O objetivo deste estudo foi avaliar o efeito da ressincronização da ovulação, iniciada 24 dias após a primeira IATF, sobre a área do corpo lúteo (CL), a concentração plasmática de progesterona (P4) e a taxa de prenhez. Exp.1 526 novilhas Brangus com idades entre 24 e 26 meses, foram submetidas a um programa de IATF no início da estação de acasalamento. O protocolo de sincronização para a primeira IATF começou com a inserção de um implante intra-vaginal contendo 750 mg de P4 e a administração de 2 mg de benzoato de estradiol (BE) intramuscular (i.m.) no dia -9 (D-9). Depois de sete dias (D-2), os implantes de P4 foram removidos, e 150 μg de D-cloprostenol (PGF), i.m., e 1 mg de cipionato de estradiol (CE), i.m., foram administrados. A IATF foi realizada entre 48 e 54 horas após a remoção do implante de P4 (D0). Vinte e quatro dias após a primeira IATF (D24), as novilhas foram divididas aleatoriamente nos seguintes grupos experimentais: controle (n = 167, sem tratamento), BE (n = 208, 1 mg de BE, i.m.) e hCG (n = 151, 1000 UI de hCG, i.m.). Novilhas dos grupos BE e hCG receberam um novo implante intra-vaginal contendo 750 mg de P4 na D24. No dia 31 (D31), os implantes de P4 foram removidos e o diagnóstico de prenhez foi realizado por ultrassonografia. As taxas de prenhez da primeira IATF no D31 foram 58,7% (98/167), 53,4% (111/208) e 52,9% (80/151), respectivamente, para os grupos controle, BE e hCG. Novilhas diagnosticadas como não gestantes receberam 150 μg de PGF, i.m., e 1 mg de CE, i.m., sendo a segunda IATF realizada 48 a 54 horas após a remoção do implante (D33). No D31, os subgrupos de novilhas prenhes de cada grupo experimental foram aleatoriamente divididos, sendo realizado exame por ultrassonografia para determinar a área do CL e coleta de uma amostra de sangue para determinar a concentração sérica de P4: Controle (n = 13), BE (n = 26), e hCG (n = 24). A área de CL foi significativamente maior (P<0,05) no grupo hCG (3,42±0,76 cm2), em comparação aos grupos de BE (2,44±0,57 cm2) e controle (2,61±0,61 cm2). Da mesma forma, a concentração sérica de P4 foi significativamente maior (P<0,05) no grupo hCG (12,43±3,48 ng/ml) em comparação aos grupos BE (6,92±3,04 ng/ml) e controle (7,29±2,45 ng/ml). O uso do BE e do hCG em programas de ressincronização da ovulação 24 dias após a IATF não interferiu na taxa de prenhez da primeira IATF. É provável que o mecanismo de ação do BE não afete a atividade do CL, a produção de P4, e consequentemente, não tenha efeito negativo na manutenção da prenhez em protocolos de ressincronização da ovulação. O tratamento com hCG resultou no aumento da área de CL e da produção de P4, porém, este efeito não favoreceu a taxa de prenhez da primeira IATF. Exp.2 184 novilhas Brangus com idade entre 24 a 26 meses e peso corporal médio de 361±29,2 kg foram submetidas a dois programas de IATF. O protocolo de sincronização para a primeira IATF foi o mesmo utilizado no Exp.1. Vinte e quatro dias após a primeira IATF (D24), as novilhas foram aleatoriamente divididas conforme os hormônios utilizados para ressincronização, formando os seguintes grupos experimentais: BE (n = 83, 1 mg de BE, i.m.) e hCG (n = 101, 1000 UI de hCG, i.m.). Novilhas dos grupos BE e hCG receberam um novo dispositivo intravaginal contendo 750mg de progesterona no D24. No D31, os implantes foram removidos e o diagnóstico de gestação por ultrassonografia foi realizado. As taxas de prenhez da primeira IATF no D31 foram de 63,9% (53/83) e 64,9% (65/101), respectivamente, para os grupos BE e hCG. Novilhas diagnosticadas como não gestantes (n=66) receberam 150 μg de PGF, i.m., e 1 mg de CE, im; a segunda IATF foi realizada no D33. Trinta dias após a segunda IATF (D63), foi realizado o segundo diagnóstico de gestação. As perdas gestacionais entre o D31 e D63, das novilhas prenhes da primeira IATF foram 9,4% (5/53) e 6,2% (4/65) respectivamente para os grupos BE e hCG. As taxas de prenhez da segunda IATF foram 40,0% (12/30) e 22,2% (8/36), respectivamente, para os grupos BE e hCG. As taxas de prenhez acumulada para os grupos BE e hCG foram, respectivamente, 72,3% (60/83) e 68,3% (69/101). O uso do BE e hCG para ressincronização da ovulação 24 dias após a primeira inseminação não afetou a taxa de prenhez da primeira IATF. As taxas de prenhez obtidas na segunda IATF foram inferiores às expectativas, considerando a resposta da primeira IATF. Entretanto, as taxas de prenhez acumulada foram similares e satisfatórias para os primeiros 33 dias da estação de acasalamento. / The aim of this study was to evaluate the effect of resynchronization of ovulation, which began 24 days after the first TAI, on the corpus luteum area (CL), plasma progesterone production (P4) and pregnancy rates. Exp.1 526 Brangus heifers between 24 and 26 months of age were submitted to a TAI program at the beginning of the breeding season. The protocol synchronization for the first TAI started with the insertion of an intravaginal implant containing 750 mg progesterone (P4) and the administration of 2 mg of estradiol benzoate (EB) intramuscular (i.m.) on day -9 (D-9). After seven days (D-2), P4 implants were removed, and 150 μg D-cloprostenol (PGF), and 1 mg estradiol cypionate (EC), were administered, i.m. The TAI was carried out between 48 and 54 hours after removal of the P4 implant (D0). Twenty-four days after the first TAI (D24), heifers were divided randomly into the following groups: control (n = 167, untreated), EB (n = 208, 1 mg EB, i.m.) and hCG (n = 151, 1000 IU hCG, i.m.). Heifers of the EB and hCG groups received a new intravaginal implant containing 750 mg of P4 on D24. On day 31 (D31), P4 implants were removed and the pregnancy diagnosis was performed by ultrasonography. Pregnancy rates for the first TAI, on D31, were 58.7% (98/167), 53.4% (111/208) and 52.9% (80/151), respectively, for the control, EB and hCG groups. Non-pregnant heifers received 150 μg PGF, i.m., and 1 mg EC, i.m., and the second TAI was performed 48 to 54 hours after removal of the P4 implant (D33). On D31, subgroups of pregnant cows from each experimental groups were randomly divided to determine the surface area of the CL by ultrasound and blood samples were collected to determine P4 concentrations: control (n = 13), BE (n = 26), and hCG (n = 24). The surface area of the CL was significantly higher (P<0.05) in the hCG group (3.42±0.76 cm2) compared to the EB (2.44±0.57 cm2) and control (2.61±0.61 cm2) groups. Also, P4 concentrations were significantly higher (P<0.05) in the hCG group (12.43±3.48 ng/mL) compared to the EB groups (6.92±3.04 ng/mL) and control (7.29±2.45 ng/mL). The use of EB and hCG in ovulation resynchronization programs 24 days after TAI did not affect the pregnancy rates of the first TAI. It is likely that EB mechanism of action does not affect the activity of the CL and P4 production, consequently having no negative effect on the maintenance of pregnancy. Nevertheless, the