Vitamin D status in female dogs with mammary gland tumors.

BACKGROUND: Little information exists about vitamin D status in bitches with mammary tumors. OBJECTIVES: To determine whether low plasma vitamin D concentrations are found in bitches with mammary tumors. ANIMALS: Eighty-five client-owned bitches with mammary tumors (n = 21 benign, n = 64 malignant) and 39 age-matched healthy bitches. METHODS: Case-control study. Plasma ionized and total calcium, phosphorus, magnesium, urea, creatinine, albumin, total proteins, alanine aminotransferase, alkaline phosphatase, parathyroid hormone (PTH), calcitriol (1,25-dihydroxyvitamin D), and 25-hydroxyvitamin D concentrations were measured in all bitches at the time of clinical diagnosis and before any treatments. Statistical analysis was performed to compare variables among groups (control, benign, and malignant). RESULTS: No significant differences were found when plasma 25-hydroxyvitamin D concentrations in bitches with malignant (148.9 [59.9] ng/mL) and benign mammary tumors (150.1 [122.3] ng/mL) were compared with control group (129.9 [54.5] ng/mL). Parathyroid hormone was significantly higher in bitches with malignant (19.9 [20.5] pg/mL), and benign mammary tumors (14.6 [14.9] pg/mL) compared with control group (7.5 [7.5] pg/mL; P < .01). Only the presence of mammary tumors (P < .01) and age (P = .04; adjusted R2 = .22) was significant in predicting PTH. CONCLUSIONS: Bitches with mammary tumors do not have low 25-hydroxyvitamin D concentrations thus vitamin D supplementation is unlikely to be useful for prevention of mammary tumors in bitches.

Analysis of cell-free DNA concentration, fragmentation patterns and TP53 gene expression in mammary tumor-bearing dogs: A pilot study.

Introduction: Liquid biopsy based on the analysis of circulating cell-free DNA (cfDNA), as well as on detection of point mutations by digital droplet PCR (ddPCR), has revolutionized the research in oncology. In recent years, this technique has been pioneering in veterinary medicine since it is a minimally invasive approach with very promising results for characterization of tumors. Methods: The aim of this study was, firstly, to analyze the concentration and the fragmentation pattern of cfDNA of dogs with mammary tumors (n = 36) and healthy dogs (n = 5) and its correlation with clinicopathological data. Secondly, analysis of TP53 gene expression and the point mutation in the codon 245 were performed in cfDNA and in tumor tissues to assess their potential as plasma biomarkers. Results and discussion: Our results highlighted that those dogs with worse clinicopathological characteristics (simple or undifferentiated carcinomas, higher histological grade and presence of peritumoral inflammation) shown higher cfDNA concentration and higher concentrations of short-fragments (<190 bp) than healthy dogs. In addition, although no detection of the point mutation in codon 245 of TP53 gene could be detected neither in plasma nor tumor tissue, an increased TP53 expression was detected in animals with tumors bearing malignant characteristics. Finally, a high concordance with TP53 gene expression in plasma and tumor tissue and cfDNA concentration was also found. The results derived from this work confirm the valuable potential of cfDNA and its fragments, as well as the analysis of TP53 expression in plasma as useful liquid biomarkers for clinical application in veterinary oncology.

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models.

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR-2, PDGFRs and c-Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c-Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.

Immunohistochemical expression of p63 protein and calponin in canine mammary tumours.

The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in canine mammary tumours. The studied material included 10 benign and 32 malignant mammary tumours from female dogs treated with mastectomy. Primary mouse monoclonal antibodies directed against p63 protein clone 4A4 and calponin clone CALP were used in single- and doublestain system of immunohistochemical reaction. The investigations have shown that majority of myoepithelial cells in benign tumours and carcinomas in situ exhibited strong positive labelling for both markers. In other malignant tumours strong immunoreactivity was observed in resting myoepithelial cells (MECs) and hypertrophic myoepithelial cells (HMECs), while the immunoreactivity in spindle-stellate myoepithelial cells (SMECs) and rounded myoepithelial cells (RMECs) was moderate. The granular-diffuse nuclear expression of p63 protein was observed only in myoepithelial cells. In terms of calponin, diffuse cytoplasmic expression was noted not only in myoepithelial cell but also in some stromal fibroblasts and vascular smooth muscle cells. The epithelial cells did not exhibit specific expression of the investigated markers. The obtained results indicate that p63 is a sensitive and more specific marker of myoepithelial cells in canine mammary tumours compared with calponin. These findings suggest that the immunohistochemical analysis peformed with the use of p63 can be a valuable complement of routine histological examinations of canine mammary tumours facilitating identification of tumours with myoepithelial component.

