O estradiol (E2) é requerido para a luteólise de fêmeas bovinas e injeções de E2 estimulam a liberação de prostaglandina F2α (PGF2α). É possível que o E2 estimule a síntese e/ou a atividade de moléculas envolvidas na cascata geradora de PGF2α, como enzimas e receptores para outros ligantes. O cálcio é um conhecido cofator para a proteína quinase C e fosfolipase A2, enzimas envolvidas na produção PGF2α. O objetivo geral desse estudo foi investigar os mecanismos endócrinos e moleculares estimulados pelo E2 durante a luteólise. No experimento 1, vacas holandesas não lactantes foram tratadas com 3mg de E2 nos dias 13 (n=2), 15 (n=2), 17 (n=3) e 19 (n=5) do ciclo estral e a produção de PGFM (metabólito plasmático da PGF2α) mensurada por radioimunoensaio. Concluiu-se que a administração de E2 no 17º dia do ciclo estral constitui um modelo experimental adequado para determinar os mecanismos envolvidos na síntese de PGF2α. O experimento 2, foi realizado para investigar o papel do E2 na cascata produtora de PGF2α. Novilhas cruzadas de corte, cíclicas, não lactantes, foram pareadas no dia 17 de um ciclo estral sincronizado, injetadas com 0 (n=6) ou 3mg de E2 (n=7) e abatidas 2 horas após. Explantes endometriais foram tratados com os seguintes estimuladores da síntese de PGF2α: ionóforo de cálcio (CI), melitina ou ocitocina. Explantes foram incubados em quadruplicata e amostras de meio foram coletadas imediatamente e 60 minutos após o início da cultura. As concentrações de PGF2α foram mensuradas por radioimunoensaio. Explantes endometriais tratados in vitro com CI tiveram um incremento na síntese de PGF2α de 48,41% quando foram previamente tratados com o E2 (P≤0,01). No experimento 3, células endometriais bovinas (células BEND) foram tratadas com 0, 10-7, 10-6 ou 10-5M CI por 12h em triplicata, em três experimentos independentes. As concentrações de 10-6 e 10-5M de CI estimularam a produção de PGF2α em comparação às outras concentrações (P≤0,05). No experimento 4, células BEND receberam 0 ou 10-13M E2 e 0 ou 10-6M CI em um arranjo fatorial 2 x 2, durante 12h em triplicata, em três experimentos independentes. A produção de PGF2α foi de 33,1; 32,5; 92,4 e 145,6 (EPM: 21,8) pg/mL para células não tratadas, tratadas com E2, CI e E2 + CI, respectivamente. Houve uma tendência do tratamento com CI estimular a produção de PGF2α (P≤0,08), entretanto, na presença de E2 o CI estimulou significativamente a síntese de PGF2≤ (P≤0,01). Conclui-se que em fêmeas bovinas o E2 potencializou os efeitos do CI na síntese de PGF2α endometrial. Propõe-se que o E2 ativa a síntese de enzimas que, estimuladas pelo cálcio, atuam na síntese de PGF2α endometrial / Estradiol (E2) is required for luteolysis in bovine female and E2 injections stimulate prostaglandin F2α (PGF2α) release. It is possible that E2 stimulates synthesis and/or activity of molecules in the cascade of PGF2α production, such as enzymes and receptors to other ligands. Calcium is a known cofactor for protein kinase C and phospholipase A2, enzymes involved in PGF2α production. The main goal of this study was to investigate endocrine and molecular mechanisms stimulated by E2 during luteolysis. In experiment 1, non-lactating Holstein cows were treated with 3mg E2 on days 13 (n=2), 15 (n=2), 17 (n=3) or 19 (n=5) of the day estrous cycle and production PGFM (a PGF2α plasma metabolite) was evaluated by radioimmunassay. It was concluded that E2 administration on day 17 of the cycle was an adequate experimental model to determine mechanisms involved in endometrial PGF2α synthesis. Experiment 2 was designed in order to investigate the role of E2 in the enzymatic cascade of PGF2α production. Cyclic, cross-bred beef heifers were paired on day 17 of a synchronized estrous cycle, injected with 0 (n=6) or 3mg of E2 (n=7) and slaughtered after two hours. Endometrial explants were treated with stimulators of the cascade of PGF2α synthesis, i.e., calcium ionophore (CI), melittin or oxytocin. Explants were incubated in quadruplicate and medium samples were collected immediately and 60min after to begin culture. The concentrations PGF2α were measured by radioimmunoassay. Endometrial explants in vitro treatment with CI production the PGF2α was 48,41% higher in from cows treated with E2 (P≤0,01). In experiment 3, bovine endometrium cells (BEND cells) were treated with 0, 10-7, 10-6 or 10-5M CI for 12h in triplicate, in three independent experiments. The 10-6 and 10-5M concentrations of CI stimulated production of PGF2α in comparison to other concentrations (P≤0,05). In experiment 4, BEND cells received 0 or 10-13M E2 and 0 or 10-6M CI in a 2 x 2 factorial arrangement, for 12h in triplicate cultures in three independent experiments. Production of PGF2α was 33.1, 32.5, 92.4 and 145.6 (pooled SEM: 21.8) pg/mL for cells treated with nothing, E2, CI and E2 + CI, respectively. Treatment with CI alone tended to stimulate PGF2α production (P≤0,08), however, in the presence of E2, CI significantly stimulated PGF2α synthesis (P≤0,01). It was concluded that in bovine female the E2 improved the CI effects in endometrial PGF2α synthesis. It proposes that E2 active the enzyme synthesis that, calcium stimulated, actuate in endometrial PGF2α synthesis

