Pathogenesis of the crosstalk between reproductive function and stress in animals - part 2: Prolactin, thyroid, inflammation and oxidative stress.

Stress has a significant impact on reproductive health and fertility in both humans as well as various animal species. In particular, chronic stress can disrupt the delicate balance of the hormonal system that regulates reproductive function, leading to a variety of reproductive disorders and fertility issues. Beside the action of the hypothalamic-pituitary-adrenal (HPA) system and the sympatho-adrenomedullary system (SAM), other subsequent mechanisms have been incriminated. Thus, stress has also been associated with increased prolactin level, resulting in an inhibition of the hypothalamo-pituitary-gonadal (HPG) system leading to several reproductive disorders. Thyroid function is inhibited during chronic stress, and therefore considered an important regulator of reproductive function. Thus, and in particular by interfering with the HPA system, stress-induced immune dysregulation can have adverse effects on reproduction. In addition, oxidative stress and inflammation have been proposed as potential mechanisms by which chronic stress affects reproductive function. This is caused by an increase in reactive oxygen species (ROS) production that has a harmful effect on cells. Furthermore, inflammation can lead to tissue damage and scarring, which can affect fertility. The present review completes the complex mechanism linking stress and reproduction through the current knowledge in various animal species in a comparative point of view.

Pathogenesis of the crosstalk between reproductive function and stress in animals-part 1: Hypothalamo-pituitary-adrenal axis, sympatho-adrenomedullary system and kisspeptin.

Stress is defined as a disruption of the body homeostasis in response to modest as well as perceived challenge. Two main physiological routes, the hypothalamic-pituitary-adrenal system (HPA) and the sympatho-adrenomedullary system (SAM), aim to maintain or restore homeostasis by mutual interaction. SAM is quickly-reacting as it primarily works through the nervous system-the sympathetic nervous system. In response to stress, signals are sent to activate the adrenal medulla which releases catecholamines (primarily adrenaline and norepinephrine). The catecholamines have a momentary effect on the body's organs that are prepared for a fight situation. At the same time, the stressor activates the HPA axis by signals from the brain causing secretion of the pituitary hormone adrenocorticotropic hormone (ACTH). ACTH acts on the adrenal cortex, which secretes glucocorticoids, including cortisol. Since HPA primarily works through hormones, the system is slightly slower than SAM and gives rise to a metabolic effect. While short-term stress response is an adaptive and beneficial process, chronic or excessive stress can lead to a range of negative health outcomes including reproductive disorders and infertility. Several mechanisms have been proposed to explain the link between stress and reproduction. This includes in particular kisspeptin, which is closely related to reproduction, as it is a powerful stimulator of the Hypothalamic-pituitary-gonadal (HPG) system. The present review, through current knowledge in various male and female species, deals with the role of the SAM and the HPA, including the major action of kisspeptin and glucocorticoids that trigger the consequences of psychological or physiological stress on reproductive function.

Metabolomics and lipidomics to identify biomarkers of effect related to exposure to non-dioxin-like polychlorinated biphenyls in pigs.

Recent epidemiological studies show that current levels of exposure to polychlorinated biphenyls (PCBs) remain of great concern, as there is still a link between such exposures and the development of chronic environmental diseases. In this sense, most studies have focused on the health effects caused by exposure to dioxin-like PCBs (DL-PCBs), although chemical exposure to non-dioxin-like PCB (NDL-PCB) congeners is more significant. In addition, adverse effects of PCBs have been documented in humans after accidental and massive exposure, but little is known about the effect of chronic exposure to low-dose PCB mixtures. In this work, exposure to Aroclor 1260 (i.e. a commercially available mixture of PCBs consisting primarily of NDL-PCB congeners) in pigs is investigated as new evidence in the risk assessment of NDL-PCBs. This animal model has been selected due to the similarities with human metabolism and to support previous toxicological studies carried out with more frequently used animal models. Dietary exposure doses in the order of few ng/kg body weight (b.w.) per day were applied. As expected, exposure to Aroclor 1260 led to the bioaccumulation of NDL-PCBs in perirenal fat of pigs. Metabolomics and lipidomics have been applied to reveal biomarkers of effect related to Aroclor 1260 exposure, and by extension to NDL-PCB exposure, for 21 days. In the metabolomics analysis, 33 metabolites have been identified (level 1 and 2) as significantly altered by the Aroclor 1260 administration, while in the lipidomics analysis, 39 metabolites were putatively annotated (level 3) and associated with NDL-PCB exposure. These biomarkers are mainly related to the alteration of fatty acid metabolism, glycerophospholipid metabolism and tryptophan-kynurenine pathway.