Immunohistochemical and quantitative RT-PCR methods to assess RANK expression in normal and neoplastic canine mammary gland.

The receptor activator of nuclear factor-kB ( RANK) gene is found in both human and murine mammary epithelial cells and in human cancer cell lines. We analyzed RANK expression in normal and proliferative canine mammary tissue samples ( n = 47) and cell lines ( n = 10), and identified its expression in epithelial cell populations. The correlation of RANK protein with clinicopathologic parameters was also studied. A double immunohistochemical method using RANK and p63 antibodies was applied to 33 tissue samples to analyze RANK protein expression and its possible co-expression with p63 protein, the latter used to identify myoepithelial (ME) cells (p63-positive) or luminal epithelial (LE) cells (p63-negative). RANK protein expression was found in ~75% of the tissue samples analyzed, at a similar level in all of the histologic types studied: dysplasias (4 of 4, 100%), malignant tumors (13 of 17, 76%), normal glands (12 of 17, 70%), and benign tumors (6 of 9, 67%). ME and LE cells expressed RANK protein at a similar level. A higher level of RANK protein expression was found in older animals (≥10 y, p = 0.027). Quantitative RT-PCR was applied to 6 ME (1 normal and 5 neoplastic) and 4 LE (1 normal and 3 neoplastic) primary cell lines. The RANK gene was found at similar expression levels in all canine mammary ME and LE cell lines studied. We found RANK expression in normal, dysplastic, and neoplastic canine mammary tissues and cell lines, in both ME and LE cell populations.

Myoepithelial cells in canine mammary tumours.

Mammary tumours are the most common neoplasms of female dogs. Compared to mammary tumours of humans and cats, myoepithelial (ME) cell involvement is common in canine mammary tumours (CMT) of any subtype. Since ME cell involvement in CMT influences both histogenetic tumour classification and prognosis, correct identification of ME cells is important. This review describes immunohistochemical methods for identification of canine mammary ME cells used in vivo. In addition, phenotypic and genotypic methods to isolate ME cells for in vitro studies to analyse tumour-suppressor protein production and gene expression are discussed. The contribution of ME cells to both histogenetic classifications and the prognosis of CMT is compared with other species and the potential use of ME cells as a method to identify carcinoma in situ is discussed.

Increased expression of insulin-like growth factor-1 receptor is correlated with worse survival in canine appendicular osteosarcoma.

Insulin-like growth factor 1 receptor (IGF-1R) is a cell membrane receptor widely expressed in tissues and involved in different cancers in humans. IGF-1R expression in human osteosarcoma has been associated with the development of tumour metastasis and with prognosis, and represents an attractive therapeutic target. The goal of this study was to investigate the expression of IGF-1R in canine osteosarcoma tissues and cell lines and assess its role and prognostic value. Samples from 34 dogs were examined by immunohistochemistry for IGF-1R expression. IGF-1R/AKT/MAPK signalling was evaluated by western blot and quantitative polymerase chain reaction in the cell lines. In addition, the in vitro inhibition of IGF-1R with pycropodophillin (PPP) was used to evaluate molecular and biological effects. Immunohistochemical data showed that IGF-1R was expressed in 71% of the analysed osteosarcoma samples and that dogs with higher levels of IGF-IR expression (47% of cases) had decreased survival (P < 0.05) when compared to dogs with lower IGF-IR expression. Molecular studies demonstrated that in canine osteosarcoma IGF-IR is activated by IGF-1 mostly in a paracrine or endocrine (rather than autocrine) manner, leading to activation of AKT/MAPK signalling. PPP caused p-IGF-1R dephosphorylation with partial blocking of p-MAPK and p-AKT, as well as apoptosis. It was concluded that IGF-1R is expressed and plays a role in canine osteosarcoma and that its expression is correlated with a poor prognosis. As in humans, IGF-1R may represent a good therapeutic target and a prognostic factor for canine osteosarcoma.

Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma.

BACKGROUND: Progesterone receptors play a key role in the development of canine mammary tumours, and recent research has focussed on their possible value as therapeutic targets using antiprogestins. Cloning and sequencing of the progesterone receptor gene has shown that the receptor has two isoforms, A and B, transcribed from a single gene. Experimental studies in human breast cancer suggest that the differential expression of progesterone receptor isoforms has implications for hormone therapy responsiveness. This study examined the effects of the antiprogestin aglepristone on cell proliferation and mRNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n = 22) or vehicle (n = 5) twice before surgery. RESULTS: Formalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor and both isoforms by RT-qPCR and Ki67 antigen labelling. Both total progesterone receptor and isoform A mRNA expression levels decreased after treatment with aglepristone. Furthermore, a significant decrease in the proliferation index (percentage of Ki67-labelled cells) was observed in progesterone-receptor positive and isoform-A positive tumours in aglepristone-treated dogs. CONCLUSIONS: These findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A.

The antiprogestins mifepristone and onapristone reduce cell proliferation in the canine mammary carcinoma cell line CMT-U27.

Canine mammary tumours (CMTs) represent nearly half of all tumours in female dogs and some 50% have malignant behaviour. Simple epithelial carcinomas have shorter disease free periods after surgery and a higher reduction of the proliferation index reduction after antiprogestin aglepristone treatment in vivo related to the expression of progesterone receptors (PR). These findings make simple carcinomas good candidates for endocrine therapy. To further explore this possibility, the effects of the antiprogestins mifepristone (RU486) and onapristone (ZK299) on cell viability and PR expression of the canine mammary carcinoma cell line isolated from a simple epithelial carcinoma CMT-U27 were studied. Twenty five percent of CMT-U27 control cells expressed PR. RU486 (p<0.05) and ZK299 (p<0.05) reduced the number of viable cells (WST-8 test) at 24h but only the latter treatment reduced significantly PR expression in viable tumour cells at 24h of incubation. The results suggest that both RU486 and ZK299 induce a decrease in the number of viable CMT-U27 tumour cells with different effects on PR expression. The canine mammary carcinoma cell line CMT-U27 is sensitive to the effects of antiprogestins and may serve to further explore the role of these drugs in canine mammary carcinomas.

Activation of mammalian target of rapamycin (mTOR) in triple negative feline mammary carcinomas.

BACKGROUND: Triple negative breast cancer (TNBC) in humans is defined by the absence of oestrogen receptor (ER), progesterone receptor (PR) and HER2 overexpression. Mammalian target of rapamycin (mTOR) is overexpressed in TNBC and it represents a potential target for the treatment of this aggressive tumour. Feline mammary carcinoma (FMC) is considered to be a model for hormone-independent human breast cancer. This study investigated mTOR and p-mTOR expression in FMC in relation to triple negative (TN) phenotype. RESULTS: The expression of mTOR, p-mTOR, ERα, PR and HER2 was evaluated in 58 FMCs by immunohistochemistry and in six FMC cell lines by Western blot analysis. 53.5% of FMC analyzed were ER, PR, HER2 negative (TN-FMC) while 56.9% and 55.2% of cases expressed mTOR and p-mTOR respectively. In this study we found that m-TOR and p-mTOR were more frequently detected in TN-FMC and in HER2 negative samples. CONCLUSIONS: In this study, we demonstrate that there is also a FMC subset defined as TN FMC, which is characterised by a statistically significant association with m-TOR and p-mTOR expression as demonstrated in human breast cancer.

Liver stiffness using transient elastography is applicable to canines for hepatic disease models.

BACKGROUND: The objectives of this study were to evaluate the best position and best exploration probe for determining liver stiffness (LS) in dogs using transient liver elastography (TE). Thirteen dogs were used in the study. METHODOLOGY/PRINCIPAL FINDINGS: Morphometric measurements taken were thoracic circumference, weight and height. Elastographic measurements were taken in 4 anatomical positions using two different probes: medium (M) and small (S). The exploration was considered correct when the success rate was above 60% and the interquartile range of the measurements did not exceed 30%. The best measurements were obtained in the middle of the 6th-9th intercostal spaces, with the dog in the left lateral position and using probe M for preference in adults and probe S mandatory for animals <2 years. The correlation between probes was 99%. Intra-observer variability showed an intra-class correlation of 97.6%. CONCLUSIONS/SIGNIFICANCE: TE is a technique that is reproducible in dogs.