Made available in DSpace on 2014-06-11T19:27:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-09Bitstream added on 2014-06-13T19:56:26Z : No. of bitstreams: 1 lopes_cn_me_botfmvz.pdf: 505921 bytes, checksum: 14aad0f6b2df70711c09f6e1b5b4acc9 (MD5) / Universidade Estadual Paulista (UNESP) / Foram realizados quatro experimentos com o objetivo de avaliar possíveis mecanismos relacionados ao aumento da prenhez com a utilização de ácidos graxos poliinsaturados (PF). No exp. 1 foram utilizadas 51 vacas multíparas Nelore não lactantes, ovuladas, para avaliar se PF alteram a produção de progesterona (P4) e o momento da luteólise. No exp. 2 foram utilizadas 43 vacas multíparas Nelore não lactantes, ovuladas, para avaliar se PF alteram a sensibilidade do corpo lúteo de seis dias a aplicação de prostaglandina (i.m. 12,5mg de dinoprost trometamina, Lutalyse). No exp. 3 foram utilizadas 27 vacas multíparas Nelore, ovuladas, com 30 a 40 dias pós parto para avaliar se PF diminuem a incidência de ciclo curto. Os tratamentos utilizados foram: grupo controle (100g mineral + 100g milho + 100g caolin vaca dia); grupo SF (100g mineral + 100g milho + 100g/vaca/dia de Megalac (7-9% C18:2; Arm&Hammer a Church&Dwight Company, EUA); grupo PF (100g mineral + 100g milho + 100g/vaca/dia de Megalac-E QGN, Brasil: 40-42% de C18:2; 2-3% de C18:3). No exp. 4 (1457 vacas multíparas Nelore) foi avaliado se a suplementação com PF pós IATF por diferentes períodos altera a taxa de prenhez. Nos experimentos 1, 2 e 3 não foi detectado efeito de PF nas concentrações de P4 durante o período avaliado, no momento da luteólise e na incidência de ciclo curto (P>0,1). Ao se agrupar os dados do Exp 1 e 2, a concentração de P4 (P=0,01) no dia 6 foi maior nos animais suplementados com PF em relação ao SF e controle (4,45; 3,25; 3,48 ng/ml, respectivamente; EPM=0,278). No experimento 4 vacas recebendo PF por 21 e 28 dias após a IATF tiveram maior (P<0,05) taxa de prenhez (50,38%) quando comparadas com os outros tratamentos agrupados (42,38%). Não foi detectado diferença entre os tratamentos PF21 (50,99%) e PF28 (49,81). Os resultados em conjunto mostram que apesar de suplementação... / Four experiments were designed to evaluate the possible mechanisms related to the increased pregnancy rates in cows supplemented with PF. In Experiment 1, 51 ovulated non-lactating Nelore multiparous cows were used to evaluate if PF supplementation affects circulating progesterone (P4) concentrations and timing of luteolysis. In Experiment 2, 43 ovulated non-lactating Nelore multiparous cows were used to evaluate if PF supplementation alters the sensitivity of a 6-d corpus luteum to exogenous prostaglandin treatment. In Experiment 3, 27 ovulated postpartum Nelore cows were used to evaluate if PF supplementation affects the incidence of premature luteolysis. Beginning at the d of estrus, the daily treatments in these 3 experiments were: Control (0.1 Kg Mineral + 0.1 Kg corn + 0.1 Kg kaolin); SF (0.1 Kg Mineral + 0.1 Kg Megalac-S® [7-9% linoleic acid] + 0.1 Kg corn), this group was used just in experimental 1 and 2; PF (0.1 Kg Mineral + 0.1 Kg Megalac-E® [40-42% linoleic acid; 2-3% linolenic acid] + 0.1 Kg corn). In Experiment 4, we evaluated if the length of PF supplementation in different times after timed-AI alters the pregnancy rate. No effect was detected on P4 concentration, luteolysis or short cycle (P>0.1), but when the cows of exp. 1 and 2 were grouped had higher (P=0.01) concentration of P4) on cows that were supplemented with PF compared with Sf or control (4.45; 3.25; 3.48 ng/ml, respectively; SEM=0,278. Cows supplemented with PF during 21 (PF21,) or 28 d post- AI (PF28) had higher pregnancy rates (50.38%; P < 0.05) than cows from other treatments (42.38%). There was no difference between PF21 (50,99%) and PF28 (49,81%) treatments. These experiments indicated that the possible mechanism for greater conception with PF supplementation post-AI may be due to effects on embryo development, animals supplemented for more than 21d had greater pregnancy rates.

