Comprendre les événements paracriniens qui régulent la fertilité chez la vache est nécessaire non seulement en raison de l'importance agricole de cette espèce, mais aussi pour son utilisation potentielle comme modèle chez l’humain. L'oxyde nitrique (NO), un gaz de radicaux libres, a été impliqué dans la croissance folliculaire et l'ovulation chez les rongeurs et d'autres espèces, mais chez la vache c’est une énigme fascinante : le NO est produit par les cellules de la granulosa bovine et est régulé par la FSH, mais la présence et le profil d'expression des enzymes responsables de la synthèse de NO (NOS) dans les cellules de la granulosa tout au long de la croissance folliculaire ne sont pas claires. Les objectifs de cette thèse sont: (1) élucider le mécanisme de contrôle des NOS et les conséquences de la production d'oxyde nitrique pour le fonctionnement des cellules de la granulosa au cours du développement folliculaire chez la vache et (2) déterminer la régulation des NOS pendant la cascade ovulatoire induite par LH chez les cellules de la granulosa bovine et si l'activité des NOS pour l’expression des gènes critiques dans la cascade ovulatoire chez cette espèce. Les résultats sont séparés en 2 articles. Dans le premier article, la régulation de NOS2 dans les cellules de la granulosa bovine a été explorée. L'abondance des ARNm codant pour NOS2 a été stimulée par la FSH et l’IGF1 en augmentant l’estradiol, et un blocage de l'action de l’estradiol a conséquemment réduit les niveaux d'ARNm codant pour NOS2. De plus, l'inhibition de l'activité des NOS a augmenté l'apoptose dans les cellules de la granulosa in vitro. Dans le second article, il a été démontré que le pic de LH induit une activation des NOS dans les cellules de la granulosa, et que l'activité de NOS induit la production de NO, ce qui est essentiel pour l’expression des gènes critiques dans la cascade ovulatoire induite par LH comme EREG/AREG/PTGS2. Ensemble, les résultats présentés dans ces 2 articles suggèrent que les niveaux physiologiques d'activité des NOS peuvent contribuer à la croissance et la survie des cellules de la granulosa et indiquent également que NO peut être essentiel pour l'ovulation chez les bovins. / Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth and ovulation in rodents and other species, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the presence and the expression pattern in granulosa cells of the enzymes responsible for NO synthesis (NOS) throughout follicular growth are unclear. The objectives of the present thesis were (1) to elucidate the mechanism of control of NOS and the consequences of nitric oxide production for granulosa cell function during follicle development in cattle; and (2) to determine the regulation of NOS during the LH-induced ovulatory cascade in bovine granulosa cells and whether NOS activity is critical for the ovulatory cascade in this species. The results are separated in 2 articles. In the first article, the regulation of NOS2 in bovine granulosa cells was explored. Abundance of mRNA encoding NOS2 was stimulated by FSH and IGF1 through increased estradiol, and a blockade of estradiol action consequently lowered NOS2 mRNA levels. Further, inhibition of NOS activity increased apoptosis in granulosa cells in vitro. In the second article, it was demonstrated that the LH surge induces NOS activation in granulosa cells, and that NOS activity induces the production of NO, which is essential for EREG/AREG/PTGS2 expression, critical genes in the LH-induced ovulatory cascade. Together, the results presented in these 2 articles suggest that physiological levels of NOS activity may contribute to growth and survival of granulosa cells, and also indicate that NO may be essential for ovulation in cattle.

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel. / Identification of regulatory elements in the genome is of paramount importance to understanding the mechanisms governing the expression of specific genes in a given cell type. Following the LH surge, the ovarian peri-ovulatory follicle enters an intensive program of cellular differentiation, orchestrated by major changes in the transcriptional profile of granulosa cells, ultimately triggering ovulation and luteinization, processes essentials for fertility in females. In the mouse, several genes essential to the success of this program are induced 2 to 6 hours after the ovulatory stimulus. Using a standard protocol for superovulation in mice, the regulatory elements were isolated and identified in granulosa cells 4h after administration of hCG using the method FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combined with next generation sequencing. The results of this analysis demonstrate that after the ovulatory stimulus, granulosa cells undergo a major reprogramming of regulatory elements, which is correlated with the extensive changes in their biological functions. In addition, this study showed that most regulatory elements were associated with distal intergenic regions and introns, indicating that these regions are important in transcriptional regulation in granulosa cells. A variety of transcriptional regulators known to be essential for ovarian function, and their binding sites were also identified in this analysis, demonstrating that the FAIRE method has the power to predict molecular events that have correlates in the known physiology of ovarian processes.

