Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias / La hembra canina presenta características reproductivas particulares comparado con las otras especies de mamíferos domésticos, donde destaca el arresto meiótico prolongado del ovocito reanudando la meiosis posterior a la ovulación. El desarrollo del ovocito es regulado por señales entre el ovocito y las células foliculares que le rodean mediante conexones constituidos por conexinas, de ellas la Conexina 43 es la proteína con mayor expresión en el ovario. El objetivo de este trabajo fue estudiar la expresión génica de Cx43 y de su proteína en células foliculares de perra durante el desarrollo folicular y a lo largo del ciclo estral. Usando un lupa estereoscópica se aislaron células de folículos preantrales y antrales de ovarios de perra en anestro, proestro, estro y diestro. Se utilizó q-PCR para la evaluación de los niveles de mARN y Western Blot e lnmunohistoquímica para la cuantificación y localización de la proteína respectivamente. Los valores de expresión génica y densidad óptica de las bandas de Western Blot se analizaron con ANOVA; además, se hicieron correlaciones de Pearson. La Cx43 se encontró en todos los folículos desde folículo primordial hasta preovulatorio, la expresión del gen de Cx43 y su proteína codificante varió a lo largo del ciclo estral, presentando los mayores niveles de mARN en anestro (P<0,05) y los mayores niveles de Cx43 en diestro (P<0,05), adicionalmente la expresión de mRNA y de Cx43 no disminuyeron en folículos preovulatorios, sugiriendo que la comunicación entre el ovocito y las células foliculares se mantienen, lo que podría explicar el arresto meiótico prolongado de la perra. La correlación entre mARN y proteína fue negativa en la mayoría de etapas del ciclo, lo que se podría asociar a los mecanismos de control traduccional a las que son sometidas las conexinas. / The female canine has particular reproductive characteristics compared to the other species of domestic mammals, where it stands out the prolonged meiotic arrest of the oocyte that resumes meiosis after ovulation. Oocyte development is regulated by signals between the oocyte and follicular cells that sorround it by connexons constituted of connexins, of these, Connexin 43 is the protein with the highest expression in the ovary. The objective of this work was to study the gene expression of Cx43 and its protein in dog follicular cells during follicular development and throughout the estrous cycle. Using a stereoscopic magnifying glass, cells were isolated from preantral and antral follicles of the ovaries of bitches in anestrus, proestrus, estrus and diestrus. q-PCR was used for the evaluation of mRNA levels and Western Blot and lmmunohistochemistry for the quantification and localization of the protein respectively. The values of gene expression and optical density of the Western Blot bands were analyzed with ANOVA; in addition , Pearson correlations were made. The Cx43 was found in all the follicles from primordial to preovulatory, the expression of the Cx43 gene and its coding protein varied throughout the estrous cycle, presenting the highest levels of mRNA in anoestrus (P <0.05) and the highest levels of Cx43 in diestrus (P <0.05), additionally the expression of mRNA and Cx43 did not decrease in preovulatory follicles, suggesting that communication between the oocyte and follicular cells is maintained, which could explain the prolonged meiotic arrest of the dog. The correlation between mRNA and protein was negative in most stages of the cycle, which could be associated with the mechanisms of translational control to which connexins are subjected. / Financiamiento: Proyecto Fondecyt 1171670

El objetivo del trabajo fue diseñar un protocolo de inducción de ciclos fértiles en la perra utilizando eCG y hCG. En el experimento uno se evaluaron las características farmacocinéticas de eCG, la concentración maxima observada Cmax) fue semejante entre las vías im e iv (6.1±0.9 vs. 8.6±0.5 IU/mL, P>0.08), mientras que el tiempo en el que alcanzó la concentración máxima observada (Tmax) fue más largo para la via im que iv.m (17.5±0.5 vs. 11.6±0.3 h, P<0.01). El área bajo la curva (AUC) fue similar para las vías im e iv (P>0.48). Así mismo eCG se detectó en suero por al menos 144h. En el experimento dos se buscó la dosis efectiva de eCG. Pudo observarse que con 50 UI/Kg (TRT 50) de eCG se pudieron observar signos externos, comportamiento y citología vaginal de estro en todos los animales tratados La media del intervalo tratamiento - estro fue de 4.0±0.4 d. En el experimento tres se estudiaron las fluctuaciones de androstenediona, prolactina e IGF1 en relación al desarrollo folicular en las perras inducidas. Pudo observarse que las concentraciones séricas de estas sustancias varían a lo largo del desarrollo folicular. En el experimento cuatro se evaluó la eficacia reproductiva del uso combinado de eCG y hCG. Todas las perras que recibieron tratamiento respondieron manifestando comportamiento, signos externos y citología vaginal compatible con estro. El 94% de las perras recibieron servicio natural o inseminación artificial y el 80% quedaron preñadas y parieron 3,62 ± 0,41 Cachorros. No se observan efectos adversos en ninguna de las perras tratadas. / The aim of this study was to design a protocol combining eCG followed by hCG for estrus induction in the bitch. In Experiment one, we study pharmacokinetics analyses of eCG. Maximum observed concentration (Cmax) eCG were similar between im and iv routes (6.17±0.97 vs. 8.69±0.50 IU/mL, P > 0.08), whereas Tmax was longer for im compared to iv delivery route (17.5±0.5 vs. 11.66±0.33 h, P<0.002). Elimination half-live was shorter for im than for iv delivery routes (38.19± 2.81 vs. 59.08± 2.81 h; P < 0.03). The AUC concentrations were similar im and iv delivery routes (307.70±5.71 vs. 381.10±70.90 IU h/mL; P > 0.48) and eCG was detectable in serum for at least 144 h for both routes. In Experiment two, ovarian stimulatory effects of eCG was studied using one of five doses of eCG (5, 10, 15, 20, 44, or 50 IU/kg eCG im; [TRT5-TRT50]). Three to five days after treatment, all bitches in TRT50 had higher scores compared to TRT5-44 animals (P < 0.01). In TRT50, mean interval from treatment to estrus was 4.0±0.4 d. No detectable clinical side effects were observed in bitches during treatment. In experiment three studied hormonal response and follicular development after eCG administration. We observed that prolactin, IGF1 and androstenedione serum concentration change with follicular growth induced by a single dose of 50 IU/kg of eCG similar to dose occurring during spontaneous oestrous cycle. In experiment four reproductive efficacies of eCG and hCG in oestrus induction was studied. In treatment group all bitches responded to treatment by coming into clinical estrus within 3-5 d after eCG-hCG administration. Ninety four percent of animals were bred (15/16; AI, n=7; natural mating, n=8), and 80% (12/15) became pregnant. The average litter size was 3.62 ± 0.41. None of the bitches showed any side-effects to the eCG and hCG therapy.