Caracterización morfológica e histoquímica del hígado de la baba (Caiman Crocodilus Crocodilus) / Morphological and Histochemical Characterization of the Liver of the Spectacled Cayman (Caiman crocodilus crocodilus)

La morfología, organización y los componentes intracitoplasmáticos del hepatocito de la baba (Caiman crocodilus crocodilus) son aspectos que se han estudiado parcialmente hasta el momento. Con el fin de generar información sobre la morfología microscópica del hígado de esta subespecie, se realizó un estudio descriptivo de las características morfológicas e histoquímicas del hígado de la baba, lo que permitirá la comprensión de algunas funciones hepáticas en estos ejemplares. De esta manera, se podrán realizar en el futuro, investigaciones utilizando a dicho órgano como modelo experimental para el estudio de las rutas metabólicas de los metabolitos procesados a este nivel. Se tomaron ocho muestras de hígado, se fijaron en formol al 10%  y se procesaron por los métodos histológicos de coloraciones de rutina (Hematoxilina y Eosina). Asimismo, se utilizaron las coloraciones especiales de Tricrómico de Gallegos, el método de Wilder y el aceite rojo “O”. Los hallazgos del presente trabajo revelaron la presencia de un hígado bilobulado, ubicado en la cavidad celómica caudal, con los  lóbulos derecho e izquierdo unidos por un istmo. El órgano se encuentra rodeado por una delgada cápsula de tejido conectivo, su parénquima se dispone en cordones tortuosos con un patrón acinar, con presencia de células pigmentadas compatibles con melanóforos, dispersas en el tejido hepático, y con gotas lipídicas intracitoplasmáticas de diversos tamaños, que en algunos casos ocupaban casi la totalidad del citoplasma de los hepatocitos.

The morphology, organization and intracytoplasmic components of the liver of the spectacled cayman (Caiman crocodilus crocodilus) are aspects that have been partially studied so far. A descriptive study of the morphological and histochemical characteristics of the liver of this subspecies was performed. This investigation would enable researchers to understand the liver functions in these animals, thus allowing future research using this organ as an experimental model. Eight liver samples were fixed in 10% v/v formalin and processed by conventional histology using routine hematoxylin and eosin staining. Additionaly, special stains, such as the Trichrome of Gallegos method and the red oil “O” and Wilder, were also used. The findings revealed the presence of a bilobed liver located in the midline of the caudal coelomic cavity, surrounded by a thin capsule of connective tissue; the parenchyma showed the layout of the classic hepatic lobule, the hepatocytes were arranged in tortuous cords and an acinar pattern. The presence of pigment-laden cells compatible with melanophores scattered in liver tissue was evidenced. Intracytoplasmic lipid droplets of various sizes occupying almost the entire cytoplasm of the hepatocytes were observed.

Immunoneutralization of inhibin in cycling rats increases follicle-stimulating hormone secretion, stimulates the ovary and attenuates progesterone receptor-dependent preovulatory luteinizing hormone secretion.