hCG treatment on D24 increased the area of CL and P4 plasma levels, but this effect neither improves nor compromised pregnancy rate of the first TAI. Exp.2 184 aged 24-26 months Brangus heifers old with mean body weight of 361±29.2 kg were submitted to two consecutive TAI programs. The synchronization protocol to the first TAI was the same as in Exp.1. Twenty-four days after the first TAI (D24), heifers were randomly divided according to the hormones used for resynchronization, according to the following groups: BE (n = 83, 1 mg EB, i.m.) and hCG (n = 101, hCG 1000 IU, i.m.). Heifers of the EB and hCG groups received a new intravaginal device containing 750 mg of progesterone on D24. On D31, P4 implants were removed and pregnancy diagnosis was performed by ultrasonography. The first TAI pregnancy rates on D31 were 63.9% (53/83) and 64.9% (65/101), respectively, for the EB and hCG groups. Heifers diagnosed as open received 150 μg PGF, i.m., and 1 mg EC, i.m.; the second TAI was performed on D33. Thirty days after the second TAI (D63), the second pregnancy diagnosis was performed. Pregnancy loss rates from D31 to D63 were 9.4% (5/53) and 6.2% (4/65) respectively for the EB and hCG groups. Heifers diagnosed as open received 150 μg PGF, i.m., and 1 mg EC, i.m.; the second TAI was performed on D33. Thirty days after the second TAI (D63), the second pregnancy diagnosis was performed. Pregnancy loss rates from D31 to D63 were 9.4% (5/53) and 6.2% (4/65) respectively for the EB and hCG groups. Pregnancy rates for the second TAI were 40.0% (12/30) and 22.2% (8/36), for the EB and hCG groups respectively. The cumulative pregnancy rates for EB and hCG groups were, respectively, 72.3% (60/83) and 68.3% (69/101). The use of hCG and EB for resynchronization of ovulation 24 days after the first insemination did not affect pregnancy rates of the first TAI. Pregnancy rates obtained in the second TAI were below expected values, considering the first TAI response. However, cumulative pregnancy rates were similar and satisfactory for the first 33 days of the breeding season.

A inseminação artificial em tempo fixo (IATF), juntamente com a inseminação artificial pós-cervical (IAPC), permite uma redução expressiva da mão de obra e uma melhor utilização dos machos geneticamente superiores. O objetivo deste estudo foi avaliar a eficiência do eCG associado agonista de GnRH (buserelina) ou apenas buserelina para indução e sincronização da ovulação em porcas desmamadas e submetidos a apenas uma IATF. Um total de 495 femeas suínas desmamadas foram utilizadas, cuja detecção de estro foi realizada uma vez ao dia (07:30). As fêmeas foram divididas em três tratamentos: Controle (n = 165) - a primeira IA foi realizada no início do estro (0 h) e repetida a cada 24 h, posteriormente, durante o estro; Tratamento 2: eCG + GnRH (n = 165) – femeas receberam uma injeção intramuscular de 600 UI de eCG após o desmame, e uma injeção intramuscular de buserelina (10 μg) 86-89 h após a administração de eCG; Tratamento 3: GnRH - fêmeas receberam uma injeção intramuscular de buserelina (10 μg) 86-89 h após o desmame. Nos três grupos era realizada a exposição ao macho no dia seguinte ao desmame uma vez ao dia, e aquelas fêmeas que demostrassem estro eram retiradas dos protocolos de IATF. Os grupos tratados receberam uma única IA, 118-120 h após o desmame (30-33 h após a buserelina). A IAPC contou com doses homospérmicas (1,5 x 109 células de espermáticas/50 ml) que foram utilizadas nas femeas em todos os três tratamentos. Não houve diferenças entre os tratamentos quanto ao número de femeas em estro até o terceiro dia (P>0,05). O intervalo entre o desmame e a ovulação foi significativamente maior (P< 0,05) no grupo controle (141,5 ± 1,58 h) comparativamente aos tratamentos eCG+GnRH (133,3 ± 1,60 h) e GnRH (135,9 ± 1,57 h). As femeas do tratamento eCG+GnRH ovularam em média antes (P<0,05) em relação ao grupo GnRH (44,5 ± 1,74 vs 48,2 ± 1,73 h). A taxa de parto foi superior (P<0,05) no grupo de controle, comparativamente aos tratamentos eCG + GnRH e GnRH, mas não houve diferença (P>0,05) em leitões nascidos totais entre os tratamentos. O uso do GnRH, com ou sem a administração prévia de eCG, antecipa a ovulação nas fêmeas suínas desmamadas. Nas fêmeas tratadas com eCG+GnRH ou GnRH, há em maior percentual de fêmeas inseminadas fora do momento ideal, em relação à ovulação, o que pode prejudicar o desempenho reprodutivo, sendo necessários ajustes no momento de aplicação dos hormônios ou no momento da inseminação para estudos subsequentes. / Fixed-time artificial insemination (FTAI) together with post-cervical artificial insemination (PCAI), allows an expressive reduction in labor requirements and a wider use of higher indexing boars. The aim of this study was to evaluate the efficiency of eCG agonist of GnRH (buserelin) or just buserelin for induction and synchronization of ovulation in weaned sows and submitted to FTAI .A total of 495 weaned sows whose estrus detection was performed once daily (07:30 A.M), starting a day after weaning. The sows were allocated into three treatment groups: Control (n=165) – the first AI was performed at estrus onset (0 h) and repeated every 24 h thereafter during estrus; Treatment 2: eCG + GnRH (n=165) – sows received an intramuscular injection of 600 UI eCG after weaning, and an intramuscular injection of buserelin (10 μg) 86-89 h after eCG administration; Treatment 3: GnRH - sows received an intramuscular injection of buserelina (10 μg) 86-89 h after weaning. . In the three groups it was performed boar exposure in the following day to weaning once a day, and those sows that demonstrated oestrus were removed from FTAI protocols. Treated groups received a single AI, 118-120 h after weaning (30-33 h after buserelin). PCAI with homospermic doses (1.5 x 109 total of sperm cells/50 ml) were respectively performed in sows in all treatment group. There were no differences (P> 0,05) in number of sows that showed oestrus until the third day between treatments. The interval between weaning and ovulation was significantly higher (P< 0,05) in the control group (141,5 ± 1,58 h) comparatively the treatments eCG+GnRH (133,3 ± 1,60 h) and GnRH (135,9 ± 1,57 h). Sows of treatment eCG+GnRH ovulated earlier (P< 0,05) comparing to the GnRH group (44,5 ± 1,74 vs 48,2 ± 1,73 h). Higher (P<0,05) farrowing rate in control group comparatively to treatments eCG+GnRH e GnRH was observed, but there were no differences (P> 0,05) in total piglets born between treatments. Use of GnRH, with or without previous administration of eCG anticipates ovulation in sows weaned, in the sows treated with eCG + GnRH or GnRH, there is a greater percentage of females inseminated outside the ideal time, with respect to ovulation, it can harm the reproductive performance, with necessary adjustments at the time of application hormones or at the time of insemination for subsequent studies.

