Induction of fertile estrus without the use of steroid hormones in seasonally anestrous Suffolk ewes.

OBJECTIVE: To evaluate the efficacy of treatments based on GnRH, GnRH-PGF2α, and/or intense exposure to novel rams to induce fertile estrus without the use of steroid hormones in seasonally anestrous Suffolk ewes. METHODS: In the first experiment, ewes were treated with one injection of GnRH, two injections of GnRH administered 7 days apart, or a sequence of GnRH-PGF2α-GnRH. In the second experiment anestrous ewes were exposed, for 36 days starting on the day of weaning, to groups of four rams of three different breeds that were alternated every day. Besides exposure to the males, the ewes were injected with saline solution (ME group, n=20), with GnRH (ME-GnRH group, n=20) or with a sequence of GnRH-PGF2α-GnRH (ME-GPG group, n=20). The rams used for male-effect were fitted with aprons to prevent mating, and ewes detected in estrus were bred to selected fertile rams. Ovarian activity was monitored by progesterone determinations in both experiments. RESULTS: In the first experiment sustained induction of ovarian activity was not achieved and no ewe was detected in estrus. In the second experiment induction of sustained ovarian activity was achieved in all groups. Most of the ewes were detected in estrus, 76.7 % of the ewes were mated during a 36-d breeding period and 71.7 % of all the ewes became pregnant during that period. No significant differences between groups were found for any of these variables. However, estrus detection efficiency was higher in the ME-GnRH group than in the ME group (p<0.05). CONCLUSION: An intense male-effect, that included the continuous presence and frequent alternation of several rams of different breeds, was sufficient to induce ovarian activity and fertile estrus in Suffolk ewes during the period of deep anestrus without the use of hormones, although addition of GnRH improved the efficiency of estrus detection.

In vivo dual targeting of the oncogenic Ether-à-go-go-1 potassium channel by calcitriol and astemizole results in enhanced antineoplastic effects in breast tumors.

BACKGROUND: The oncogenic ether-à-go-go-1 potassium channel (EAG1) activity and expression are necessary for cell cycle progression and tumorigenesis. The active vitamin D metabolite, calcitriol, and astemizole, a promising antineoplastic drug, target EAG1 by inhibiting its expression and blocking ion currents, respectively. We have previously shown a synergistic antiproliferative effect of calcitriol and astemizole in breast cancer cells in vitro, but the effect of this dual therapy in vivo has not been studied. METHODS: In the present study, we explored the combined antineoplastic effect of both drugs in vivo using mice xenografted with the human breast cancer cell line T-47D and a primary breast cancer-derived cell culture (MBCDF). Tumor-bearing athymic female mice were treated with oral astemizole (50 mg/kg/day) and/or intraperitoneal injections of calcitriol (0.03 µg/g body weight twice a week) during 3 weeks. Tumor sizes were measured thrice weekly. For mechanistic insights, we studied EAG1 expression by qPCR and Western blot. The expression of Ki-67 and the relative tumor volume were used as indicators of therapeutic efficacy. RESULTS: Compared to untreated controls, astemizole and calcitriol significantly reduced, while the coadministration of both drugs further suppressed, tumor growth (P < 0.05). In addition, the combined therapy significantly downregulated tumoral EAG1 and Ki-67 expression. CONCLUSIONS: The concomitant administration of calcitriol and astemizole inhibited tumor growth more efficiently than each drug alone, which may be explained by the blocking of EAG1. These results provide the bases for further studies aimed at testing EAG1-dual targeting in breast cancer tumors expressing both EAG1 and the vitamin D receptor.

Calcitriol reduces thrombospondin-1 and increases vascular endothelial growth factor in breast cancer cells: implications for tumor angiogenesis.

Calcitriol, a potent antineoplastic vitamin D metabolite, inhibits proliferation, induces apoptosis and slows the growth of tumors. Calcitriol also may exert either antiangiogenic or proangiogenic effects depending on the tissue. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (Tsp-1) are key factors involved in promoting and inhibiting angiogenesis, respectively. The effects of calcitriol on Tsp-1 have not been studied in the mammary gland, while VEGF regulation is not clear, since opposite outcomes have been demonstrated. Therefore, the present study was undertaken to investigate the effects of calcitriol on VEGF and Tsp-1 expression in primary breast tumor-derived cells and a panel of established breast cancer cell lines. In vivo studies in athymic mice were also performed in order to gain further insight into the biological effects of calcitriol on angiogenesis. Real time-PCR and ELISA analyses showed that calcitriol stimulated VEGF mRNA expression and protein secretion while elicited the opposite effect on Tsp-1 in 7 out of 8 cell lines studied, independently of the cell phenotype (P<0.05 in n=5). In vivo, calcitriol significantly inhibited the relative tumoral volume after 4 weeks of treatment; however, serum VEGF was higher in calcitriol-treated animals compared to controls (P<0.05). The integrated fluorescence intensity analysis of CD31, a vessel marker, showed that xenografted breast cancer cells developed tumors with similar vascular density regardless of the treatment. Nevertheless, larger necrotic areas were observed in the tumors of calcitriol-treated mice compared to controls. Since the antineoplastic activity of calcitriol has been consistently demonstrated in several studies including this one, our results suggest that the antitumoral effect of calcitriol in vivo involve different mechanisms not necessarily related to the inhibition of tumor vascularization. Overall, our findings indicate that calcitriol can impact the angiogenic process in breast cancer by regulating VEGF and Tsp-1 expression. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Learning curve in a Western training center of the circumferential en bloc esophageal endoscopic submucosal dissection in an in vivo animal model.