Passive immunization against inhibin with an anti-inhibin serum (AIS) during the diestrous phase in cycling rats increased follicle-stimulating hormone secretion, stimulated the ovaries and reduced the magnitude of the luteinizing hormone (LH) surge in the afternoon of proestrus. The involvement of gonadotrope progesterone receptor (PR) expression/action in the inhibitory effects of the follicle-stimulating hormone-dependent putative ovarian factor gonadotropin surge-attenuating factor on preovulatory LH secretion was studied in the absence of circulating free inhibin. Proestrous pituitaries from rats injected with AIS or a non-immune serum (NIS) were studied for determination of PR-AB and PR-B mRNAs by RT-PCR and PR-B and PR-A isoform proteins by Western blot. In addition, pituitaries from AIS- and NIS-injected rats were incubated and studied for PR-dependent LH secretion parameters: LH-releasing hormone (LHRH)-stimulated LH secretion, progesterone-potentiated LHRH-stimulated LH secretion and LHRH self-priming. Also, the effects of the antiprogestagen RU486 on these LH secretion parameters were evaluated and compared with those of AIS. Finally, gonadotrope PR phosphorylation was evaluated by immunohistochemistry. Results showed that the hyperstimulated ovaries of AIS-injected rats produce a factor, different from inhibin, that blocked LHRH self-priming and P-potentiation of LHRH-stimulated LH secretion. These effects were not due to decreased pituitary PR mRNAs, PR protein expression or PR protein B/A ratio. The inhibitory effect of AIS on PR-dependent LH secretion seemed to be due to gonadotrope PR dephosphorylation. Taken together, the findings indicated that the putative gonadotropin surge-attenuating factor affected LH surge through an inhibition of PR phosphorylation/action but not PR expression.

Activation of estrogen receptor-alpha induces gonadotroph progesterone receptor expression and action differently in young and middle-aged ovariectomized rats.

BACKGROUND: We attempted to define the effect of estrogen receptor (ER)alpha activation on gonadotroph progesterone receptor (PR) expression (mRNA and protein) and action (GnRH-stimulated and GnRH self-priming) in short- and long-term ovariectomized (OVX) rats. METHODS: Two weeks or 1 year after OVX, rats were injected over 3 days with 125 microg/kg of estradiol benzoate (EB), 7.5 mg/kg of the selective ERalpha agonist propylpyrazole triol (PPT), or 15 mg/kg of the selective ER modulator tamoxifen (TX). Controls were given 0.2 ml oil. The last day of ER analog treatment, half of the rats in each group received 25 mg/kg of progesterone (P). The next day, anterior pituitaries were removed and analyzed for PR-AB mRNA and protein. Gonadotrophin secretion in incubated pituitaries was also measured. RESULTS: (i) PR mRNA expression was higher in young than in middle-aged OVX rats although PR protein was absent in pituitaries from both groups of OVX rats; (ii) activation of ERalpha reduced gonadotroph hypertrophy and increased PR mRNA and protein expression (EB > PPT > TX) more efficiently in young than in middle-aged rats, (iii) ER agonists elicited GnRH-stimulated LH and FSH secretion in young but only FSH secretion in middle-aged OVX rats, (iv) evaluated by peak LH concentrations, GnRH self-priming was observed in both groups of OVX rats and (v) P down-regulated PR protein expression in young, and to a lesser extent, in middle-aged OVX rats, in close association with PR-dependent GnRH self-priming. CONCLUSIONS: Middle-aged OVX rats exhibited clear-cut LH, but not FSH, secretory defects in pituitary sensitivity to estrogen and P.

Anatomía del sistema biliar del hígado en el canino / Biliary system anatomy of the canine liver

Debido a que las enfermedades hepáticas y biliares representan un alto porcentaje del motivo de consulta en la clínica veterinaria se realizó un estudio descriptivo de la anatomía del sistema biliar del hígado en el canino, en un total de cuarenta (40) perros adultos. Los animales fueron heparinizados y anestesiados vía endovenosa, la eutanasia se practicó por desangramiento, los hígados de cada perro fueron perfundidos con acetato de vinil rojo, a nivel de la arteria hepática, azul por las venas porta y cava caudal y amarillo por el conducto biliar común. Los hígados se maceraron en agua por dos semanas obteniendo así los moldes de corrosión para estudiar el origen, trayecto y distribución de los conductos biliares. En 39 especímenes se observó la presencia de una vesícula biliar, con un tamaño variable en relación al tamaño del animal y solo en un especímen se encontró la presencia de dos vesículas biliares. Existen muchas variaciones en cuanto a la forma como los conductos hepáticos principales se unen al conducto cístico y en cuanto a la procedencia de los conductos que forman al conducto hepático común (colédoco), aunque sí se demuestra que es un solo conducto que llega al duodeno para drenar la bilis.