Orientador: Maria Emilia Franco Oliveira / Banca: Erika da Silva Carvalho Morani / Banca: Wilter Ricardo Russiano Vicente / Resumo: Um dos grandes entraves da MOTE é a ocorrência de falhas no tratamento superovulatório, sendo que uma das causas pode ser a alta dose de FSH, podendo promover regressão prematura dos corpos lúteos e consequentemente uma resposta ineficaz ao tratamento superovulatório. O objetivo deste estudo foi avaliar o efeito da dose de FSH exógeno no tratamento superestimulatório sobre a dinâmica de desenvolvimento e funcionalidade de corpos lúteos em fêmeas ovinas doadoras de embrião. Foram utilizadas 29 ovelhas Santa Inês, as quais foram submetidas à sincronização do estro com um dispositivo intravaginal de progesterona (CIDR®), inserido no Dia 0, permanecendo até o Dia 8. Concomitantemente ao início do protocolo e retirada do dispositivo intravaginal foi administrado um análogo da PGF2α. O tratamento gonadotrófico teve início 48 horas antes da retirada da progesterona (D6), quando as fêmeas foram aleatoriamente divididas em três grupos experimentais de acordo com a dose total de FSH suíno exógeno: G200 (controle, n=8) - 200 mg de FSH; G133 (n=8) - 133 mg de FSH; e G100 (n=8) - 100 mg de FSH. No D6 (1ª aplicação de FSH), as fêmeas receberam também 300 UI de eCG. As avaliações ultrassonográficas Modo-B e Doppler foram realizadas durante o período de luteogênese (D11) até o dia correspondente a videolaparoscopia (D15), com o propósito de acompanhar o desenvolvimento das estruturas lúteas (dimensões), avaliar os índices do fluxo sanguíneo da artéria ovariana (VPS,VDF,IR e IP) e, de avalia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: One of the major obstacles of MOTE is occurrence of failures in superovulation, and one reason may be the high dose of FSH, which can promote premature regression of the corpus luteum and therefore ineffective response to superovulation. The aim of the present study was to evaluate the effect of exogenous FSH dose in superstimulatory treatment on development dynamic and function of corpora lutea in embryo donor ewes. Twenty-nine Santa Inês ewes were submitted to estrus synchronization with an intravaginal progesterone device (CIDR®), which was inserted on Day 0 and was remain until the Day 8. At the same time the beginning of the protocol and intravaginal device withdrawal was administered an analog of PGF2α. The gonadotropin treatment was commenced 48 hours before the CIDR withdrawal (D6), when females are randomly divided into three groups according to the total dose of exogenous porcine FSH: G200 (control, n = 9) - 200 mg of FSH ; G133 (n = 10) - 133 mg FSH; and G100 (n = 10) - 100 mg of FSH. The total doses were administered in 8 to applications at 12 hour intervals (20, 20, 15, 15, 10, 10, 5 and 5% FSHp). In D6 (1st application FSH), females also received 300 IU of eCG. Evaluations ultrasound mode-B and Doppler were performed during the luteogênese period (D11) until the corresponding day of videolaparoscopy (D15), in order to follow the development of luteal structures (dimensions), evaluate the contents of the artery blood flow ovarian (VPS, EDV, RI and PI), and macros... (Complete abstract click electronic access below) / Mestre

O 17&beta;-estradiol (E2) desempenha um papel importante na função reprodutora e na fertilidade feminina, porém, sua ação é estendida para a maioria dos tecidos. Sabendo que o E2 tem funções pleiotrópicas em diferentes tecidos e órgãos, e que pode estar envolvido tanto na proliferação como na morte celular, nossa hipótese é que o E2 seja um dos iniciadores de regressão luteínica em cadelas não prenhes. Para testar nossa hipótese, foram usados corpos lúteos (CL) provenientes de 28 cadelas nos dias 10, 20, 30, 40, 50, 60, 70 e >70 após a ovulação (n = 4/grupo) para os experimentos ex vivo. Nesta etapa foram analisadas as proteínas pró-apoptóticas (CASPASES, 3, 8, 9 e BAX) por imuno-histoquímica e western blotting, além da expressão dos genes CASP3, 8, 9, BAX, FAS, MKI67, ESR1, ESR2m por PCR em tempo real. Na segunda etapa deste trabalho, utilizamos CL de 12 cadelas nos dias 20, 40 e 60 após a ovulação (n=4 por grupo), cujas células foram cultivadas e divididas em seis tratamentos: Controle, E2 (tratado com E2), bloqueador de ER&alpha; (tratado com MPP), bloqueador de ER&beta; (tratado com PHTPP), E2 + bloqueador de ER&alpha; (tratado com E2 + MPP) e E2 + bloqueador de ER&beta; (tratado com E2 + PHTPP). Foram avaliados os mesmos genes do experimento ex vivo, bem como os genes CYP19A1, CYP11A1, HSD3B1 e SLC2A4. De modo geral a expressão dos fatores pró-apoptóticos foi mais alta a partir do dia 40 e atingiu valores máximos nos dias 60 e 70 após a ovulação, assim como a expressão de ERS2. Essa correlação foi observada também nas células do grupo E2 + bloqueador de ER&alpha;, que também apresentaram regulação negativa de HSD3B1. Quando do bloqueio do ER&beta;, as células luteínicas responderam com aumento dos genes relacionados à esteroidogênese e à proliferação celular, principalmente quando oriundas dos dias 20 e 40 p.o. Dessa forma, conclui-se que o bloqueio do ER&alpha; levou ao aumento dos genes pró-apoptóticos, e o bloqueio do ER&beta; possibilitou aumento dos genes luteotróficos. Estes achados confirmam o papel pleiotrópico do estradiol no CL canino e incluem este hormônio, assim como o balanço entre seus receptores, dentre os atores principais da regulação da meia vida do CL canino. / The 17&beta;-estradiol (E2) plays an important role in the reproductive function and female fertility, however, its action is extended to most tissues. Knowing that E2 has pleiotropic roles in different organs and tissues, and that might be involved in both cell death and proliferation, our hypothesis is that E2 is one of the triggers of luteal regression in non-pregnant bitches. To test our hypothesis, corpora lutea (CL) from 28 dogs on days 10, 20, 30, 40, 50, 60, 70 and >70 after ovulation (n = 4/group) were used for ex vivo experiments. Pro-apoptotic proteins expression (CASPASES, 3, 8, 9 e BAX) were analyzed by immunohistochemistry and western blotting, and the gene expression of CASP3, 8, 9, BAX, FAS, MKI67, ESR1 and ESR2 were analyzed by real-time PCR. In the second step of this study, CL from 12 bitches on days 20, 40 and 60 (n = 4 per group) were used. Luteal cells were isolated and divided into six treatments: Control, E2 (treated with E2), ER&alpha; Blocker (treated with MPP), ER&beta; Blocker (treated with PHTPP), ER&alpha; + E2 Blocker (treated with E2 + MPP) and E2 + ER&beta; Blocker (treated with E2 + PHTPP). The expression of the same genes from the in vivo experiment was evaluated, as well as that of CYP19A1, CYP11A1, HSD3B1 and SLC2A4. In general, the expression of pro-apoptotic factors was higher from day 40 and reached highest expression on days 60 and 70 after ovulation, coinciding with increasing in expression of ERS2. This correlation was also observed in the cells from the group E2 + ER&alpha; blockade, which also showed HSD3B1down-regulation. When ER&beta; was blocked, cells of days 20 and 40 post ovulation responded increasing the expression of steroidogenesis and proliferation related genes. Thus, we deduce that the ER&alpha; blockade promotes the increase of pro-apoptotic and that o ER&beta; of luteotrophic genes expression. Our findings confirm the pleiotropic role of estradiol in canine CL and include this hormone, as well as the balance between its two receptors, among the factors controlling canine CL lifespan.