Master of Science / Department of Animal Sciences and Industry / Jeffrey S. Stevenson / Lactating dairy cows from 2 Kansas farms were used to determine the effectiveness of exogenous progesterone in the form of an intravaginal insert (controlled internal drug release; CIDR) in conjunction with an ovulation-synchronization protocol. Cows were enrolled in a Presynch + Ovsynch protocol after parturition, where they received 2 injections of PGF[subscript]2[alpha], 14 d apart (Presynch) beginning between 30 and 36 DIM. Cows (n = 155) detected in estrus after the second PGF[subscript]2[alpha] injection of Presynch were inseminated (early AI). Remaining cows were assigned randomly to be treated with the Cosynch-72 protocol (GnRH 12 d after last Presynch PGF[subscript]2[alpha] injection, PGF[subscript]2[alpha] 7 d after GnRH, and timed AI + GnRH injection 72 h later) and served as controls (n = 159), or to be treated with the Cosynch-72 protocol and receive a progesterone insert (Ovsynch + CIDR; n = 175) for 7 d between GnRH and PGF[subscript]2[alpha]. Blood was collected at d −22 and −10 (relative to TAI at d 0) to determine cycling status based on progesterone concentrations and again at d 11 post AI to determine luteal competency. Treated cows were assigned body condition scores (BCS) on d −22 and −10. Pregnancy status was confirmed by palpation of the uterus per rectum and its contents on d 38 post-timed AI and verified again 4 wk later. Treatment with the progesterone insert increased timed AI pregnancies per AI in Cosynch- 72 + CIDR-treated cows when compared with controls (38 vs. 24%), but did not differ from early AI cows (38%). Pregnancy loss was numerically less in progesterone-treated cows than in controls (4.4 vs. 11.8%). Our study shows that increased pregnancies per AI can be achieved by the use of a progesterone insert in a reduced population of cows not yet inseminated, but treated with a progesterone insert.

The overall objective was to elucidate the mechanism of action of ovulation-inducing factor/nerve growth factor (OIF/NGF) in the reproductive function of spontaneous and induced ovulators, using cow and llama as models. In Study 1, the dynamics of trkA, the high affinity receptor for OIF/NGF, were studied during periovulatory period in cows. Unilateral ovariectomies were performed by colpotomy on Days 2, 4 and 6 of the estrous cycle (Day 0= ovulation), and before and after LH administration. Ovarian samples were processed for immunofluorescent detection of trkA. The intensity and area of immuno-positive staining, and the proportion of immuno-positive cells in both the granulosa and theca layers were higher in dominant than in subordinate follicles (P<0.05). Dominant follicles displayed a different intracellular distribution of trkA from subordinate follicles. The number of positive cells was higher in the developing CL (Day 2 and 4) than in the mature or regressing CL (Day 6, Pre-LH, and Post-LH). In Study 2, the distribution of GnRH neurons in the hypothalamus was examined in female llamas (n = 4). Hypothalamic samples were processed for immunohistochemistry for GnRH. The distribution of GnRH neurons had no evident accumulation in specific hypothalamic nuclei. The majority of GnRH neurons were detected in the anterior and medio-basal hypothalamus (P<0.05). The GnRH neuron fibers were detected primarily in the median eminence and in the