Aim. Evaluate the feasibility to overcome the learning curve in a western training center of the en bloc circumferential esophageal (ECE-) ESD in an in vivo animal model. Methods. ECE-ESD was performed on ten canine models under general anesthesia on artificial lesions at the esophagus marked with coagulation points. After the ESD each canine model was euthanized and surgical resection of the esophagus and stomach was carried out according to "the Principles of Humane Experimental Technique, Russel and Burch." The specimen was fixed with needles on cork submerged in formalin with the esophagus and stomach then delivered to the pathology department to be analyzed. Results. ECE-ESD was completed without complications in the last 3/10 animal models. Mean duration for the procedures was 192 ± 35 minutes (range 140-235 minutes). All the procedures were done at the animal lab surgery room with cardio pulmonary monitoring and artificial ventilation by staff surgery members and a staff member of the Gastroenterology department trained during 1999-2001 at the Fujigaoka hospital of the Showa U. in Yokohama, Japan, length (range 15-18 mm) and 51 ± 6.99 width (range 40-60 mm). Conclusion. ECE-ESD training is feasible in canine models for postgraduate endoscopy fellows.

Endoscopic submucosal dissection in dogs in a World Gastroenterology Organisation training center.

AIM: To evaluate if canine models are appropriate for teaching endoscopy fellows the techniques of endoscopic submucosal dissection (ESD). METHODS: ESD was performed in 10 canine models under general anesthesia, on artificial lesions of the esophagus or stomach marked with coagulation points. After ESD, each canine model was euthanized and surgical resection of the esophagus or stomach was carried out according to "The Principles of Humane Experimental Technique, Russel and Burch". The ESD specimens were fixed with needles on cork submerged in a formol solution with the esophagus or stomach, and delivered to the pathology department to be analyzed. RESULTS: ESD was completed without complications using the Hook-knife in five esophageal areas, with a procedural duration of 124 +/- 19 min, a length of 27.4 +/- 2.6 mm and a width of 21 +/- 2.4 mm. ESD was also completed without complications using the IT-knife2 in five gastric areas, with a procedural duration of 92.6 +/- 19 min, a length of 32 +/- 2.5 mm and a width of 18 +/- 3.7 mm. CONCLUSION: ESD is feasible in the normal esophagus and stomach of canine models, which are appropriate for teaching this technique.

Efecto de eCG e inseminación laparoscópica sobre el comportamiento reproductivo en ovejas F1 (damara x merino) / Effect of eCG and laparoscopic insemination on reproductive performance in F1 (damarax merino) ewes

Cuarenta y cinco ovejas F1 (Damara x Merino) con 123 ± 27,6 días post parto mantenidas en clima tropical húmedo fueron utilizadas para evaluar el efecto de dosis de gonadotropina coriónica equina (eCG) en el porcentaje de sincronización del estro, fertilidas por inseminación laparoscópica (IL) prolificidad. Para sincronizar estros en la ovejas se les colocó un dispositivo intravaginal (CIDR) inpregnado con 0,03 de progesterona por 12 d. Las ovejas fueron asignadas aleatoriamente a tres tratamientos. T1: CIDR y monta natural; T2: CIDR más 150 UI de ecG e IL a las 42-46 h al retiro del dispositivo con semen de machos de la raza Damara; T3. CIDR más 300 UI de eCG e IL a las 42-46 h de retiro del dispositivo con semen de machos de la raza Damara. La fertilidad del estro sincronizado fue de 40; 40 y 46,6% para T1, T2 y T3 respectivamente, no encontrando diferencias (P> 0,05) entre ellos. El porcentaje de prolificidad presentó diferencias (P<0,05) entre tratamientos y fue de 100; 100 y 133,3% para T1, T2 y T3. Estos resultados permitirán hacer ajustes en estudios futuros sobre la metodología de programas de sincronización de estros e inseminación artificial laparoscópica a tiempo fijo con semen congelado, con el objetivo de mejorar el comportamiento reproductivo de ovejas F1 (Damara x Merino) bajo condiciones de clima tropical húmedo en México.