A inseminação artificial em tempo fixo (IATF), juntamente com a inseminação artificial pós-cervical (IAPC), permite uma redução expressiva da mão de obra e uma melhor utilização dos machos geneticamente superiores. O objetivo deste estudo foi avaliar a eficiência do eCG associado agonista de GnRH (buserelina) ou apenas buserelina para indução e sincronização da ovulação em porcas desmamadas e submetidos a apenas uma IATF. Um total de 495 femeas suínas desmamadas foram utilizadas, cuja detecção de estro foi realizada uma vez ao dia (07:30). As fêmeas foram divididas em três tratamentos: Controle (n = 165) - a primeira IA foi realizada no início do estro (0 h) e repetida a cada 24 h, posteriormente, durante o estro; Tratamento 2: eCG + GnRH (n = 165) – femeas receberam uma injeção intramuscular de 600 UI de eCG após o desmame, e uma injeção intramuscular de buserelina (10 μg) 86-89 h após a administração de eCG; Tratamento 3: GnRH - fêmeas receberam uma injeção intramuscular de buserelina (10 μg) 86-89 h após o desmame. Nos três grupos era realizada a exposição ao macho no dia seguinte ao desmame uma vez ao dia, e aquelas fêmeas que demostrassem estro eram retiradas dos protocolos de IATF. Os grupos tratados receberam uma única IA, 118-120 h após o desmame (30-33 h após a buserelina). A IAPC contou com doses homospérmicas (1,5 x 109 células de espermáticas/50 ml) que foram utilizadas nas femeas em todos os três tratamentos. Não houve diferenças entre os tratamentos quanto ao número de femeas em estro até o terceiro dia (P>0,05). O intervalo entre o desmame e a ovulação foi significativamente maior (P< 0,05) no grupo controle (141,5 ± 1,58 h) comparativamente aos tratamentos eCG+GnRH (133,3 ± 1,60 h) e GnRH (135,9 ± 1,57 h). As femeas do tratamento eCG+GnRH ovularam em média antes (P<0,05) em relação ao grupo GnRH (44,5 ± 1,74 vs 48,2 ± 1,73 h). A taxa de parto foi superior (P<0,05) no grupo de controle, comparativamente aos tratamentos eCG + GnRH e GnRH, mas não houve diferença (P>0,05) em leitões nascidos totais entre os tratamentos. O uso do GnRH, com ou sem a administração prévia de eCG, antecipa a ovulação nas fêmeas suínas desmamadas. Nas fêmeas tratadas com eCG+GnRH ou GnRH, há em maior percentual de fêmeas inseminadas fora do momento ideal, em relação à ovulação, o que pode prejudicar o desempenho reprodutivo, sendo necessários ajustes no momento de aplicação dos hormônios ou no momento da inseminação para estudos subsequentes. / Fixed-time artificial insemination (FTAI) together with post-cervical artificial insemination (PCAI), allows an expressive reduction in labor requirements and a wider use of higher indexing boars. The aim of this study was to evaluate the efficiency of eCG agonist of GnRH (buserelin) or just buserelin for induction and synchronization of ovulation in weaned sows and submitted to FTAI .A total of 495 weaned sows whose estrus detection was performed once daily (07:30 A.M), starting a day after weaning. The sows were allocated into three treatment groups: Control (n=165) – the first AI was performed at estrus onset (0 h) and repeated every 24 h thereafter during estrus; Treatment 2: eCG + GnRH (n=165) – sows received an intramuscular injection of 600 UI eCG after weaning, and an intramuscular injection of buserelin (10 μg) 86-89 h after eCG administration; Treatment 3: GnRH - sows received an intramuscular injection of buserelina (10 μg) 86-89 h after weaning. . In the three groups it was performed boar exposure in the following day to weaning once a day, and those sows that demonstrated oestrus were removed from FTAI protocols. Treated groups received a single AI, 118-120 h after weaning (30-33 h after buserelin). PCAI with homospermic doses (1.5 x 109 total of sperm cells/50 ml) were respectively performed in sows in all treatment group. There were no differences (P> 0,05) in number of sows that showed oestrus until the third day between treatments. The interval between weaning and ovulation was significantly higher (P< 0,05) in the control group (141,5 ± 1,58 h) comparatively the treatments eCG+GnRH (133,3 ± 1,60 h) and GnRH (135,9 ± 1,57 h). Sows of treatment eCG+GnRH ovulated earlier (P< 0,05) comparing to the GnRH group (44,5 ± 1,74 vs 48,2 ± 1,73 h). Higher (P<0,05) farrowing rate in control group comparatively to treatments eCG+GnRH e GnRH was observed, but there were no differences (P> 0,05) in total piglets born between treatments. Use of GnRH, with or without previous administration of eCG anticipates ovulation in sows weaned, in the sows treated with eCG + GnRH or GnRH, there is a greater percentage of females inseminated outside the ideal time, with respect to ovulation, it can harm the reproductive performance, with necessary adjustments at the time of application hormones or at the time of insemination for subsequent studies.

