37.5 µg d-cloprostenol and different doses of EB: 0.0 mg (0.0EB group; n = 3 NULL and 14 MULT); 0.5 mg (0.5EB group; n = 4 NULL and 12 MULT) or 1.0 mg (1.0EB group, n = 6 NULL and 11 MULT) 16 h before NSER. All ewes received 50 IU of oxytocin 20 min before NSER (D17). Blood samples were collected and ultrasound exams (B-mode and color Doppler) were performed at two timepoints: immediately before d-cloprostenol and EB treatments and prior to NSER. Estrous behavior, corpora lutea count and NSER success outcomes were not affected by EB treatments nor parity (P > 0.05). Embryo recovery rate was greater for ewes in the 0.5EB group and in the NULL ewes (P < 0.05). Ovarian biometrics differed between the two evaluation timepoints in all groups (P < 0.05). Plasma estradiol increased over time, reaching a significant greater level in 1.0EB ewes compared to controls on D17 (P < 0.05), whereas progesterone concentrations decreased over time in all groups (P > 0.05). In conclusion, treatments did not affect NSER success but they did affect luteal function by altering P4 and E2 concentrations. Therefore, the NSER technique can be successfully performed in ewes with or without prior treatment with EB.
Assuntos
Corpo Lúteo , Progesterona , Gravidez , Ovinos , Feminino , Animais , Estradiol/farmacologia , Cloprostenol/farmacologia , Ensaios Clínicos Veterinários como AssuntoRESUMO
This study aimed to evaluate the effects of hCG treatment during the early luteal phase on ovarian function, progesterone profile, and embryo yield in superovulated ewes. Superovulated sheep were randomly assigned to receive 300 IU hCG i.m. (GhCG, n = 24) or not (GControl, n = 25) at 96 h after the removal of the progesterone (P4) device (D13). Non-surgical embryo recovery (NSER) was performed eight days after P4 withdrawal. Ultrasound evaluations were performed on D13, D14, D16, and D17. Blood samples were collected on D14, D16, and D17. Superovulation scores were recorded based on the number of corpora lutea (CL) as follows: 1 (≤ 2), 2 (3-5), 3 (6-8), and 4 (≥ 9). NSER efficiency, superovulation response, and luteal tissue area were similar in both groups (P > 0.05). Structural luteolysis tended to be higher in GControl (P = 0.07; 47.0 %) while functional luteolysis was similar in both groups (P > 0.05; 0.0 % and 5.9 %). The recovery rate was greater (P < 0.05) in GhCG (89.8 %) compared with GControl (71.0 %), with similar overall ova/embryo numbers observed for both groups (P > 0.05). GhCG showed a higher concentration of animals with a superovulatory response score of 4 (54.5 %; P < 0.05) compared with the lowest scores. Plasma progesterone on D16 was higher (P < 0.05) in GhCG ewes (11.1 ± 1.5 vs 6.9 ± 1.5 ng/mL). In conclusion, the hCG treatment improved circulating P4 and embryo recovery rate, tended to maintain luteal functionality, and thus constitutes an additional tool for improving embryo yield in superovulated ewes.
Assuntos
Fase Luteal , Progesterona , Ovinos , Feminino , Animais , Hormônio Foliculoestimulante/farmacologia , Corpo Lúteo/fisiologia , SuperovulaçãoRESUMO
The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3⯱â¯1.2 ova/embryos per flush, nâ¯=â¯5) or poor responders (1.1⯱â¯0.3 ova/embryos per flush, nâ¯=â¯5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8â¯mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (-0.5 ± 0.2 and -0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.