O objetivo do trabalho foi verificar o efeito da antecipação de um luteolítico prévio a retirada do dispositivo intravaginal de progesterona (P4) sobre a taxa de prenhez na inseminação artificial em tempo fixo (IATF) de vacas com cria ao pé em comparação com protocolos de IATF tradicionais que utilizam o luteolítico na ocasião da retirada de P4. Também verificar a ação e eficácia dos implantes com um grama de progesterona de primeiro e segundo uso. Utilizou-se 210 vacas de corte amamentando da raça Montana com 48-98 dias pós-parto. Destas, 127 eram vacas multíparas e 83 eram vacas primíparas. Os animais foram divididos em dois lotes de 105 vacas, comparando oito grupos. O lote D6,5 foi tratado no dia zero (data:17/11/2008) com 2mg de benzoato de estradiol i.m. (BE, Estrogin®) juntamente com implante intravaginal de progesterona de 1º uso (G1- Sincrogest®, n=29; G2- Primer®, n=26) e de 2º uso (G3- Sincrogest®, n=26; G4- Primer®, n=24), no dia 6,5 foi aplicado 150mcg i.m. de Cloprostenol Sódico (Sincrocio®), sendo a retirada do implante de P4 no dia 8. Dia 9, ocorreu a aplicação de 1mg de BE i.m. e no dia 10 a tarde a IATF. O lote D8 iniciou o protocolo de IATF no dia 25/11/2008 e foi tratado da mesma forma, à exceção da aplicação do luteolítico que ocorreu no oitavo dia. Os grupos foram formados: implante de 1º uso (G5- Sincrogest®, n=27; G6- Primer®, n=28) e de 2º uso (G7-Sincrogest®, n=26; G8- Primer®, n=24). Foram realizadas duas coletas de sangue (dia 0 e dia 9 do protocolo) para dosagem de progesterona plasmática através do método de radioimunoensaio. As taxas de prenhez dos grupos do lote D6,5 foram de 55,17%(G1), 69,23%(G2), 57,67%(G3), 70,83%(G4) (p=0,263). No lote D8 os grupos G5, G6, G7, G8 deste lote apresentaram 44,44%, 67,86%, 46,15%, e 58,33% de prenhez (p=0,573). As taxas de prenhez para os lotes D6,5 e D8 foram, de 62,86% e 54,29%. Não houve diferença nas taxas de prenhez a IATF entre os lotes (p= 0,262). O teste Qui-quadrado e o T-test foram utiizados para a análise estatística dos dados. O ECC médio dos dois lotes foi de 2,66 (n=210). Não houve influência do ECC sobre as taxas de prenhez a IATF (p=0,562) . Também não houve diferença estatística na taxa de prenhez a IATF e de prenhez final dos animais conforme a idade e dias pós parto. Dezenove vacas apresentaram P4 maior que 1ng/ml no dia 0 (D6,5= 7, com 5 vacas prenhez e 2 vazias; D8= 12, com 7 vacas prenhez e 5 vacas vazias) e quatro vacas do lote D8 apresentaram P4 maior que 1ng/ml no dia 9 do protocolo (com 1 vaca prenhe). Os implantes da Primer ® e Sincrogest® de primeiro e segundo uso foram eficientes para a sincronização da ovulação das vacas com cria ao pé. A antecipação do luteolítico não aumentou as taxas de prenhez a IATF e prenhez final. / The objective of this work was to verify the effect of the anticipation of a luteolytic before the removal of the intravaginal device with progesterone (P4) on the range of pregnancy in the artificial insemination in fixed time (TAI) of post partum cows compared to the IATF traditional protocols that use the luteolytic while removing P4 and also verify the action and efficacy of the implants with a gram of progesterone of first and second use. 210 post partum cows beef cows of Montana breed were used with 48-98 days post partum. Out of these, 127 were multiparous cows and 83 were primiparous cows. The animals were divided in two lots of 105 cows, comparing 8 groups. The lot D6,5 was treated on the day zero (27/11/2008) with 2mg of estradiol benzoate i.m. (BE, Estrogin®) together with the intravaginal implant of progesterone of first use (G1- Sincrogest®, n=29; G2- Primer®, n=26) and of second use (G3- Sincrogest®, n=26; G4- Primer®, n=24), on the day 6,5 150mcg i.m of Cloprostenol (Sincrocio®), the removal of the implant of P4 on the day 8. On the day 9, there was an application 1mg of BE i.m and on the day 10 the IATF. The lot D8 (05/12/2008) was treated on the same way, except for the application of luteolitico, that occurred on the eighth day. The groups were formed this way: first use (G5- Sincrogest®, n=27; G6- Primer®, n=28) and second use (G7-Sincrogest®, n=26; G8- Primer®, n=24). Two blood sample collections were performed (Day 0 and Day 9 of protocol) for plasma progesterone dosage through the radioimunoensaio method. The ranges of pregnancy of the groups in the lot D6,5 were 55,17%(G1), 69,23%(G2), 57,67%(G3), 70,83%(G4) (p=0,263). In the lot D8 the groups G1, G2, G3, G4 of this lot presented 44,44%, 67,86%, 46,15%, e 58,33% of pregnancy (p=0,573). The ranges of pregnancy for the lots D6,5 and D8 were 62,86% e 54,29%. There was no difference in the ranges of pregnancy TAI between the lots (p= 0,262). The test Qui- Quadrado and T-test was used for the statistics analysis of the data. The average body condition of the two lots was 2,66 (n=210). There was no influence of the body condition on the ranges od pregnancy TAI (p=0,562). There was no statistic difference in the range TAI and final pregnancy of the animals according to the age and post partum days. Nineteen cows presented P4 maior que 1ng/ml no dia 0 (D6,5= 7, with 5 cows pregancy and 2 empty; D8=12, with 7 cows pregnancy and 5 cows empty) and four cows of the lot D8 presented P4 maior que 1ng/ml on the Day 9 of the protocol (with only one cow pregnant). The implants of PRIMER and SINCROGEST of first and second use were efficient for the synchronization of the ovulation of the post partum cows. The anticipation of luteolytic treatment did not increase pregnancy rates to TAI and also the final pregnancy rate.