A prevalência da obesidade tem aumentado em todo mundo, afetando também mulheres em idade reprodutiva. Estudos têm demonstrado que o acúmulo excessivo de tecido adiposo resulta em prejuízos à reprodução feminina. O mecanismo pelo qual a obesidade diminui a fertilidade não está totalmente estabelecido. Atualmente, são propostas a condição inflamatória e a indução de estresse oxidativo como potenciais mecanismos de ação para as patologias relacionadas à obesidade. Adicionalmente, a angiotensina II (Ang II) é mais um fator que tem sido relacionado com alterações associadas à obesidade, e os efeitos deste peptídeo incluem ações pró-inflamatórias e pró-oxidativas. O presente estudo analizou o efeito da obesidade na ovulação e no metabolismo oxidativo ovariano, e a participação da Ang II como um possível modulador da formação de espécies reativas de oxigênio em ovários de ratas obesas e seus efeitos na ovulação. Foram utilizadas ratas submetidas a uma dieta hipercalórica composta por alimentos palatáveis, conhecida como dieta de cafeteria, a partir do desmame até a idade adulta, por um período de 17 semanas. Os animais foram divididos em grupos: CTL (controle), CTL LOS (controle + losartan), CAF (cafeteria) e CAF LOS (cafeteria + losartan). Avaliamos o consumo de alimentos e líquidos, o peso corporal, o peso da gordura abdominal e retroperitonial, a concentração de insulina plasmática, o número de oócitos, as atividades das enzimas antioxidantes (superóxido dismutase e catalase), concentração de peróxido de hidrogênio (H2O2) e parâmetros de dano oxidativo (lipoperoxidação e oxidação de proteínas). As fêmeas obesas do grupo CAF apresentaram maior consumo de energia e reduzido consumo de água e ração padrão, hiperinsulinemia, aumento das gorduras abdominal e retroperitonial, e aumento de peso corporal. A obesidade não reduziu significativamente a ovulação, mas aumentou a atividade das enzimas antioxidantes e a concentração de H2O2 no ovário. A administração do losartan, em ratas alimentadas com a dieta de cafeteria, reduziu a ingestão energética total, a concentração plasmática de insulina e o ganho de gordura abdominal, mas não evitou o desenvolvimento da obesidade. O losartan inibiu a atividade da enzima antioxidante superóxido dismutase no ovário das ratas obesas do grupo CAF, porém não alterou a atividade da enzima antioxidante catalase. Os resultados deste estudo demonstram que a obesidade resulta em alterações no metabolismo e sugerem a participação da Ang II na formação de espécies reativas de oxigênio no ovário. / The prevalence of obesity has increased worldwide, also affecting women of reproductive age. Studies have shown that excessive accumulation of adipose tissue results in impaired female reproduction. The mechanism by which obesity reduces fertility is not fully established. Currently, proposals are the inflammatory condition and the induction of oxidative stress as a potential mechanism of action for diseases related to obesity. Additionally, angiotensin II (Ang II) is another factor that has been related to changes associated with obesity, and the effects of this peptide include pro-inflammatory and pro-oxidative actions. The present study considers the effect of obesity on ovulation and ovarian oxidative metabolism, and the participation of Ang II as a possible modulator of the formation of reactive oxygen species in ovaries of obese rats and their effects on ovulation. Female rats fed a diet consisting of high calorie foods palatable, known as cafeteria diet from weaning to adulthood, for a period of 17 weeks. The animals were divided into groups: Control (CTL) CTL LOS (control + losartan), CAF (cafeteria) and CAF LOS (losartan + cafeteria). We evaluate the consumption of food and fluids, body weight, abdominal and retroperitoneal fat weight, the plasma insulin concentration, the number of oocytes, the activities of antioxidant enzymes (superoxide dismutase and catalase), concentration of hydrogen peroxide (H2O2 ) and parameters of oxidative damage (lipid peroxidation and protein oxidation). The females of the CAF group had higher energy consumption and reduced consumption of water and standard chow, hyperinsulinemia, increased abdominal and retroperitoneal fat, and weight gain. Obesity not significantly reduced ovulation, but increased the activity of antioxidant enzymes and the concentration of H2O2 in the ovary. The administration of losartan in rats fed the cafeteria diet, reduced total energy intake, plasma insulin concentration and abdominal fat gain, but did not prevent the development of obesity. Losartan inhibited the activity of the antioxidant enzyme superoxide dismutase in the ovary of rats CAF group, but did not alter the activity of the catalase antioxidant enzyme. The results of this study demonstrate that the cafeteria diet results in changes in metabolism and suggests the involvement of Ang II in the formation of ROS in the ovary.

Made available in DSpace on 2014-07-29T15:07:30Z (GMT). No. of bitstreams: 1 Ubirajara_Bilego.pdf: 525711 bytes, checksum: 92b1baad77eca0e7758c3f7bfe166098 (MD5) Previous issue date: 2009-02-26 / The aim of this study was to describe the physiological responses to the Ovsynch protocol in Girolando heifers breeding in extensive model on Centro-Oeste of Brazil region and to determine relationships with environments factors in two stations, dry and rain. This responses were describe through the characterization of ovarian structures how: (NTL) total number of follicles; (DFOLIPR) mean of follicular diameter on protocol s begin; (DFOLOV) ovulatory diameter follicles; (DCL) corpus luteum ovulatory diameter; (SCL) ovulatory corpus luteum brightness score; pregnancy rates and ovulation rate on day 9 obtained in two year stations. Were utilized 40 heifers 18-24 months age, split in two groups of 20 animals being G1 group of dry station and G2 group of rain station. During the evaluations were found differences for the total number of follicles 8,07±0,25 e 8,83±0,26 in two stations applied. The mean of follicular diameter on protocol s begin was not show differences, but the measure of the ovulatory follicle diameter, have differences for the year stations (P<0,01), being the diameter for the dry station 11,88 ± 0,4mm whereas the rain station was 10,13 ± 0,36mm. Have in this study, higher frequency of follicles that ovulate with 11 to 12 mm on dry station and 10 to 11mm on rain station. The mean of the corpus luteum diameter on the present study not differ among the stations (P>0,05), with10,46±0,41mm for the dry station and 10,49±0,34mm for the rain station, being the higher frequency of CL 11 to 12mm in both stages. The CL brightness score not have differ(P>0,05), being 2,6 ± 0,16 for the dry and 2,31 ± 0,11 for the rain. The pregnancy rate have