medio-basal hypothalamus. In Study 3, the relationship between trkA and GnRH neurons in the llama diencephalon was examined in llama brains (n = 4) obtained in Study 2. Samples were stained using double immunofluorescence. TrkA immuno-reactivity was present in most hypothalamic areas examined; the highest density was found in the diagonal band of Broca and the periventricular nuclei. A low percentage of GnRH cells (1%) showed immuno-reactivity to trkA. Close association between immuno-reactive cells (i.e., GnRH and trkA in the same microscopic field) was detected rarely (3/160 GnRH neurons). We concluded that: 1) the high affinity receptor for OIF/NGF is expressed in greater quantities in dominant than subordinate follicles and in the developing CL; 2) GnRH neurons of llamas are concentrated in the anterior and middle hypothalamus, in close relationship to the third ventricle; and, 3) expression of trkA receptors on GnRH neurons was rare, suggesting that the ovulatory effect of OIF/NGF is not via direct interaction with GnRH neurons.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Master of Science / Department of Animal Sciences and Industry / Jeffrey S. Stevenson / We hypothesized that pregnancy outcomes may be improved by inducing luteal regression, ovulation, or both before a control CO-Synch + CIDR program (100 mcg GnRH i.m. [GnRH-1] and insertion of a progesterone-impregnated intravaginal controlled internal drug release [CIDR] insert on d -10, 25 mg PGF2alpha (PG) i.m. and CIDR insert removal on d -3, and 100 mcg GnRH i.m. [GnRH-2] and timed AI [TAI] on d 0) in suckled beef cows. This hypothesis was tested in 2 experiments, in which cows were treated with either PG or PG + GnRH before initiating a control CO-Synch + CIDR program to increase the proportion of cows starting the program in a low (< 1 ng/mL; Exp. 1) or high (≥ 1 ng/mL; Exp. 2) progesterone status, respectively. Blood was collected before each injection for later progesterone analyses. In Exp. 1, cows at 9 locations (n = 1,537) were assigned to either: (1) control or (2) PrePG (same as control with a PG injection on d -13). The PrePG cows had larger (P < 0.05) follicles on d -10 and more (P < 0.05) ovulated after GnRH-1 than controls (60.6 vs. 36.5%). Incidence of estrus between d -3 and 0 was greater (P < 0.05) for treated multiparous cows than multiparous controls and treated and control primiparous cows (74.1 vs. 64.3, 58.6, and 59.1%, respectively). In Exp. 2, cows at 4 locations (n = 803) were assigned to: (1) control (same as Exp. 1) or (2) PrePGG (same as control with PG injection on d -20 and GnRH injection on d -17. Cows with BCS > 5.0 or ≥ 70 d postpartum at TAI were more (P < 0.05) likely to become pregnant than thinner cows or those with fewer days postpartum. Treated cows in both experiments were more (P < 0.05) likely than controls to have luteolysis after initial PG injections and reduced (P < 0.05) serum progesterone. In both experiments, pregnancy rates at d 35 did not differ between treatment and control; however, cows classified as anestrous before d -10, but with elevated progesterone on d -10, had increased (P < 0.05) pregnancy outcomes than remaining anestrous cows with low progesterone concentrations. In summary, luteal regression and ovulation were enhanced by treatments before the 7 d CO-Synch + CIDR program; however, pregnancy per TAI was not improved.