Zinc-induced survival of Leydig cells in Fischer rats (Rattus norvegicus) treated with cadmium chloride.

Zinc is known to prevent cadmium-induced carcinogenesis and Leydig cell destruction in rat testes; however, the mechanism of action is not known, although it has been suggested that pituitary feedback increases the production of luteinizing hormone (LH) in response to low circulating androgen. We therefore examined the biological role of zinc in reducing cadmium toxicity in the Leydig cells of Fischer rats. Two groups of eleven 6-month-old rats were injected subcutaneously with 20 micromol CdCl2/kg weekly for 5 weeks; one of these groups also received 1 mmol/kg zinc acetate weekly for the same 5 weeks. A third group of rats received 1 mmol/kg zinc acetate weekly, and a fourth group was injected with saline weekly for 5 weeks. After 8 months of study, the animals were euthanized by CO2 inhalation. The results indicated that the number of surviving Leydig cells was significantly lower in the cadmium group (7.34% = 0.095 x 10(9)/cm3) than in the cadmium-zinc group (20.85%) or control animals (91.2%). Moreover, the concentrations of serum testosterone and LH were significantly higher in the cadmium group than in any of the other groups. This difference probably was due to the testosterone produced by a small reservoir of surviving Leydig cells and to other endocrine factors. These findings suggest that Fischer rat testis may be a good model system for testing the effects of cadmium and zinc on the production of LH and testosterone and other androgens before spontaneous cancers develop.

Administración posmonta de acetato de fluorogestona en ovejas donadoras de embriones / Posmating administration of fluorogestone acetate in ambryo donor ewes

De dos estudios realizados con ovejas donadoras de embriones tratadas posmonta con acetato de fluorogestona se analizaron el número de cuerpos lúteos normales o en regresión y el número de ovocitos o embriones recolectados. La sincronización de los estros se realizó con esponjas intravaginales impregnadas con 40 mg de acetato de fluorogestona (FGA) y para la superovulación se utilizaron 200 UI de hormona folículo estimulante en esquema decreciente. A las hembras en estro se les dio monta mientras permanecieron receptivas y doce horas después de finalizada la última monta, en la mitad de las ovejas del primer estudio y en todas las del segundo, se colocaron otras esponjas intravaginales con FGA que se retiraron el día de la recolección embrionaria. En cada oveja se clasificaron los cuerpos lúteos como normales o en regresión de acuerdo con su morfología y producción de progesterona. De las ovejas del primer trabajo tratadas con FGA se recolectaron más embriones que de las no tratadas (7.66 vs 3.87, P<0.05). Las menores concentraciones promedio de progesterona se encontraron en las ovejas con cuerpos lúteos en regresión (0.62 ng/mL). De acuerdo con los niveles de progesterona la regresión prematura de los cuerpos lúteos ocurrió al día 3.7 posestro. De las ovejas del segundo experimento que presentaron cuerpos lúteos en regresión y que fueron tratadas con FGA se recolectaron en promedio 8.0 embriones. En conclusión, la administración posmonta de FGA incrementó el número de embriones recolectados en las donadoras con regresión lútea prematura.

Efecto del líquido folicular equino y la somatotropina bovina en la sobrevivencia de embriones ovinos transferidos asincrónicamente / Effect of equine follicular fluid and bovine somatotropin in the survival of ovine embryos asynchronously transferred

La asincronía materno-embrionaria pudiera resolverse incrementando los niveles de progesterona (P4) para promover el desarrollo embrionario, o retrasando la luteólisis. Para tal fin se administró somatotropina bovina (ST), líquido folicular equino (LFE) o ambos, a receptoras de embriones en asincronía. Se recolectaron embriones en 25 donadores el día 6 posestro. Las receptoras se sincronizaron con acetato de fluorogestona y PGF2Ó, y en las asincrónicas de todos los grupos se retiró el progestágeno 3.5 días antes que en las donadoras. Se dividieron en cinco tratamientos: sincronía (sin), n=16; asincronía + LFE (asinlfe), n=19; asincronía + ST (asins), n=7; y asincronía + LFE + ST (asinlfst), n=10. Las concentraciones de progesterona (3.3 ng/ml) fueron mayores en el grupo asinlfe (P< 0.05). Las menores concentraciones (1.4 ng/ml) se presentaron en el asinst. La fase lútea de mayor duración, 16.8 días promedio, se presentó en las no gestantes del grupo asin. En cuatro ovejas del asinlfe se retrasó la luteólisis hasta el día 19. La fertilidad del grupo sin (86.6 por ciento) no fue diferente a la del asin (68.7 por ciento). Los tratamientos no mejoraron la fertilidad a pesar de inrementarse la P4 en las gestantes asinlfe y asinlfst o retrasarse la luteólisis en las asinlfe. Se propone que antes de modificar la asincronía materno-embrionaria se determine el grado de asincronía tolerable de manera natural en los ovinos