differences on two stages (P<0,01) with values 45% and 11% on station dry and rain respectively. The THI values differ among the stations dry and rain (P<0,01). The ovulation rate on D9 do not differ (P>0,05) although to have numeric differences. It was possible to conclude that in two stations evaluated, dry and rain, have differences on follicular morfometry and pregnancy rates. / Objetivou-se com este estudo descrever a resposta fisiológica do protocolo Ovsynch em novilhas Girolando, criadas extensivamente na região Centro-Oeste do Brasil em duas estações do ano, seca e águas. Tais respostas foram descritas através da caracterização das estruturas ovarianas como: (NTF) Número total de folículos; (DFOLIPR) Diâmetro folicular médio no início do protocolo; (DFOLOV) Diâmetro do folículo ovulatório; (DCL) Diâmetro do corpo lúteo; (SCL) Escore de ecogenicidade do corpo lúteo; Taxa de gestação e Taxa de ovulação no D9 obtidas em duas estações do ano. Foram utilizadas 40 novilhas girolando com idades entre 18-24 meses, divididas em dois grupos de 20 animais, sendo (G1) de estação seca e (G2) grupo de estação de águas. Durante as avaliações foram encontradas diferenças quanto ao número total de folículos 8,07±0,25 e 8,83±0,26 nas duas estações avaliadas. O diâmetro folicular médio no início do protocolo não mostrou diferenças, porém, houve diferença entre as estações do ano (P< 0,01), quanto ao diâmetro folicular ovulatório sendo o diâmetro médio para a estação seca 11,88 ± 0,4mm enquanto para a estação das águas foi 10,13 ± 0,36mm. Houve nesse estudo maior freqüência de folículos que ovularam em torno de 11 a 12mm na estação seca e 10 a 11mm na estação das águas. O diâmetro médio do corpo lúteo no presente estudo não diferiu entre as estações (P>0,05), com 10,46±0,41mm para a estação seca e 10,49±0,34mm, com maior freqüência de CLs de 11 a 12mm em ambas as estações. Os escores de corpo lúteo também não mostraram diferenças (P>0,05) sendo 2,6 ± 0,16 para seca e 2,31 ± 0,11 para águas. A taxa de gestação também mostrou diferença entre as estações com valores de 45% e 11% nas estações seca e águas respectivamente. Taxa de ovulação no D9 não diferiu (P>0,05) entre as estações apesar de ser numericamente diferente entre os tratamentos. Os valores de THI diferiram entre as estações seca e águas (P<0,01). Foi possível concluir que as duas estações avaliadas, seca e águas, mostraram diferenças quanto morfometria folicular e taxas de gestação.

Orientador: Frederico Ozanam Papa / Resumo: A superovulação é uma biotécnica utilizada nos programas de transferência de embrião e proporciona diversos benefícios devido ao maior número de ovulações por ciclo. Entretanto, os protocolos superovulatórios na espécie equina ainda não são utilizados rotineiramente, pois apresentam resultados inconsistentes relacionados a baixa taxa de recuperação embrionária frente ao número de ovulações, estando muito aquém do esperado quando comparado a espécie bovina. Atualmente, sabe-se que uma das possíveis causas responsáveis por este fato pode estar relacionada pela formação de grandes coágulos de sangue obstruindo a fossa da ovulação, dificultando assim a captação do oócito para o interior da tuba uterina. Desta forma, o objetivo desse estudo foi avaliar o efeito da utilização de dois tipos de anticoagulantes parenterais (heparina não fracionada - HNF e heparina de baixo peso molecular - HBPM) em éguas superovuladas com extrato de pituitária equina (EPE), sobre a posterior taxa de recuperação embrionária. Foram utilizados quatro ciclos estrais de 11 éguas, sendo subdivididos em quatro grupos: Grupo 1 (G1) utilizado como controle; Grupo 2 (G2) receberam 25 mg de EPE intramuscular (IM) a cada 12 horas sendo a primeira e a terceira administração associada a 5 mg de dinoprost-trometamina (IM) e solução fisiológica 0,9% única aplicação 35 horas após indução da ovulação; Grupo 3 (G3) foi utilizado mesmo protocolo superovulatório, sendo substituído a solução fisiológica por um dos anticoag... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Superovulation is a biotechnic from embryo transfer programs providing several benefits due to the higher number of ovulations per cycle. However, superovulation protocols in the equine species are not yet routinely used, as they present inconsistent results related to the low embryonic recovery rate concerning the number of ovulations, being far below expectations when compared to the bovine species. Currently, one of the possible causes responsible for this fact may be related to the development of large blood clots obstructing the ovulation fossa, making it difficult to capture the oocyte into the fallopian tube. Therefore, this study aimed to evaluate the effect of two types of parenteral anticoagulants (unfractionated heparin - HNF and low molecular weight heparin - LMWH) on equine pituitary extract (EPE) superovulated mares of embryonic recovery. Four estrous cycles of 11 mares were used and subdivided into four groups: Group 1 (G1) as a control; Group 2 (G2) received 25 mg intramuscular EPE (MI) every 12 hours being the first and third administration associated with 5 mg dinoprost-tromethamine (IM) and 0.9% saline only application 35 hours after ovulation induction; Group 3 (G3) in the same superovulation protocol, replacing the physiological solution with one of the anticoagulants, Sodium Heparin 450 IU / kg / IV, and Group 4 (G4) where the mares were superovulated by the same protocol mentioned above and received 1 mg. / Kg / IV of Enoxaparin Sodium. All animals were... (Complete abstract click electronic access below) / Mestre

L'efficacité de la reproduction bovine a considérablement diminué dans les dernières décennies et cette diminution constitue un problème économique majeur. Pour mieux contrer ce problème, la physiologie des cellules stéroïdogéniques ovariennes dont les cellules de granulosa (CG) doit être mieux comprise au cours des dernières étapes de la croissance folliculaire, de l'ovulation et de la lutéinisation. En ce sens, nous avons précédemment identifié divers gènes induits dans les CG des follicules ovulatoires bovins par la LH/hCG incluant Ankyrin-repeat and SOCS-box protein 9 (ASB9). Cependant, les mécanismes d’action d’ASB9 dans les CG étaient encore indéfinis. Les objectifs de cette étude étaient d'élucider le rôle d'ASB9 dans les CG ainsi que ses effets sur ses partenaires spécifiques PAR1, TSG6 et TAOK1. Un modèle in vivo de CG provenant de follicules à différentes phases de développement: petits follicules (SF), follicules dominants (DF) et follicules ovulatoires (OF), et un modèle in vitro de CG en culture ont été utilisées. L'inhibition d’ASB9 dans les CG via CRISPR/Cas9 a montré une augmentation significative de PAR1, PCNA, CCND2 et CCNE2 et une diminution significative