Orientador: Tereza Cristina Cardoso Silva / Coorientador: Alicio Martins Junior / Banca: Guilherme de Paula Nogueira / Banca: Claudia Maria Bertan Membrive / Resumo: O objetivo do presente trabalho foi verificar os efeitos da administração de meia dose de GnRH na submucosa da vulva sobre os níveis de LH, progesterona, dinâmica folicular ovariana e taxa de concepção em rebanho leiteiro, utilizando o protocolo ―Ovsynch‖, modificado. As vacas (n = 13) e novilhas (n = 11) foram pré-sincronizadas com duas injeções de PGF2α, com intervalo de 14 dias e 12 dias. Após a última prostaglandina, foi iniciado o protocolo ―Ovsynch‖, modificado pela inclusão de duas injeções de PGF2α. No momento da aplicação da segunda dose de GnRH (GnRH-2) os animais foram distribuídos de maneira homogênea para os grupos: controle (CN; n = 8), animais recebendo 2,5 mL de cloreto de sódio 0,9% por via im, intramuscular (IM; n = 8), injetado 10,5 µg de GnRH por via intramuscular e submucosa da vulva (SV; n = 8), recebendo 5,25 µg de GnRH. As amostras de sangue foram colhidas no D0, D7, D9 e D17 para determinação da concentração sérica de progesterona, e para o LH, foram colhidas em cinco momentos no D9 (M-40, M0, M40, M80 e M120 min). Quanto ao LH, não houve diferença significativa entre os grupos IM e SV em todos os momentos, os quais foram significativamente maiores do que o CN, enquanto no SV, maiores valores de média foram obtidos para a área sob a curva de LH (ASC-LH) em comparação com os grupos IM e CN. Não houve diferença significativa entre os grupos quanto à concentração de progesterona, independente do dia avaliado. Todos os grupos apresentaram o mesmo número d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to verify the effects of low dose of GnRH administered in the vulva submucosa on LH and progesterone levels, ovarian dynamics and conception rate in dairy cattle using a modified Ovsynch protocol. Cows (n = 13) and heifers (n = 11) were pre-synchronized with two injections of PGF2α administered with a 14-days interval. Twelve days after the last prostaglandin dose a modified Ovsynch protocol was initiated and at the time of the second dose of GnRH (GnRH-2) the animals were homogeneously distributed to groups: control (CN; n = 8), receiving 2.5 mL of 0.9% sodium chloride, im, intramuscular (IM; n = 8,) receiving 10.5 μg of GnRH intramuscular; and vulva submucosa (VS; n = 8) with administration of 5.25 μg of GnRH. Blood samples were collected to determine serum progesterone concentration on D0, D7, D9 and D17, and the level of LH at five moments on D9 (M-40, M0, M40, M80 and M120 min). Higher levels of LH were observed in IM and VS groups in comparison with CN, however no significant difference was found between IM and VS for any time of evaluation. The higher mean value for the area under LH curve (AUC-LH) was found in VS than for IM and CN. There was no difference among groups for progesterone levels measured on in the different days of the protocol. The number of ovulated animals was similar among groups, but the time elapsed to ovulation was lower in IM and VS than in control group. The conception rate was higher in IM group, followed by CN and the... (Complete abstract click electronic access below) / Mestre

Para aferir as prováveis causas da baixa taxa de recuperação de estruturas embrionárias em búfalas superovuladas, foram realizados 4 experimentos. No Exp.1, foram utilizados sistemas genitais de búfalas e de vacas tratadas para a indução de ovulações única ou múltipla (fatorial 2x2). Os sistemas genitais foram submetidos à morfometria, os ovidutos foram lavados para a recuperação dos oócitos e, posteriormente, foram encaminhados à histologia. A taxa de recuperação de oócitos e o volume dos ovários foram maiores para as vacas que para as búfalas (P&lt;0,05). A área das fímbrias foi maior para as búfalas que para as vacas (P&lt;0,05). As camadas musculares do