Assuntos
Bovinos/fisiologia , Indução da Ovulação/veterinária , Receptores do LH/metabolismo , Técnicas de Reprodução Assistida/veterinária , Superovulação/genética , Animais , Bovinos/metabolismo , Sincronização do Estro , Feminino , Células da Granulosa/metabolismo , Indução da Ovulação/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores do LH/genéticaRESUMO
O objetivo do presente estudo foi avaliar a viabilidade da técnica de transferência não cirúrgica em cabras. Quatro cabras não-lactantes pluríparas da raça Toggenburg foram utilizadas como receptoras de embriões, sendo que duas receberam um embriões e duas receberam dois embriões coletados não cirurgicamente cabras doadoras. Os corpos lúteos das receptoras foram detectados um dia antes da transferência de embriões por ultrassonografia transretal. Uma seringa de 5mL contendo 2mL de meio holding foi acoplada em um cateter tomcat, no qual os embriões foram aspirados em uma coluna central a duas outras colunas. Um espéculo Colin número 2 foi inserido na vulva e na vagina, e com o uso de uma fonte de luz, a cerviz foi localizada e imobilizada com uma pinça de Allis. Um cateter uretral número seis acoplado a um mandril e lubrificado com meio PBS foi inserido na cérvix, e assim os aneis cervicais foram gradualmente transpostos. Após perder a resistência, o cateter uretral foi movido lateralmente para o corno uterino desejado. O mandril e a pinça de Allis foram retirados e o conjunto seringa e tomcat foi acoplado ao cateter uretral e o conteúdo injetado no corno uterino ipsilateral ao corpo lúteo com posterior retirada do cateter. Cabras que ovularam em apenas um ovário foram usadas para testar a eficiência da deposição do embrião. O tempo gasto entre a inserção do espéculo e a sua remoção foi inferior a três minutos. O tempo para transpor a cérvix foi inferior a um minuto. A ultrassonografia revelou a deposição de líquido no corno desejado. Receptoras que receberam dois embriões tornaram-se gestantes e pariram três crias. Estes primeiros resultados encorajam a técnica e demonstram que a transferência de embriões em caprinos pode ser feita totalmente por procedimentos não cirúrgicos...
Assuntos
Animais , Cabras/embriologia , Colo do Útero/embriologia , Transferência Embrionária/métodos , Transferência Embrionária/veterinária , Técnicas de Reprodução Assistida/veterinária , Ultrassonografia/veterináriaRESUMO
O objetivo do presente estudo foi avaliar a viabilidade da técnica de transferência não cirúrgica em cabras. Quatro cabras não-lactantes pluríparas da raça Toggenburg foram utilizadas como receptoras de embriões, sendo que duas receberam um embriões e duas receberam dois embriões coletados não cirurgicamente cabras doadoras. Os corpos lúteos das receptoras foram detectados um dia antes da transferência de embriões por ultrassonografia transretal. Uma seringa de 5mL contendo 2mL de meio holding foi acoplada em um cateter tomcat, no qual os embriões foram aspirados em uma coluna central a duas outras colunas. Um espéculo Colin número 2 foi inserido na vulva e na vagina, e com o uso de uma fonte de luz, a cerviz foi localizada e imobilizada com uma pinça de Allis. Um cateter uretral número seis acoplado a um mandril e lubrificado com meio PBS foi inserido na cérvix, e assim os aneis cervicais foram gradualmente transpostos. Após perder a resistência, o cateter uretral foi movido lateralmente para o corno uterino desejado. O mandril e a pinça de Allis foram retirados e o conjunto seringa e tomcat foi acoplado ao cateter uretral e o conteúdo injetado no corno uterino ipsilateral ao corpo lúteo com posterior retirada do cateter. Cabras que ovularam em apenas um ovário foram usadas para testar a eficiência da deposição do embrião. O tempo gasto entre a inserção do espéculo e a sua remoção foi inferior a três minutos. O tempo para transpor a cérvix foi inferior a um minuto. A ultrassonografia revelou a deposição de líquido no corno desejado. Receptoras que receberam dois embriões tornaram-se gestantes e pariram três crias. Estes primeiros resultados encorajam a técnica e demonstram que a transferência de embriões em caprinos pode ser feita totalmente por procedimentos não cirúrgicos.(AU)
Assuntos
Animais , Cabras/embriologia , Transferência Embrionária/veterinária , Transferência Embrionária/métodos , Colo do Útero/embriologia , Técnicas de Reprodução Assistida/veterinária , Ultrassonografia/veterináriaRESUMO