de TAOK1, TSG6 et CASP3. Dans le modèle in vivo, PAR1 a été différentiellement exprimé dans DF et TSG6 et TAOK1 ont été induits dans OF. L'inhibition de l'ASB9 a aussi entraîné une diminution de l'apoptose des CG et de l'activité caspase3/7. Des analyses Western blot ont démontré que l'induction d'ASB9 dans OF, après l'injection d'hCG, était concomitante avec une diminution significative des niveaux de phosphorylation de MAPK3/1 tandis que pMAPK3/1 augmentait après l'inhibition d'ASB9. Ces résultats supportent qu'ASB9 pourrait être un régulateur de l'activité et de la fonction des CG en ciblant des protéines spécifiques qui affectent la signalisation MAPK, limitant la prolifération des CG. Ces résultats contribuent à une meilleure compréhension de l’activité ovarienne et de la reproduction bovine. / The efficiency of bovine reproduction has considerably decreased in recent decades and this decrease constitutes a major economic problem. To better counter this problem, the physiology of ovarian steroidogenic cells including granulosa (CG) cells needs to be better understood during the later stages of follicular growth, ovulation and luteinization. In this sense, we have previously identified various genes induced in the CGs of bovine ovulatory follicles by LH / hCG including Ankyrin-repeat and SOCS-box protein 9 (ASB9). However, ASB9 mechanisms of action in GC were still undefined. The objectives of this study were to elucidate the role of ASB9 in CG as well as its effects on target partners PAR1, TSG6 and TAOK1, and on MAPK signaling. An in vivo model of GC from follicles at different developmental stages: small follicles (SF), dominant follicles (DF), and ovulatory follicles (OF) and an in vitro model of cultured GC along with the CRISPR/Cas9 approach to inhibit ASB9 were used. Inhibition of ASB9 in GC resulted in significant increase in PAR1, PCNA, CCND2, and CCNE2 and significant decrease in TAOK1, TNFAIP6, and CASP3 expression. From in vivo samples, PAR1 was differentially expressed in DF as compared to OF while TSG6 and TAOK1 were induced in OF. Further analyses showed an increase in GC number and a decrease in apoptosis and caspase3/7 activity following ASB9 inhibition. Western blot analyses demonstrated that ASB9 induction in OF by hCG was concomitant with a significant decrease in MAPK3/1 phosphorylation levels while pMAPK3/1 increased following ASB9 inhibition. These results provide strong evidence that ASB9 is a regulator of GC activity and function by modulating MAPK signaling pathway likely through specific binding partners such as PAR1, therefore controlling GC proliferation. These results contribute to a better understanding of ovarian activity and bovine reproduction.

Le récepteur nucléaire orphelin facteur stéroïdogénique 1 (SF-1 ou NR5A1) est un modulateur indispensable du développement surrénal et gonadique et qui joue un rôle dans la détermination du sexe, le développent hypothalamique, la fonction hypophysaire et la stéroïdogénèse. Toutefois, les études sur SF-1 dans le milieu de la biologie de la reproduction portent majoritairement sur des modèles embryonnaires ou de mammifères immatures. L’objectif principal de cette thèse était de déterminer le rôle de SF-1 dans les évènements clés de la fonction reproductrice chez les mâles et femelles matures. Ce facteur de transcription est exprimé dans différents organes, principalement ceux de l’axe hypothalamo-hypophyso-gonadique, et dans divers types cellulaires des gonades. Nous avons donc généré 4 modèles de souris knockout conditionnels (cKO) en utilisant les allèles Cre-recombinase et flox de SF-1 (SF-1f/f) afin d’identifier son rôle dans les différentes populations cellulaires des testicules et ovaires de souris matures. Dans la première étude, nous avons présenté une analyse des souris femelles du modèle cKO du récepteur de la progestérone (PRCre/+;Nr5a1f/f), où la suppression de SF-1 est spécifique aux cellules gonadotropes de l’hypophyse et aux cellules ovariennes de type granulosa suite au déclenchement du signal ovulatoire ainsi que les cellules lutéales du corps jaune. Cette étude a révélé de nouveaux rôles in vivo de SF-1 durant l’ovulation et la lutéinisation, tout en suggérant que SF-1 est un médiateur de la synthèse et sécrétion des gonadotrophines. Les femelles PRCre/+;Nr5a1f/f cKO étaient infertiles principalement en raison de l’importante réduction de FSH et LH sécrété dans la circulation, causé par le phénotype hypophysaire. Afin de contourner cette dysfonction hypophysaire, des traitements de gonadotrophines exogènes ainsi que des transplantations d’ovaires nous ont permis de démontrer que SF-1 régule la transcription de gènes impliqués dans l’expansion du cumulus ainsi que la rupture de follicules pour induire l’ovulation. De plus, nous avons montré que l’absence de SF-1 dans les ovaires de souris matures peut mener à l’infertilité, indépendamment du phénotype hypophysaire. D’autre part, nos trouvailles indiquent que, malgré la basse expression de SF-1 dans le corpus luteum chez la souris, sa déplétion dans les cellules lutéales conditionnée par recombinase Cre inhibe la production de progestérone. Aucun phénotype reproductif a été observé chez les souris PRCre/+;Nr5a1f/f cKO mâles. La deuxième étude a démontré le rôle essentiel de SF-1 dans la fonction du testicule mature. La souris mâle P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f) cKO, où la suppression de SF-1 est spécifique aux cellules de Leydig, était fertile malgré la taille réduite de ses testicules, la malformation de ses tubes séminifères, la perturbation de la spermiogénèse, ainsi que la réduction d’expression de gènes de la stéroïdogénèse. Bien que les mâles aromatase (Cyp19Cre/+;Nr5a1f/f) cKO, supprimant SF-1 dans les cellules de Sertoli, étaient fertiles et démontraient des capacités reproductives similaires aux mâles contrôle, les souris Cyp17Cre/++Cyp19Cre/+; Nr5a1f/f cKO (dKO) étaient soit infertiles ou montrait une fertilité affaiblie. La dysgénésie sévère du cordon testiculaire ainsi que la spermatogénèse perturbée chez la souris dKO étaient causées par la déplétion simultanée de SF-1 chez les cellules de Leydig et Sertoli, suggérant que les cellules de Sertoli peuvent compenser pour l’absence de SF-1 dans les cellules de Leydig et vice versa. Ces données démontrent que SF-1 est requis pour une stéroïdogénèse testiculaire et une spermatogénèse ainsi qu’une fertilité normale, bien que savoir si la régulation de ces fonctions par SF-1 est directe ou indirecte reste à élucider. De façon intéressante, les femelles des trois lignées cKO étudié dans ce deuxième article étaient fertiles et la sous expression de SF-1 dans les cellules ovariennes de type granulosa ou de la thèque a produit des effets mineurs sur leur fonction reproductive. En