infundíbulo e do istmo foram mais espessas para as búfalas que para as vacas (P&lt;0,05) e, na ampola, não foi verificado diferença nesta medida entre as espécies (P&gt;0,05). No Exp.2 foram utilizados ovidutos de búfalas e de vacas, tratadas para a indução de ovulação única. Os ovidutos foram abertos, colocados em meio de cultura com ou sem E2 (fatorial 2x2) e incubados por 30 minutos. Após o período de incubação, foram colocadas microesferas na superfície dos ovidutos para a aferição do movimento ciliar. As microesferas no infundíbulo direcionaram-se para o útero (P&gt;0,05). Na ampola, direcionaram-se para o útero e para o ovário (P&gt;0,05). No istmo das vacas, direcionaram-se para o ovário e no das búfalas, para o útero (P&lt;0,05). A presença de E2 no meio de cultura não interferiu na direção do deslocamento das microesferas em nenhuma porção dos ovidutos de búfalas e de vacas. No Exp.3 foram utilizados ovidutos de búfalas e de vacas tratadas para a indução de ovulação única. Os ovidutos foram colocados em meio de cultura com ou sem E2 e receberam oócitos de búfalas ou de vacas (fatorial 2x2x2). Posteriormente, foram incubados por 24 horas e, após isso, foram lavados para a recuperação e contagem dos oócitos. O número e a taxa de recuperação de oócitos foi maior para as vacas que para as búfalas (P&lt;0,05) e, estas variáveis não foram influenciadas pelo tratamento (P&gt;0,05). Não foi verificada diferença no número de oócitos de búfalas ou de vacas recuperados (P&gt;0,05). Os dados são indicativos de que o transporte de oócitos pelo oviduto de búfalas e de vacas independe da espécie do oócito e não é influenciado pelo E2. No Exp.4, búfalas e vacas foram tratadas para a indução de ovulações única ou múltipla. As fêmeas foram inseminadas e submetidas à laparotomia para a inserção no interior do oviduto de oócitos de búfalas ou de vacas (fatorial 2x2x2). Os sistemas genitais foram lavados 5 (oócitos de búfala) e 6 dias (oócitos de vaca) após a laparotomia para a recuperação das estruturas embrionárias. O número de estruturas embrionárias recuperadas foi maior para as vacas que para as búfalas (P&lt;0,05) e, o número e a taxa de recuperação de estruturas embrionárias não foram influenciados pelo tratamento ou pela espécie dos oócitos (P&gt;0,05). A espécie dos oócitos e o tratamento parecem não influenciar no transporte dos oócitos pelos ovidutos de búfalas e de vacas / In order to study the probable causes of the low embryo recovery rates in superovulated buffaloes, 4 experiments were performed. The experiment 1, consisted of treating buffaloes and cows in order to induce single or multiple ovulation (2x2 factorial). Genital systems were morphometrycally evaluated; oviducts were flushed in order to recover the oocytes, and subsequently submitted to histological examination. The oocyte recovery rate and the ovarian volume were higher for cows when compared to buffaloes (P&lt;0.05). In contrast, fimbria area was higher for buffaloes than cows (P&lt;0.05). Likewise, infundibulum and isthmus muscle layers were thicker in buffalo than cows (P&lt;0.05). In addition, histological measurements were similar in cows and buffaloes (P&gt;0.05). In the experiment 2, oviducts of buffaloes and cows treated to show a single ovulation, were dissected and placed in two media (with or without estradiol [E2] in a 2x2 factorial) and incubated for 30 minutes. After this period, microesferes were used to assess of the cilia movements. When placed in the infundibulum, microesferes moved toward the uterus (P&gt;0.05). The microesferes in the ampulla moved towards the uterus and ovary in a similar fashion in both genetic groups (P&gt;0.05). The microesferes in the isthmus, moved towards the ovary in cows; whereas, they moved towards the uterus in buffalo (P&lt;0.05). There was no effect of media in the cilia movements at any portion of the oviduct neither in buffaloes or