O objetivo do trabalho foi verificar o efeito da antecipação de um luteolítico prévio a retirada do dispositivo intravaginal de progesterona (P4) sobre a taxa de prenhez na inseminação artificial em tempo fixo (IATF) de vacas com cria ao pé em comparação com protocolos de IATF tradicionais que utilizam o luteolítico na ocasião da retirada de P4. Também verificar a ação e eficácia dos implantes com um grama de progesterona de primeiro e segundo uso. Utilizou-se 210 vacas de corte amamentando da raça Montana com 48-98 dias pós-parto. Destas, 127 eram vacas multíparas e 83 eram vacas primíparas. Os animais foram divididos em dois lotes de 105 vacas, comparando oito grupos. O lote D6,5 foi tratado no dia zero (data:17/11/2008) com 2mg de benzoato de estradiol i.m. (BE, Estrogin®) juntamente com implante intravaginal de progesterona de 1º uso (G1- Sincrogest®, n=29; G2- Primer®, n=26) e de 2º uso (G3- Sincrogest®, n=26; G4- Primer®, n=24), no dia 6,5 foi aplicado 150mcg i.m. de Cloprostenol Sódico (Sincrocio®), sendo a retirada do implante de P4 no dia 8. Dia 9, ocorreu a aplicação de 1mg de BE i.m. e no dia 10 a tarde a IATF. O lote D8 iniciou o protocolo de IATF no dia 25/11/2008 e foi tratado da mesma forma, à exceção da aplicação do luteolítico que ocorreu no oitavo dia. Os grupos foram formados: implante de 1º uso (G5- Sincrogest®, n=27; G6- Primer®, n=28) e de 2º uso (G7-Sincrogest®, n=26; G8- Primer®, n=24). Foram realizadas duas coletas de sangue (dia 0 e dia 9 do protocolo) para dosagem de progesterona plasmática através do método de radioimunoensaio. As taxas de prenhez dos grupos do lote D6,5 foram de 55,17%(G1), 69,23%(G2), 57,67%(G3), 70,83%(G4) (p=0,263). No lote D8 os grupos G5, G6, G7, G8 deste lote apresentaram 44,44%, 67,86%, 46,15%, e 58,33% de prenhez (p=0,573). As taxas de prenhez para os lotes D6,5 e D8 foram, de 62,86% e 54,29%. Não houve diferença nas taxas de prenhez a IATF entre os lotes (p= 0,262). O teste Qui-quadrado e o T-test foram utiizados para a análise estatística dos dados. O ECC médio dos dois lotes foi de 2,66 (n=210). Não houve influência do ECC sobre as taxas de prenhez a IATF (p=0,562) . Também não houve diferença estatística na taxa de prenhez a IATF e de prenhez final dos animais conforme a idade e dias pós parto. Dezenove vacas apresentaram P4 maior que 1ng/ml no dia 0 (D6,5= 7, com 5 vacas prenhez e 2 vazias; D8= 12, com 7 vacas prenhez e 5 vacas vazias) e quatro vacas do lote D8 apresentaram P4 maior que 1ng/ml no dia 9 do protocolo (com 1 vaca prenhe). Os implantes da Primer ® e Sincrogest® de primeiro e segundo uso foram eficientes para a sincronização da ovulação das vacas com cria ao pé. A antecipação do luteolítico não aumentou as taxas de prenhez a IATF e prenhez final. / The objective of this work was to verify the effect of the anticipation of a luteolytic before the removal of the intravaginal device with progesterone (P4) on the range of pregnancy in the artificial insemination in fixed time (TAI) of post partum cows compared to the IATF traditional protocols that use the luteolytic while removing P4 and also verify the action and efficacy of the implants with a gram of progesterone of first and second use. 210 post partum cows beef cows of Montana breed were used with 48-98 days post partum. Out of these, 127 were multiparous cows and 83 were primiparous cows. The animals were divided in two lots of 105 cows, comparing 8 groups. The lot D6,5 was treated on the day zero (27/11/2008) with 2mg of estradiol benzoate i.m. (BE, Estrogin®) together with the intravaginal implant of progesterone of first use (G1- Sincrogest®, n=29; G2- Primer®, n=26) and of second use (G3- Sincrogest®, n=26; G4- Primer®, n=24), on the day 6,5 150mcg i.m of Cloprostenol (Sincrocio®), the removal of the implant of P4 on the day 8. On the day 9, there was an application 1mg of BE i.m and on the day 10 the IATF. The lot D8 (05/12/2008) was treated on the same way, except for the application of luteolitico, that occurred on the eighth day. The groups were formed this way: first use (G5- Sincrogest®, n=27; G6- Primer®, n=28) and second use (G7-Sincrogest®, n=26; G8- Primer®, n=24). Two blood sample collections were performed (Day 0 and Day 9 of protocol) for plasma progesterone dosage through the radioimunoensaio method. The ranges of pregnancy of the groups in the lot D6,5 were 55,17%(G1), 69,23%(G2), 57,67%(G3), 70,83%(G4) (p=0,263). In the lot D8 the groups G1, G2, G3, G4 of this lot presented 44,44%, 67,86%, 46,15%, e 58,33% of pregnancy (p=0,573). The ranges of pregnancy for the lots D6,5 and D8 were 62,86% e 54,29%. There was no difference in the ranges of pregnancy TAI between the lots (p= 0,262). The test Qui- Quadrado and T-test was used for the statistics analysis of the data. The average body condition of the two lots was 2,66 (n=210). There was no influence of the body condition on the ranges od pregnancy TAI (p=0,562). There was no statistic difference in the range TAI and final pregnancy of the animals according to the age and post partum days. Nineteen cows presented P4 maior que 1ng/ml no dia 0 (D6,5= 7, with 5 cows pregancy and 2 empty; D8=12, with 7 cows pregnancy and 5 cows empty) and four cows of the lot D8 presented P4 maior que 1ng/ml on the Day 9 of the protocol (with only one cow pregnant). The implants of PRIMER and SINCROGEST of first and second use were efficient for the synchronization of the ovulation of the post partum cows. The anticipation of luteolytic treatment did not increase pregnancy rates to TAI and also the final pregnancy rate.