somme, la recherche présentée dans cette thèse contribue à l’avancement des connaissances sur SF-1 et son rôle dans la régulation d’événements reproductifs cruciaux dans l’hypophyse, l’ovaire et le testicule de souris matures. Les lignes de souris produites dans ce projet vont servir d’outil indispensable pour élucider les mécanismes de régulation de SF-1 sur la fonction gonadique and présenter de nouvelles avenues de recherches pour ce récepteur orphelin. / The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable modulator of adrenal and gonadal development, playing key roles in sex determination, hypothalamic development, pituitary function and steroidogenesis. Yet, studies to date of SF-1 in reproductive biology mostly focus on embryonic and immature mammalian models. The overall objective of this thesis was to determine the role of SF-1 in key events of mature male and female mouse reproductive function. This transcription factor is expressed in a variety of organs, mainly those of the hypothalamic-pituitary-gonadal axis, as well as in multiple cell types of the gonads. Therefore, we generated four conditional KO (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (SF-1f/f) to identify its role in different cell types of the testes and ovaries of mature mice. Our first study presents an analysis of female mice from the progesterone receptor (PRCre/+;Nr5a1f/f) cKO model, where SF-1 depletion is specific to gonadotropes in the pituitary gland as well as granulosa cells of the peri-ovulatory follicle and luteal cells of the corpus luteum. This research highlighted new in vivo roles for SF-1 in ovulation and luteinization, and provided further evidence that SF-1 is a mediator of gonadotropin synthesis and secretion. PRCre/+;Nr5a1f/f cKO females were infertile, due in large part to the reduced secretion of FSH and LH, caused by the pituitary phenotype. Exogenous gonadotropin treatments and ovarian transplantation experiments allowed us to circumvent the pituitary dysfunction to demonstrate that SF-1 in granulosa cells regulates the transcription of cumulus expansion and follicle rupture genes to induce ovulation. In addition, we showed that the absence of SF-1 in ovaries of mature mice can lead to female infertility, independent of the pituitary phenotype. Moreover, the data showed that, though SF-1 expression is reduced in mouse corpus luteum, its Cre-mediated depletion in luteal cells abrogates progesterone production. No reproductive phenotype was observed in PRCre/+;Nr5a1f/f cKO males. Results from our second study demonstrated that SF-1 plays an essential role in mature testicular function. The P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f) cKO male mouse, where the SF-1 depletion is specific to Leydig cells, were fertile, though showed reduced testis size with disrupted seminiferous tubules and impaired spermiogenesis, in addition to reduced expression of steroidogenic genes. While the aromatase (Cyp19Cre/+;Nr5a1f/f) cKO males were fertile and showed reproductive capacities comparable to control males, the Cyp17Cre/++Cyp19Cre/+; Nr5a1f/f cKO (dKO) model were either infertile or showed significantly impaired fertility. The dKO males displayed severe testis cord dysgenesis and impaired spermatogenesis caused by the depletion of SF-1 in both Leydig and Sertoli cells, suggesting that Sertoli cells can compensate for the absence of SF-1 in Leydig cells and vice versa. These data provide strong evidence that SF-1 is required for normal testicular steroidogenesis, spermatogenesis and male fertility, though whether the regulation of these functions is direct or indirect remains to be elucidated. Interestingly, the females of the three cKO mouse lines studied in this second article were fertile and the depletion of SF-1 in granulosa cells of antral follicles or in theca cells produced minor effects on their steroidogenic capacities. Collectively, the research presented in this thesis contributes to advance our understanding of the role of SF-1 in the regulation of essential reproductive events in the pituitary, ovary and testis of mature mouse gonads. The mouse lines generated for this project will serve as valuable tools to elucidate the mechanisms underlying SF-1 regulation of gonad function and present novel directions for the investigation of this nuclear receptor.

Conura torvina (Cresson) (Hymenoptera: Chalcididae) is a solitary pupal endoparasite of numerous insect species. In Brassica crops it acts as a parasite of Plutella xylostella (L.) (Lepidoptera: Plutellidae) and was found as a hyperparasite of Cotesia rubecula (Marshall) (Hymenoptera: Braconidae) and several other parasitoid species. Cotesia rubecula was introduced into Virginia in 1987 as a biological control agent for Pieris rapae (L.) (Lepidoptera: Pieridae), and because C. torvina was thought to have eliminated this population of C. rubecula, studies of C. torvina's reproductive biology and behavior were initiated. A study using plants laden with "trap hosts" to detect C. torvina activity in the spring indicated no activity until late June, but proved trap host sampling to be an efficient and effective method of monitoring C. torvina activity. Studies of C. torvina's ability to reproduce in C. rubecula pupae of different ages indicated that C. torvina can successfully parasitize pupae at all stages of development, but was most successful in young to middle aged pupae. Studies of C. torvina's host species preference indicated the larger host species such as P. xylostella were preferred. Equal numbers of P. xylostella and C. rubecula were parasitized, but a greater proportion of fertile eggs were laid in P. xylostella. Smaller host species were often ignored. Host dissection studies indicated that caged C. torvina were inefficient at host finding and oviposition. Superparasitism was common, but declined as the females gained oviposition experience. Experienced C. torvina produced an average of 8.25 progenies per day for a period of 12 days when provided with 13 P. xylostella hosts each day. Conura torvina produced up to 14 progenies a day when provided 3 26 hosts. Dissection of C. torvina ovaries indicated three ovarioles per ovary with a mean of 9.2 and maximum of 15 mature eggs per female. Host dissection indicated that a mean of 18 and maximum of 30 eggs could be laid per day. New eggs were produced as oviposition occurred. Significantly larger eggs were laid in P. xylostella than in C. rubecula, and significantly more eggs were laid in C. rubecula than in P. xylostella. From these data and data from earlier studies I concluded that C. torvina has a poor reproductive ability and its impact as a hyperparasite is limited to the summer months. This makes C. torvina an unlikely cause of C. rubecula's disappearance. / Ph. D.