cows. In the experiment 3, oviducts of buffaloes and cows in the same conditions as in the previous experiment (single ovulation in with or without E2), received oocytes originated from buffaloes or cows (2x2x2 factorial). These oviducts were incubated for 24h and then flushed for oocyte recovery. The total number and the recovery rate of oocytes were higher for cows than for buffaloes (P&lt;0.05). Interestingly, there was no effect of treatment in these same variables (P&gt;0.05). The number of oocytes from buffaloes and cows that were recovered was similar (P&gt;0.05). These results indicate that oocytes transport through the oviduct of buffaloes or cows does not depend on the oocyte specie and is not influenced by the E2. In the experiment 4, buffaloes and cows were treated in order to induce single or multiple ovulation. Females were inseminated and submitted to laparotomy in order to insert, inside the oviduct, oocytes of buffaloes or cows (2x2x2 factorial). Genital tracts were flushed on day 5 (oocytes from buffaloes) and on day 6 (oocytes from cows) after laparotomy for recovery of embryonic structures. The number of embryonic structures recovered was higher for cows when compared to the buffaloes (P&lt;0.05). No effect of treatment or oocyte origin were found for the number or the rate of embryonic structures recovery (P&gt;0.05). In conclusion, it is likely that type of oocyte and treatment do not affect transport of oocytes in the oviducts of buffaloes and cows

Objetivando reduzir o manejo em protocolos de inseminação artificial em tempo fixo (IATF), quatro experimentos foram realizados para avaliar a dinâmica folicular (Experimentos 1A e 1B) e a taxa de concepção (TC; Experimentos 2, 3 e 4) em vacas de corte tratadas com Cipionato (CE) ou Benzoato (BE) de estradiol como indutores da ovulação. No Experimento 1A, 51 animais receberam 2mg de BE e um dispositivo intravaginal de progesterona (DIB) novo no D0. No D8, os animais foram distribuídos entre quatro tratamentos (G-BE8, G-BE8,5, G-BE9 e G-CE8). Neste dia, o dispositivo foi removido, 0,530mg de Cloprostenol e 300UI de eCG foram administrados [manhã (M) no G-BE8, G-CE8 e G-BE9; tarde (T) no G-BE8,5]. As vacas do G-CE8 receberam 1,0mg de CE, as do G-BE8 e G-BE8,5 receberam 1,0mg de BE na retirada do DIB, e aquelas do G-BE9 receberam 1,0mg de BE 24 horas após. Análises ultra-sonográficas foram realizadas a cada 12 horas, da retirada do dispositivo até a ovulação. Foram utilizados os PROC GLM e GLIMMIX do SAS para análise estatística. Nos experimentos não foram observadas interações. Os resultados para G-BE8, G-CE8, G-BE8,5 e G-BE9 foram, respectivamente: diâmetro do folículo ovulatório (FO;11,9±0,4b; 14,3±0,4a; 12,3±0,4b; e 13,3±0,abmm; P=0,01), taxa de ovulação (TO;100%; 90,0%; 100% e 91,7%; P=0,99] e intervalo retirada/ovulação (IRO;58,3±2,1b; 72,0±2,0a; 57,6±1,3b e 72,0±0,0ah; p<0,001). No Experimento 1B (n=35), foi utilizado arranjo fatorial 3x3 [números de uso do DIB vs protocolos de indução da ovulação (PIO)]. No D0, os animais foram divididos em três grupos [DIB novo (DIBN), usado 8 (DIB8) e 16 dias (DIB16)]. Na retirada do DIB, os animais foram redistribuídos para os PIOs descritos no Experimento 1A (exceto G-BE8). Os resultados para G-CE8, G-BE8,5 e G-BE9 foram, respectivamente: FO (14,0±0,6; 13,8±0,6 e 13,6±0,5mm; P=0,88), TO [81,8%; 83,3% e 83,3%; P=0,93] e IRO (72,0±2,0a; 59,6±1,6b e 73,2±1,2ah; p<0,01). Os resultados para DIBN, DIB8 e DIB16 foram respectivamente: FO (13,8±0,7; 13,5±0,6 e 13,9±0,5mm; P=0,99), TO [90,9%; 83,3% e 75,0%; P=0,93] e IRO (74,4±1,6a; 72,0±0,0ab e 68,0±2,0bh; P=0,02). No Experimento 2, 584 animais foram alocados em fatorial 3x2 [PIO (CE8, BE8,5 e BE9) e período de IATF (IATF-M ou IATF-T)], utilizando DIBN. Não houve diferença na TC: CE8 (57,5%), BE9 (59,9%) e BE8,5 (49,5%; P=0,09) e IATF M (56,6%) ou T (54,8%; P=0,66). No Experimento 3, 521 vacas foram divididas