O objetivo do trabalho foi verificar o efeito da antecipação de um luteolítico prévio a retirada do dispositivo intravaginal de progesterona (P4) sobre a taxa de prenhez na inseminação artificial em tempo fixo (IATF) de vacas com cria ao pé em comparação com protocolos de IATF tradicionais que utilizam o luteolítico na ocasião da retirada de P4. Também verificar a ação e eficácia dos implantes com um grama de progesterona de primeiro e segundo uso. Utilizou-se 210 vacas de corte amamentando da raça Montana com 48-98 dias pós-parto. Destas, 127 eram vacas multíparas e 83 eram vacas primíparas. Os animais foram divididos em dois lotes de 105 vacas, comparando oito grupos. O lote D6,5 foi tratado no dia zero (data:17/11/2008) com 2mg de benzoato de estradiol i.m. (BE, Estrogin®) juntamente com implante intravaginal de progesterona de 1º uso (G1- Sincrogest®, n=29; G2- Primer®, n=26) e de 2º uso (G3- Sincrogest®, n=26; G4- Primer®, n=24), no dia 6,5 foi aplicado 150mcg i.m. de Cloprostenol Sódico (Sincrocio®), sendo a retirada do implante de P4 no dia 8. Dia 9, ocorreu a aplicação de 1mg de BE i.m. e no dia 10 a tarde a IATF. O lote D8 iniciou o protocolo de IATF no dia 25/11/2008 e foi tratado da mesma forma, à exceção da aplicação do luteolítico que ocorreu no oitavo dia. Os grupos foram formados: implante de 1º uso (G5- Sincrogest®, n=27; G6- Primer®, n=28) e de 2º uso (G7-Sincrogest®, n=26; G8- Primer®, n=24). Foram realizadas duas coletas de sangue (dia 0 e dia 9 do protocolo) para dosagem de progesterona plasmática através do método de radioimunoensaio. As taxas de prenhez dos grupos do lote D6,5 foram de 55,17%(G1), 69,23%(G2), 57,67%(G3), 70,83%(G4) (p=0,263). No lote D8 os grupos G5, G6, G7, G8 deste lote apresentaram 44,44%, 67,86%, 46,15%, e 58,33% de prenhez (p=0,573). As taxas de prenhez para os lotes D6,5 e D8 foram, de 62,86% e 54,29%. Não houve diferença nas taxas de prenhez a IATF entre os lotes (p= 0,262). O teste Qui-quadrado e o T-test foram utiizados para a análise estatística dos dados. O ECC médio dos dois lotes foi de 2,66 (n=210). Não houve influência do ECC sobre as taxas de prenhez a IATF (p=0,562) . Também não houve diferença estatística na taxa de prenhez a IATF e de prenhez final dos animais conforme a idade e dias pós parto. Dezenove vacas apresentaram P4 maior que 1ng/ml no dia 0 (D6,5= 7, com 5 vacas prenhez e 2 vazias; D8= 12, com 7 vacas prenhez e 5 vacas vazias) e quatro vacas do lote D8 apresentaram P4 maior que 1ng/ml no dia 9 do protocolo (com 1 vaca prenhe). Os implantes da Primer ® e Sincrogest® de primeiro e segundo uso foram eficientes para a sincronização da ovulação das vacas com cria ao pé. A antecipação do luteolítico não aumentou as taxas de prenhez a IATF e prenhez final. / The objective of this work was to verify the effect of the anticipation of a luteolytic before the removal of the intravaginal device with progesterone (P4) on the range of pregnancy in the artificial insemination in fixed time (TAI) of post partum cows compared to the IATF traditional protocols that use the luteolytic while removing P4 and also verify the action and efficacy of the implants with a gram of progesterone of first and second use. 210 post partum cows beef cows of Montana breed were used with 48-98 days post partum. Out of these, 127 were multiparous cows and 83 were primiparous cows. The animals were divided in two lots of 105 cows, comparing 8 groups. The lot D6,5 was treated on the day zero (27/11/2008) with 2mg of estradiol benzoate i.m. (BE, Estrogin®) together with the intravaginal implant of progesterone of first use (G1- Sincrogest®, n=29; G2- Primer®, n=26) and of second use (G3- Sincrogest®, n=26; G4- Primer®, n=24), on the day 6,5 150mcg i.m of Cloprostenol (Sincrocio®), the removal of the implant of P4 on the day 8. On the day 9, there was an application 1mg of BE i.m and on the day 10 the IATF. The lot D8 (05/12/2008) was treated on the same way, except for the application of luteolitico, that occurred on the eighth day. The groups were formed this way: first use (G5- Sincrogest®, n=27; G6- Primer®, n=28) and second use (G7-Sincrogest®, n=26; G8- Primer®, n=24). Two blood sample collections were performed (Day 0 and Day 9 of protocol) for plasma progesterone dosage through the radioimunoensaio method. The ranges of pregnancy of the groups in the lot D6,5 were 55,17%(G1), 69,23%(G2), 57,67%(G3), 70,83%(G4) (p=0,263). In the lot D8 the groups G1, G2, G3, G4 of this lot presented 44,44%, 67,86%, 