La croissance folliculaire anormale, l'anoestrus et l'anovulation sont parmi les principales causes de la baisse de fertilité chez les bovins laitiers à haut rendement. Lors de la croissance folliculaire et de l'ovulation, les cellules stéroïdogènes, y compris les cellules de granulosa (GC), jouent un rôle crucial dans la maturation et la libération de l'ovocyte. Il est donc essentiel de décrire en détail les processus physiologiques et pathologiques régulant la fonction ovarienne. Des études précédentes de notre laboratoire ont identifié et caractérisé pour la première fois un membre de la famille des pseudo-kinases Tribbles (à savoir TRIB2) dans les cellules ovariennes de granulosa. La famille Tribbles comprend des protéines de type sérine-thréonine kinase largement exprimées (TRIB1, TRIB2 et TRIB3) qui peuvent jouer des rôles cruciaux dans divers processus biologiques tels que la coordination de la mitose et de la morphogenèse, la maturation des ovocytes et la prolifération cellulaire. Cependant, leurs rôles exacts dans la fonction des cellules de granulosa et leurs effets sur les voies de signalisation impliquées dans la croissance folliculaire et l'ovulation restent inconnus. Nous avons émis l'hypothèse que les pseudo-kinases TRIB jouent des rôles cruciaux dans la régulation de la fonction et de l'activité des cellules de granulosa au cours des dernières étapes du développement folliculaire et peuvent activer des voies de signalisation distinctes. L'objectif de la présente étude était donc d'examiner les fonctions des membres TRIB dans les cellules de granulosa bovine avec les objectifs spécifiques suivants : 1) Analyser l'expression et la régulation des membres TRIB dans différents types cellulaires folliculaires et modèles de culture ; 2) Étudier les fonctions des membres TRIB dans les cellules GC en utilisant l'approche CRISPR/Cas9. En utilisant un modèle d'étude in vivo composé de cellules GC obtenues à partir de follicules à différents stades de développement, nous avons confirmé la régulation négative de TRIB2 par l'hormone lutéinisante (LH) / hormone chorionique gonadotrope humaine (hCG) et la régulation positive de TRIB1 et TRIB3 aux niveaux de l'ARN et des protéines par la LH. Nous avons montré que TRIB2 est presque exclusivement présent dans les cellules GC et dans les follicules dominants (DF) alors qu'il est absent dans les cellules de la thèque (TC) et régulé négativement dans les follicules ovulatoires (OF) par hCG, tandis que TRIB1 et TRIB3 sont présents dans les cellules TC des DF ou OF. TRIB1 et TRIB3 sont exprimés dans les cellules GC uniquement dans les OF post-hCG et non dans les DF. Les résultats suggèrent que la contribution des cellules TC à l'expression de TRIB2 dans l'ovaire bovin est très limitée par rapport aux cellules GC. Nos données ont montreé que TRIB1 et TRIB3 sont induits par hCG, suggérant des rôles significatifs, respectivement, dans le processus d'ovulation ou la formation et la fonction du corps jaune. Les résultats d'immunohistochimie ont montré la présence de TRIB2 dans la couche GC des DF par rapport à la couche TC et absente dans les OF post-hCG. L'analyse par Western blot a montré que TRIB2 et TRIB3 sont induits par la FSH mais à des moments différents. De plus, les données in vitro ont confirmé que la LH supprime l'expression de TRIB2, tandis que l'expression de TRIB3 est induite par la LH, en particulier 6 heures après le traitement avec LH. L'inhibition de TRIB3 via CRISPR/Cas9 suggère que, bien que TRIB3 puisse avoir un effet positif sur la voie de signalisation AKT dans les cellules GC, il a des effets négatifs sur la voie de signalisation P38MAPK. Nos données actuelles fournissent des preuves que TRIB2 pourrait être impliqué dans la prolifération des cellules GC et le développement folliculaire, tandis que TRIB1 et TRIB3 pourraient être impliqués respectivement dans les processus d'ovulation et de lutéinisation. En conclusion, les TRIB jouent des rôles cruciaux dans la régulation de la fonction et de l'activité des cellules de granulosa au cours du développement folliculaire et peuvent activer des voies de signalisation distinctes. / Abnormal follicular growth, anestrus and anovulation are among the main causes of declining fertility in high-yielding dairy cattle. During follicular growth and ovulation, steroidogenic cells, including granulosa cells (GC), play a crucial role in the maturation and release of the oocyte. It is therefore essential to describe in more details the physiological and pathological processes regulating the ovarian function. Previous studies from our laboratory identified and characterized for the first time a tribbles pseudokinase family member (namely TRIB2) in ovarian GC. The Tribbles family includes widely expressed serine-threonine kinase-like proteins (TRIB1, TRIB2 and TRIB3) that may play crucial roles in various biological processes such as coordination of mitosis and morphogenesis, oocyte maturation and cell proliferation. However, their exact roles in GC function and effects on the signaling pathways involved in follicular growth and ovulation remained to be defined. We hypothesized that TRIB pseudokinases play crucial roles in regulating the function and activity of granulosa cells during the final stages of follicular development and may activate separate signaling pathways. The aim of the present study was therefore to investigate the functions of TRIB members in bovine GC with the following specific objectives: 1) Analyze the expression and regulation of TRIB members in different follicular cell types and culture models; 2) to study TRIB members functions in GC cells using the CRISPR/Cas9 approach. Using an in vivo study model consisting of GC obtained from follicles at different stages of development, we confirmed down-regulation of TRIB2 by luteinizing hormone (LH) and human chorionic gonadotropin (hCG) and up-regulation of TRIB1 and TRIB3 at both RNA and protein levels. We showed that, TRIB2 is almost exclusively present in GC and in dominant follicles (DF) while it is absent in theca cells (TC) and downregulated in ovulatory follicles (OF) by hCG while TRIB1 and TRIB3 are present in TC whether of DF or OF. Both TRIB1 and TRIB3 are expressed in GC only in OF post-hCG and not in DF. The results suggest that TC contribution to TRIB2 expression in the ovary is very limited as compared to GC. Our data show that TRIB1 and TRIB3 are induced by hCG suggesting significant roles in the ovulation process (TRIB1) or the formation and function of the corpus luteum. Results from immunohistochemistry, showed the presence of TRIB2 in the GC layer of DF as compared to TC layer and absent in OF post hCG injection. Western blot analysis showed that TRIB2 and TRIB3 are induced by FSH but at different times. Moreover, in vitro data confirmed that LH suppresses TRIB2 expression, whereas TRIB3 expression is induced by LH, particularly 6 hours post-LH treatment. Inhibition of TRIB3 via CRISPR-Cas9 suggested that while TRIB3 might have positive effect on AKT signaling pathway in GC, it has negative effects on P38 signaling pathway. Our current data provide evidence that TRIB2 could be involved in GC proliferation and follicular development while TRIB1 and TRIB3 could be involved in the ovulation and luteinization processes, respectively. Overall, TRIBs play crucial roles in regulating the function and activity of granulosa cells follicular development and may activate separate signaling pathways.