como no Experimento 2, porém com DIB8. Não houve diferença na TC nos PIOs: CE8 (47,3%), BE9 (53,3%) e BE8,5 (55,2%; P=0,10). Porém, houve diferença na TC para IATF-M (60,7%) ou IATF-T (48,3%; P=0,01). No Experimento 4, 1192 vacas foram distribuídas em fatorial 2x3x2 (DIBN e DIB8; BE8,5, BE9 e CE8; IATF-M e IATF-T). Não houve diferença na TC para: IATF-M (64,6%) ou IATF-T (59,5%; P=0,06) e PIOs; CE8 (65,8%), BE9 (61,8%) e BE8,5 (58,6%; P=0,12). Entretanto, houve diferença na TC (P=0,04) para uso do DIB; DIBN (65,0%) e DIB8 (59,4%). Esses resultados indicam que os PIOs apresentam mesma eficiência. Com DIB8, a IATF-T apresentou menor TC e o uso de DIB8 resultou em menor TC do que DIBN. / Aiming to minimize the number of handling during protocols for fixed-time artificial insemination (FTAI), four experiments were performed to evaluate the follicular dynamics (Experiments 1A and 1B) and conception rate (CR; Experiments 2, 3 e 4) in beef cows treated with estradiol cypionate (EC) or benzoate (EB) as ovulation inducers. In Experiment 1A, 51 animals received 2mg of EB and a new intravaginal progesterone device (DIB) on D0. On D8, animals were assigned into four treatments (G-EB8, G-EB8.5, G-EB9 and G-EC8). On the same day, the device was removed, 0.530mg of Cloprostenol and 300UI of eCG were administered [morning (AM) - G-EB8, G-EC8 and G-EB9; afternoon (PM) - G-EB8.5]. Cows of G-EC8 received 1.0mg of EC, while G-EB8 and G-EB8.5 received 1.0mg of EB at DIB removal, and those of G-EB9 received 1.0mg of EB 24h later. Ultrasonography was performed every 12h from DIB removal until ovulation. Statistical analysis was performed using PROC GLM and PROC GLIMMIX (SAS). No interactions were observed between treatments. Results of G-EB8, G-EC8, G-EB8.5 and G-EB9 were, respectively: diameter of ovulatory follicle (OF;11.9±0.4b; 14.3±0.4a; 12.3±0.4b; and 13.3±0.6abmm; P=0.01), ovulation rate (OR; 100%; 90.0%; 100% and 91.7%; P=0.99) and interval between device removal-ovulation (IRO; 58.3±2.1b; 72.0±2.0a; 57.6±1.3b and 72.0±0.0ah; p<0.001). Experiment 1B (n=35) was performed using a fatorial design 3x3 [DIB reutilization and ovulation inducer (OI)]. On D0, animals were allocated into three groups [new DIB (DIBN), used 8 (DIB8) and 16 days (DIB16)]. At DIB removal, animals were reallocated into the same OIs described on Experiment 1A, except by the G-EB8. The results of G-EC8, G-EB8.5 and G-EB9 were, respectively: OF (14.0±0.6; 13.8±0.6 and 13.6±0.5mm; P=0.88), OR [81.8%; 83.3% and 83.3%; P=0.93], and IRO (72.0±2.0a; 59.6±1.6b and 73.2±1.2ah; p<0.01). Results of DIBN, DIB8 and DIB16 were, respectively: OF (13.8±0.7; 13.5±0.6 and 13.9±0.5mm; P=0.99), OR (90.9%; 83.3% and 75.0%; P=0.93), and IRO (74.4±1.6a; 72.0±0.0ab and 68.0±2.0bh; P=0.02). At Experiment 2, 584 animals were allocated into a 3x2 fatorial design [OI (EC8, EB8.5 and EB9) and FTAI period (FTAI-AM or FTAI-PM)], being treated with DIBN. There was no difference on CR: EC8 (57.5%), EB9 (59.9%) and EB8.5 (49.5%; P=0.09) and IATF AM (56.6%) or PM (54.8%; P=0.66). On Experiment 3, 521 cows were allocated into the same experimental design of Experiment 2, however using DIB8. There was no difference on CR between different OIs: EC8 (47.3%), EB9 (53.3%) and EB8.5 (55.2%; P=0.10). However, there was difference on CR between FTAI-AM (60.7%) and FTAI-AM (48.3%; P=0.01). On Experiment 4, 1192 cows assigned into a 2x3x2 fatorial design (DIBN and DIB8; EB8.5, EB9 and C8; FTAI-AM and FTAI-PM). No differences were found on CR between FTAI-AM (64.6%) or FTAI-PM (59.5%; P=0.06), and OIs; EC8 (65.8%), EB9 (61.8%) and EB8.5 (58.6%; P=0.12). Cows treated with DIBN showed higher CR when compared to those treated with DIB8 (65.8 vs. 59.4%, respectively; P=0.04). These results indicate a similar efficiency using different OIs. Using DIB8, the FTAI-PM showed lower CR. Also the protocol using DIB8 resulted in lower CR than using DIBN.