This thesis examines American Far Eastern policy from the beginning of the Franklin Roosevelt presidency through the early months of 1938. This study is chiefly concerned with the attack by Japanese aircraft on the USS Panay and its effect on the course of U.S. foreign policy. Particular attention is paid to the Anglo-American dialogue which occurred throughout the Far Eastern Crisis. Prior to the end of 1938, the U.S. administration’s position in Asia was dictated both by policies inherited from preceding administrations and by the extreme isolationism of the American people. This foundation effectively inhibited any cooperation with foreign powers. Relying on a reactive policy in the Far East, Washington remained aloof from entanglement as the President sought a plan which would permit U.S. involvement without inviting isolationist wrath. This paper traces an evolution in American Far Eastern policy, highlighting the Panay incident as a distinctly identifiable turning point whereby isolationism gave way to internationalism.

This thesis examines American Far Eastern policy from the beginning of the Franklin Roosevelt presidency through the early months of 1938. This study is chiefly concerned with the attack by Japanese aircraft on the USS Panay and its effect on the course of U.S. foreign policy. Particular attention is paid to the Anglo-American dialogue which occurred throughout the Far Eastern Crisis. Prior to the end of 1938, the U.S. administration’s position in Asia was dictated both by policies inherited from preceding administrations and by the extreme isolationism of the American people. This foundation effectively inhibited any cooperation with foreign powers. Relying on a reactive policy in the Far East, Washington remained aloof from entanglement as the President sought a plan which would permit U.S. involvement without inviting isolationist wrath. This paper traces an evolution in American Far Eastern policy, highlighting the Panay incident as a distinctly identifiable turning point whereby isolationism gave way to internationalism.

This thesis examines American Far Eastern policy from the beginning of the Franklin Roosevelt presidency through the early months of 1938. This study is chiefly concerned with the attack by Japanese aircraft on the USS Panay and its effect on the course of U.S. foreign policy. Particular attention is paid to the Anglo-American dialogue which occurred throughout the Far Eastern Crisis. Prior to the end of 1938, the U.S. administration’s position in Asia was dictated both by policies inherited from preceding administrations and by the extreme isolationism of the American people. This foundation effectively inhibited any cooperation with foreign powers. Relying on a reactive policy in the Far East, Washington remained aloof from entanglement as the President sought a plan which would permit U.S. involvement without inviting isolationist wrath. This paper traces an evolution in American Far Eastern policy, highlighting the Panay incident as a distinctly identifiable turning point whereby isolationism gave way to internationalism.

This thesis examines American Far Eastern policy from the beginning of the Franklin Roosevelt presidency through the early months of 1938. This study is chiefly concerned with the attack by Japanese aircraft on the USS Panay and its effect on the course of U.S. foreign policy. Particular attention is paid to the Anglo-American dialogue which occurred throughout the Far Eastern Crisis. Prior to the end of 1938, the U.S. administration’s position in Asia was dictated both by policies inherited from preceding administrations and by the extreme isolationism of the American people. This foundation effectively inhibited any cooperation with foreign powers. Relying on a reactive policy in the Far East, Washington remained aloof from entanglement as the President sought a plan which would permit U.S. involvement without inviting isolationist wrath. This paper traces an evolution in American Far Eastern policy, highlighting the Panay incident as a distinctly identifiable turning point whereby isolationism gave way to internationalism.