The objective of this study was to evaluate the effect of a PGF2α-analogue (PGF) on ovulation and pregnancy rates after timed artificial insemination (TAI) in cattle. In experiment 1, crossbred dual-purpose heifers, in a crossover design (3 × 3), were given an intravaginal progesterone-releasing insert (controlled internal drug release [CIDR]) plus 1 mg estradiol benzoate (EB) intramuscularly (im) and 250 µg of a PGF-analogue im on Day 0. The CIDR inserts were removed 5 days after follicular wave emergence, and the heifers were randomly divided into three treatment groups to receive the following treatments: (1) 1 mg of EB im (EB group, n = 13); (2) 500 µg of PGF im (PG group, n = 13); or (3) saline (control group, n = 13), 24 hours after CIDR removal. Ovulation occurred earlier in EB (69.81 ± 3.23 hours) and PG groups (73.09 ± 3.23 hours) compared with control (83.07 ± 4.6 hours; P = 0.01) after CIDR removal. In experiment 2, pubertal beef heifers (n = 444), 12 to 14 months of age were used. On Day 0, the heifers were given a CIDR insert plus 2 mg EB im. On Day 9, the CIDR was removed and the heifers were given 500 µg of PGF im. Heifers were randomly assigned into one of three treatment groups: (1) 1 mg of EB (EB group; n = 145); (2) 500 µg of PGF (PG group; n = 149), both 24 hours after CIDR removal; or (3) 600 µg of estradiol cypionate (ECP group; n = 150) at CIDR removal. Timed artificial insemination occurred 48 hours after CIDR removal in the ECP group and 54 hours in the PG and EB groups. The percentage of heifers ovulating was higher in the PG group compared with the other groups (P = 0.08). However, the pregnancy rates did not differ among groups (47.6%, 45%, and 46.6%, for EB, PG, and ECP, respectively; P = 0.9). In experiment 3, 224 lactating beef cows, 40 to 50 days postpartum with 2.5 to 3.5 of body condition score were treated similarly as described in experiment 2, except for the ECP group, which was excluded. The treatments were as follows: 1 mg EB (EB group; n = 117) or 500 µg PGF (PG group; n = 107), 24 hours after CIDR removal. The calves were temporarily separated from their dams from Days 9 to 11. No difference was detected on the pregnancy rate between the EB and PG groups (58.1% vs. 47.6%, respectively; P = 0.11). Taken together, the combined results suggested that PGF2α could be successfully used to induce and synchronize ovulation in cattle undergoing TAI, with similar pregnancy rates when compared with other ovulatory stimuli (ECP and EB).
Assuntos
Bovinos/fisiologia , Dinoprosta/farmacologia , Inseminação Artificial/veterinária , Indução da Ovulação/veterinária , Administração Intravaginal , Animais , Brasil , Corpo Lúteo/diagnóstico por imagem , Estudos Cross-Over , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Sincronização do Estro/métodos , Feminino , Inseminação Artificial/métodos , Lactação , Folículo Ovariano/diagnóstico por imagem , Indução da Ovulação/métodos , Gravidez , Taxa de Gravidez , Progesterona/administração & dosagem , Fatores de Tempo , Ultrassonografia , Útero/diagnóstico por imagemRESUMO
Ultrasound-guided transvaginal follicle aspiration is used to recover cumulus-oocyte complexes (for IVF) and to synchronize follicular wave emergence (ablation of dominant follicle). Although aspirated follicles are generally supposed to undergo immediate atresia, there are indications that they may remain active. The objective was to evaluate the occurrence and characteristics of residual follicles (RF) after transvaginal follicle aspiration in cattle. Ovarian follicular wave emergence was synchronized in Holstein cows (N = 13) in the presence (groups 1 and 3) or absence (groups 2 and 4) of norgestomet implants. The largest follicle was aspirated at a diameter of 8 mm (groups 1 and 2) or 12 mm (groups 3 and 4). Ovarian follicles were visualized (transrectal ultrasonography) every 12 h after wave emergence. Follicular fluid samples were collected from the largest follicle and from the ensuing RF and concentrations of estradiol and progesterone were determined. After aspiration, 73.2% (52/71) of the follicles refilled with fluid, and a new antrum was detected 12 to 24 h later. Norgestomet did not affect (P > 0.05) RF occurrence or diameter, but in RF from group 4, concentrations of estradiol decreased (-530.7 ± 133.9 ng/mL; P < 0.01) whereas progesterone increased (+429.6 ± 171.7 ng/mL; P < 0.05) relative to preaspiration. In RF, there were three steroidogenesis patterns: (1) high estradiol concentration and high estradiol:progesterone ratio (estradiol-active RF); (2) low estradiol, but high progesterone concentrations (luteinized RF); and (3) low estradiol and low progesterone concentrations (inactive RF). Estradiol-active RF were more likely (P < 0.05) from follicles with high estradiol concentrations (regardless of diameter). In conclusion, fluid-filled structures (RF) with variable steroid production patterns are frequently formed after ultrasound-guided follicle aspiration. The occurrence and features of these RF depended on the diameter and status of these follicles before aspiration.