Because of the hepatic and biliary diseases represent a high percentage of the cause for veterinary consulting was accomplished a descriptive study of the biliary system anatomy of the liver in a total of forty (40) adult dogs. The animals were heparinized and anesthetized for intravenous route. The euthanasia was practiced by bleeding, the livers of each dog were injected with red vinyl acetate at level of the hepatic artery, blue vinyl acetate by the caudal cava and portal veins and yellow vinyl acetate by the common bile duct. The livers macerated in water by two weeks obtaining thus, the corrosion cast in order to study the origin, passage and distribution of the biliary ducts. In 39 specimens the presence of a gallbladder was observed, with a variable size in relation to the size of the animal and only in one specimen was the presence of two biliary vesicles. There are variations in the manner as main the hepatic ducts are joined to the cystic duct and of the origin of the ducts that form to the common hepatic (ductus choledochus), although was demonstrated that it is a single duct that arrives at the duodenum in order to drain the bile.

Occurrence and fate of alkylphenols and alkylphenol ethoxylates in sewage treatment plants and impact on receiving waters along the Ter River (Catalonia, NE Spain).

The partitioning of alkylphenols in the dissolved and particulate matter of influents, effluents, accumulation onto sludge and the impact of sewage treatment plant upon receiving waters was studied along the Ter River basin (Catalonia, NE Spain). A solid-phase extraction or pressurized liquid extraction followed by liquid chromatography-mass spectrometry was developed and permitted to determine target compounds with high efficiency in waters, particulate material and sludge. Nonylphenol mono- and diethoxylate, nonylphenol and octylphenol partitioned preferably upon particulate matter and sludge, whereas long chain NPE(3-15)O prevailed in the dissolved phase and was released by effluents. Within the treatment process, a net accumulation of alkylphenols in sludge was found, producing up to 148g/t/month. The removal efficiency of alkylphenols was of 37-90% and depended on the treatment. Assessment on the fate of these contaminants within STPs is discussed in terms of flow rates, biological oxygen demand and tons of sludge produced.

Irrigación arterial del ovario en la oveja y la vaca durante el ciclo estral / Arterial blood supply of ovaries in ewes and cows during estrous cycle

Las diferencias de la anatomía vascular de los ovarios se han asociado a diferencias significativas en las respuestas fisiológicas de estos órganos durante el ciclo estral. El objetivo de esta investigación fue describir comparativamente el patrón vascular arterial del ovario en oveja y vaca durante el ciclo estral en las diferentes especies. La irrigación arterial del ovario de 18 ovejas y 10 vacas reproductivamente maduras, durante las diferentes fases del ciclo estral, se evaluó a través de disección simple, clarificación de tejidos, moldes de corrosión, microscopía óptica, microscopía electrónica y arteriograma. Los resultados mostraron que las arterias ováricas penetraban en número de dos, en la zona vascular del órgano, formando un complejo ovillo arterial muy tortuoso. En cambio en la vaca, las arterias ováricas penetraban al órgano en número de cinco o seis, sin formar ningún ovillo. Esto sugiere que la oveja tiene un mayor aporte sanguíneo al ovario en comparación con la vaca. Se observó una red arterial con origen en la arteria uterina y ovárica en la superficie de la vena uterina en ambas especies. Durante las fases del ciclo estrual se observaron diferencias en la irrigación del ovario. En ovarios de animales en el día 7 del ciclo estral, con cuerpos lúteos, se evidenció una irrigación bien definida a diferencia de los ovarios con cuerpos lúteos a los 13 días del ciclo estral (oveja) o a los 17 del ciclo estral (vaca), en los cuales las arterias disminuían tanto en número como en diámetro.

Differences in ovarian vascular anatomy have been associated to significant differences in physiologic responses of ovaries during estrous cycle in different species. The objective of this study was to describe on a comparative basis the ovarian arterial vascular pattern from 18 ewes and 10 cows in reproductive age, during the different stages of the estrous cycle. The above mentioned pattern was evaluated through simple dissection, tissue clarification, corrosion templates, optic microscopy, electron microscopy and arteriograms. Results showed that in ewes, two ovarian arteries entered into the ovarian vascular zone, forming a very contorted and complex arterial mesh (glomus). By contrast, in cows five o six, ovarian arteries entered into the ovaries, without forming mesh. This suggests that ewes might have a better blood supply to the ovary than cows. A network was observed originating from uterine and ovarian arteries on the surface of uterine vein in both species. Differences in ovarian blood supply were seen during the different stages of estrous cycle. In animals having corpora lutea at 7 days of the estrous cycle was noticed a well defined ovarian blood supply, whereas in animals at 14 (ewes) or 18 (cows) post-heat, having corpora lutea as well, ovarian arteries diminished in size and number.