46,15%, e 58,33% of pregnancy (p=0,573). The ranges of pregnancy for the lots D6,5 and D8 were 62,86% e 54,29%. There was no difference in the ranges of pregnancy TAI between the lots (p= 0,262). The test Qui- Quadrado and T-test was used for the statistics analysis of the data. The average body condition of the two lots was 2,66 (n=210). There was no influence of the body condition on the ranges od pregnancy TAI (p=0,562). There was no statistic difference in the range TAI and final pregnancy of the animals according to the age and post partum days. Nineteen cows presented P4 maior que 1ng/ml no dia 0 (D6,5= 7, with 5 cows pregancy and 2 empty; D8=12, with 7 cows pregnancy and 5 cows empty) and four cows of the lot D8 presented P4 maior que 1ng/ml on the Day 9 of the protocol (with only one cow pregnant). The implants of PRIMER and SINCROGEST of first and second use were efficient for the synchronization of the ovulation of the post partum cows. The anticipation of luteolytic treatment did not increase pregnancy rates to TAI and also the final pregnancy rate.

The mechanisms causing luteolysis of the primate corpus luteum are unknown. There is an increase in expression of liver x receptor (LXR) target genes and reduced low density lipoprotein receptor (LDLR) during spontaneous luteolysis in primates. The LXRs belong to the nuclear receptor superfamily and increase cholesterol efflux by inducing transcription of their target genes. Uptake of cholesterol into primate luteal cells occurs primarily via LDL, and LDLR transcription is regulated by sterol regulatory element binding transcription factor 2 (SREBF2). Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) maintain luteal function by binding to the LH/CG receptor (LHCGR), which stimulates progesterone (P4) synthesis via protein kinase A (PKA). It has also been previously reported that there is an increase in 27-hydroxycholesterol (27OH) concentrations during spontaneous luteolysis in primates. Pregnenolone and P4 inhibit the enzyme activity of CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1), which converts cholesterol into 27OH, an oxysterol that is a natural LXR agonist and SREBF2 inhibitor. Therefore, the overall hypothesis is that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediate luteolysis of the human CL. The objective of study 1 is to determine the effects of LXR activation and SREBF2 inhibition on P4 production, cholesterol metabolism and gene expression; and how hCG signaling via PKA regulates these effects in human luteinized granulosa cells. Basal and hCG-stimulated P4 secretion were significantly decreased by the combined actions of the LXR agonist T0901317 (T09) and the SREBF2 inhibitor fatostatin, which was associated with alterations in cholesterol metabolism leading to reduced intracellular cholesterol storage. Expression of LXR target genes in the presence of T09 was significantly reduced by hCG, while hCG significantly increased LDLR expression. These effects of hCG were reversed by a specific PKA inhibitor. Chronic hCG exposure had similar effects on LXR target gene and LDLR expression without an exogenous LXR agonist. The objective of study 2 is to determine the effects of 27OH on P4 production and cholesterol metabolism; and to determine if inhibiting the conversion of cholesterol into pregnenolone increases LXR and decreases SREBF2 target gene expression via CYP27A1 in human luteinized granulosa cells. During luteolysis in primates and sheep, CYP27A1 expression significantly increased. 27OH significantly decreased hCG-stimulated P4 secretion and enhanced cholesterol efflux. Aminoglutethimide, which inhibits the conversion of cholesterol to pregnenolone, significantly increased ABCA1 and decreased LDLR. Knock-down of CYP27A1 resulted in a significant increase in P4 secretion, but did not prevent aminoglutethimide-induced effects on ABCA1 and LDLR. Knock-down of steroidogenic acute regulatory protein (STAR), which controls cholesterol transport into the mitochondria where CYP27A1 resides, significantly decreased LDLR transcription. Collectively, the data from study 1 support the hypothesis that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediates luteolysis in primates, which is reversed by hCG. Data from study 2 indicates that 27OH produced via CYP27A1 may contribute to reductions in P4 synthesis during luteolysis, partially by serving as a dual LXR agonist and SREBF2 inhibitor, although other oxysterols are also likely involved.