Assuntos
Bovinos , Folículo Ovariano/fisiologia , Coleta de Tecidos e Órgãos/veterinária , Ultrassonografia/veterinária , Animais , Bovinos/anatomia & histologia , Bovinos/fisiologia , Células do Cúmulo , Estradiol/análise , Feminino , Líquido Folicular/química , Oócitos , Folículo Ovariano/diagnóstico por imagem , Folículo Ovariano/cirurgia , Pregnenodionas/administração & dosagem , Progesterona/análise , Sucção/veterinária , Coleta de Tecidos e Órgãos/métodosRESUMO
Ovarian blood supply is directly related to follicle developmental potential and to oocyte quality, and color Doppler ultrasonography might be a valuable tool to predict in vitro fertilization outcomes. In most studies in large domestic animals, however, the evaluation of follicle blood flow is qualitative (presence or absence of color signal) or dependent on the analysis of a single image. The objective of the present study was to first describe the use of a three-dimensional (3D) modeling of color Doppler images for a quantitative assessment of vascularization in bovine ovarian follicles. Follicular wave emergence was synchronized in Holstein and Gir heifers (n = 20), and follicular dynamics were assessed every 12 h using a color Doppler ultrasound device. The recorded cine-loop of the dominant follicle was decomposed into frames and medical image processing software was used to isolate the Doppler signal, generate the 3D model and calculate the volume of vascularization. In experiment 1, the model was validated by comparing the expected and calculated volumes and was used to predict possible variations in the results of the 2D approach. In experiment 2, vascularization was analyzed during follicular development. In both breeds, the volume of vascularization increased after follicle deviation and was positively correlated (P < 0.05) to follicular diameter (r = 0.65 and 0.54 for Holstein and Gir heifers, respectively). Spatial analysis of the three-dimensional model showed an uneven distribution of vascularization in the follicular wall, with a more intense blood flow being detected in the basal (nearest the ovarian hilus) and lateral regions of the dominant follicles. These results demonstrate the potential of this technique as a new tool for in vivo studies of ovarian physiology in large animals.(AU)
Assuntos
Animais , Fase Folicular/fisiologia , Ovário/anatomia & histologia , Ecocardiografia Doppler , Bovinos/classificaçãoRESUMO
Doppler ultrasonography allows the characterization and measurement of blood flow, and can be used to indirectly make inference regarding the functionality of different organs, including the ovaries. Several studies highlighted the importance of an adequate blood flow for follicle development and acquisition of ovulatory potential, and for progesterone secretion by the corpus luteum. Due to some particularities of the ovarian vascular anatomy, however, different strategies had to be developed to measure the blood flow detected by Doppler imaging. Some of these approaches were successful to characterize the patterns of blood flow throughout the estrous cycle, pregnancy, and early postpartum, but require post acquisition image processing and do not allow real time decisions to be taken. The subjective evaluation of blood flow has been alternatively used in field conditions aiming to early detect non pregnant animals, select embryo recipients, or predict artificial insemination and in vitro fertilization outcomes. In summary, color Doppler imaging provides important information about the function of corpus luteum and follicles, supporting clinical diagnoses and management decisions. Nonetheless, the adoption of Doppler ultrasonography as a routine exam, instead of a limited use in an individual basis, requires further development of feasible blood flow evaluation procedures and the establishment of reference values.(AU)
Assuntos
Animais , Folículo Ovariano/anatomia & histologia , Ovário/anatomia & histologia , Bovinos/classificação , Fluxo Sanguíneo RegionalRESUMO
Doppler ultrasonography allows the characterization and measurement of blood flow, and can be used to indirectly make inference regarding the functionality of different organs, including the ovaries. Several studies highlighted the importance of an adequate blood flow for follicle development and acquisition of ovulatory potential, and for progesterone secretion by the corpus luteum. Due to some particularities of the ovarian vascular anatomy, however, different strategies had to be developed to measure the blood flow detected by Doppler imaging. Some of these approaches were successful to characterize the patterns of blood flow throughout the estrous cycle, pregnancy, and early postpartum, but require post acquisition image processing and do not allow real time decisions to be taken. The subjective evaluation of blood flow has been alternatively used in field conditions aiming to early detect non pregnant animals, select embryo recipients, or predict artificial insemination and in vitro fertilization outcomes. In summary, color Doppler imaging provides important information about the function of corpus luteum and follicles, supporting clinical diagnoses and management decisions. Nonetheless, the adoption of Doppler ultrasonography as a routine exam, instead of a limited use in an individual basis, requires further development of feasible blood flow evaluation procedures and the establishment of reference values.