Irrigación arterial y venosa del útero y los ovarios de la perra (Canis familiaris) y su relación con la actividad ovárica / Arterial and venous supply of the uterus and ovaries in the bitch (Caninis familiaris) and its relationship with ovarian activity

Con el objetivo de describir las caracetrísticas anatómicas de la vascularización arterial y venosa del útero y los ovarios, el ciclo estral de nueve perras fue monitoreado utilizando citologías vaginales y ultrasonografía para inferir el mecanismo por el cual el útero ejerce su efecto sobre el ovario mediante técnicas radiográficas, clarificación de tejidos y maceración tisular. El estudio comprobó que en todas las fases del ciclo estral, el útero y los ovarios de la perra están irrigados principalmente por las arterias ovárica, uterina y vaginal, las cuales establecen anastomosis ipsilaterales entre ellas y contralaterales entre las arterias uterinas y vaginal. En el ligamento ancho, la vena uterina forma un circuito de dos ramas que se anastomosan craneal y caudalmente para permitir la conexión venosa entre el ovario y el cuerno ipsilateral al mismo. Pudo observarse a simple vista que en los especímenes provenientes de perras en fases de proestro y estro, el número de grosor de los vasos sanguíneos que irrigan el tracto reproductor es mayor en comparación con los especímenes provenientes de perras en fases de diestro y anestro. Estos estudios sugieren la necesidad de realizar investigaciones más avanzadas con el uso de hormonas marcadas para dilucidar el mecanismo de luteólisis en esta especie.

Chemical monitoring and occurrence of alkylphenols, alkylphenol ethoxylates, alcohol ethoxylates, phthalates and benzothiazoles in sewage treatment plants and receiving waters along the Ter River basin (Catalonia, N. E. Spain).

This study presents a quantitative estimation of the analysis and fate of several emerging pollutants, some of them endocrine-disrupting compounds, in surface water samples collected at several locations along the Ter River and two of its tributaries. Influent and effluent waters and particulate matter from five sewage treatment plants (STP) that discharge into these rivers were also studied. The target compounds analyzed were: nonylphenol ethoxylates (NPEO), nonylphenol (NP), octylphenol (OP), bisphenol A (BPA), phthalates, alcohol ethoxylates (AEO) and benzothiazoles. Chemical analysis by liquid chromatography-mass spectrometry using an electrospray interface (LC-ESI-MS) revealed the presence of low amounts (between 0.06 and 17.5 microg L(-1)) of the target compounds NPE(1+2)O and NP, which were detected in 100% and 84% of the samples respectively. Maximum concentrations occurred in the STPs associated with the municipalities of Vic and Girona. From the fate and behavior data obtained for the various compounds analyzed in the STP influent and effluent, we can conclude that the STPs are effective at removing large amounts (more than 70%) of the compounds studied from the water.

Distribution of endocrine disruptors in the Llobregat River basin (Catalonia, NE Spain).

The Llobregat basin is a Mediterranean fluvial system with major agricultural, urban and industrial impacts. We combined chemical quantification by liquid chromatography-mass spectrometry with electrospray interface (LC-ESI-MS) and the recombinant yeast assay (RYA) assays to estimate the loads of endocrine disrupting compounds (EDCs) along the basin. Chemical analysis revealed maximum concentrations (at microg l(-1) level) of alkylphenols at the lower course of the Llobregat River, which correlated with high levels of estrogenic activity detected by RYA. Analysis by RYA and LC-ESI-MS of influent and effluent waters from four sewage treatment plants (STP) discharging into the basin showed the removal of 80-95% of EDCs by STP treatment. Chemical analysis data and RYA data showed a quasi-linear correlation, demonstrating the complementariness of both methods. Our data suggest that the concentrations of the analysed compounds were enough to explain the total estrogenicity of water and STP samples from the Llobregat basin.