In ruminants, prostaglandin F2 alpha (PGF2α) is released from the endometrium in a pulsatile pattern at the time of luteolysis. The luteolytic PGF2α pulses are transported from the uterus to the corpus luteum (CL) through the utero-ovarian plexus (UOP) to cause luteolysis. At the time of establishment of pregnancy, interferon tau (IFNT) secreted by the conceptus suppresses the pulsatile release of PGF2α and thereby rescues the CL and maintains its secretion of progesterone. However, basal concentrations of PGF2α are higher in pregnant ewes than in cyclic ewes. The pulsatile release of PGF2α likely requires selective carrier-mediated transport and cannot be supported by a simple diffusion mechanism. The molecular and functional aspects of carrier mediated transport of PGF2α from the uterus to the ovary through the utero- ovarian plexus (UOP) at the time of luteolysis and recognition/establishment of pregnancy are largely unknown ruminants. Results indicate that intrauterine inhibition of (PGT) prevents the pulsatile release of PGF2α independently of spatial expressions of estrogen receptor (ESR-1) and oxytocin receptor (OXTR) proteins by the endometrium at the time of luteolysis in sheep. PGT protein is expressed in the UOP during the estrous cycle and pharmacological inhibition of PGT prevents transport of luteolytic PGF2α pulse through the UOP in sheep. IFNT activates novel JAK-SRC-EGFR-RAS-RAF-ERK1/2-EGR-1 signaling modules in endometrial luminal epithelial (LE) cells and regulates PGT- mediated release of PGF2α through these novel cell-signaling pathways. IFNT stimulates ERK1/2 pathways in endometrial LE cells and inhibition of ERK1/2 inhibits IFNT action and restores spatial expression of OXTR and ESR-1 proteins in endometrial LE cells and restores endometrial luteolytic pulses of PGF2α in sheep. Collectively, the results of the present study provide the first evidence to indicate that transport of endometrial luteolytic PGF2α pulses from the uterus to the ovary through the UOP is controlled by a PGT-mediated mechanism in sheep, new mechanistic insight into molecular mechanisms regulating cellular and compartmental transport of PGF2α at the time of luteolysis, and new mechanistic understanding of IFNT action and release of PGF2α from the endometrial LE cells and thus opens a new arena of research in IFNT signaling and PGT function.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to verify if prostaglandin F2α (PGF2α) administered by intravulvosubmucous (IVSM) injection induces luteolysis by reaching the corpus luteum (CL) directly by a local absorption rote, preventing its metabolism in the lungs, or after absorption and distribution via the systemic circulation. In a first trial, the estrus rate of 1,937 beef cows was monitored for 5 days (25.6% of estrus). At day 5, the cows that did not show estrus received 1/5 of the standard dose of dinoprost IVSM (5 mg; n = 1440) and resulted in 68.2% of estrus in the next 5 days. However, in a second trial, the number of heifers detected in estrus after dinoprost injected via the IVSM (47.4%; n = 97) or intramuscular route (IM; 54.7%; n = 95) at day 5 was not different (P > 0.05). Based on serum progesterone concentrations, animals treated with 5 mg dinoprost at day 5 of the estrous cycle do not present functional luteolysis regardless of the administration route. At day 10 of the estrous cycle, luteolysis was variable in cows treated with 5 mg of dinoprost. Nevertheless, luteolysis occurred in all animals treated with 25 mg dinoprost independent of the estrous cycle day. After treatment, the PGF2α concentration did not differ in serum from uterine and jugular veins. This was further confirmed by measuring the concentration of 13,14-dihydro-15-keto- PGF (PGFM) after 5 mg dinoprost injection via the IM or IVSM route. Dinoprost IM- and IVSM-administered resulted in a similar PGFM serum pattern over time, suggesting the same absorption rate for both routes. Although anatomical evidences suggest that PGF2α injected IVSM could be taken direct to the ovaries, avoiding the systemic circulation, the results do not support this hypothesis. In summary, the route of PGF2α administration (IVSM or IM) resulted in similar serum concentrations of PGF2α, PGFM, and luteolysis. Taking all the results together, the PGF2α injection via IVSM reached the systemic circulation before reaching the ovary, and the effectiveness of low doses of PGF2α was dependent on luteal phase and not on the route of administration. / O presente trabalho teve por objetivo verificar se a prostaglandina F2α (PGF2α) administrada na submucosa vulvar (IVSM) induz a luteólise ao alcançar o corpo lúteo (CL) diretamente por uma via local, evitando sua metabolização nos pulmões, ou após a absorção e distribuição através da circulação sistêmica. Em um primeiro estudo, o estro de 1937 vacas de corte foi monitorado durante 5 dias (25,6% de estro). No dia 5, as vacas que não apresentaram estro receberam 5mg de dinoprost via IVSM (1/5 da dose padrão; n=1440), resultando em 68,2% de estro nos 5 dias seguintes. Todavia, em outro experimento utilizando a mesma dose, o número de novilhas detectadas em estro após o tratamento IVSM (47,4%, n=97) ou intramuscular (IM; 54,7%, n=95) no dia 5 não diferiu entre os grupos (P>0,05). Com base na concentração sérica de progesterona, os animais tratados com 5mg de dinoprost no dia 5 do ciclo estral não apresentaram luteólise funcional, independentemente da via de administração. Após o tratamento com 5mg dinoprost IM ou IVSM no dia 10 do ciclo, 3/5 e 2/5 animais apresentaram luteólise, respectivamente. Entretanto, a luteólise ocorreu em todos os animais tratados com 25mg de dinoprost, independente do dia do ciclo estral (dia 5 ou 10). A concentração de PGF2α não diferiu no soro das veias uterina e jugular. O mesmo foi observado quanto ao padrão de 13,14-dihidro-15-ceto prostaglandina F2α (metabólito de PGF2α; PGFM) sérico ao longo do tempo após a aplicação de 5 mg de dinoprost via IM ou IVSM. Em resumo, a via de administração de PGF2α (IVSM ou IM) resultou em concentrações séricas semelhante de PGF2α, PGFM e luteólise. Embora evidências anatômicas permitam sugerir que a PGF2α injetada via IVSM possa ser transportada diretamente aos ovários, a injeção de PGF2α via IVSM atinge a circulação sistêmica antes de chegar ao ovário, e a eficácia de baixas doses de PGF2α é dependente da fase luteal e não da via de administração.