Evaluation of the potential benefits of iron supplementation in organic pig farming.

Background: Iron from the stock acquired during foetal life and the ingestion of milk is not sufficient to cover the needs of the piglets during their first weeks of life. In organic farming, systematic supplementation with iron is problematic due to a strong limitation in pharmaceutic treatments. Methods: Erythroid parameters around weaning were measured in piglets from organic outdoor and indoor farms, and related to indicators of the inflammatory status. Blood samples were collected from 28.9±2.6 piglets/herd at 42.0±3.2 days of age and 11.9±3.0 kg live weight (mean ± SD) in 21 farms from the west part of France. Among the 11 outdoor farms, only one had supplemented piglets with 200 mg iron while among the 10 indoor farms, only one had not supplemented piglets, one had supplemented them with 100 mg, 8 with 200 mg and one with 400 mg. Results: Compared to outdoor piglets without supplementation, piglets kept indoors and receiving 200 mg iron had lower haemoglobin concentration (105 vs 118±2 g/l, mean ± SE) and red blood cell volume (56 vs 60±1 fl) (P<0.005). The reduction in haemoglobin concentration and red blood cell volume was more pronounced in indoor piglets supplemented with 100 mg of iron and even more when they had not received iron. The plasma concentration of haptoglobin was lower in outdoor than in indoor piglets (0.51±0.06 vs 0.78±0.09 g/l) whereas no effect of housing was observed for markers of oxidative stress (dROM, BAP). In the 14 farms where sow parity was known, the haemoglobin concentration was lower in piglets from primiparous than from multiparous sows (109 versus 114±2 g/l, P < 0.001). Conclusion: With the exception of soils where the content of bioavailable iron is very low, piglets from outdoor farms do not require iron supplementation, unlike those raised indoors.

Inflammatory and oxidative status in European captive black rhinoceroses: A link with Iron Overload Disorder?

Iron Overload Disorder (IOD) is a syndrome developed by captive browsing rhinoceroses like black rhinoceroses (Diceros bicornis), in which hemosiderosis develops in vital organs while free iron accumulates in the body, potentially predisposing to various secondary diseases. Captive grazing species like white rhinoceroses (Ceratotherium simum) do not seem to be affected. The authors hypothesized that inflammation and oxidative stress may be implicated in the pathogenesis of IOD in captive black rhinoceroses, making this syndrome a potential common denominator to various diseases described in captivity in this species. In this prospective study, 15 black (BR) and 29 white rhinoceroses (WR) originating from 22 European zoos were blood-sampled and compared for their iron status (serum iron), liver/muscle biochemical parameters (AST, GGT, cholesterol), inflammatory status (total proteins, protein electrophoresis) and oxidative stress markers (SOD, GPX, dROMs). Results showed higher serum iron and liver enzyme levels in black rhinoceroses (P < 0.01), as well as higher dROMs (P < 0.01) and a trend for higher GPX (P = 0.06) levels. The albumin/globulin ratio was lower in black rhinoceroses (P < 0.05) due to higher α2-globulin levels (P < 0.001). The present study suggests a higher inflammatory and oxidative profile in captive BR than in WR, possibly in relation to iron status. This could be either a consequence or a cause of iron accumulation. Further investigations are needed to assess the prognostic value of the inflammatory and oxidative markers in captive black rhinoceroses, particularly for evaluating the impact of reduced-iron and antioxidant-supplemented diets.

Plasma cell leukemia with plasmablastic morphology in a dog.

A 5-y-old female Golden Retriever was presented with a 2-wk history of hyporexia, vomiting, diarrhea, lethargy, weight loss, polyuria, and polydipsia. Clinical examination and ultrasonography revealed multiple organ enlargement with gallbladder and kidney nodules suggestive of disseminated neoplasia. Hematologic and biochemical analyses revealed pancytopenia, hypercalcemia, and monoclonal IgA gammopathy suspicious for a plasma cell neoplasm. Bone marrow and blood smear examination revealed neoplastic atypical cells highly suggestive of lymphoid origin. Autopsy confirmed the presence of homogeneous white masses and multifocal pale infiltrates in the spleen, kidney, small intestine, gallbladder, and urinary tract. Histologic features were consistent with a multicentric atypical plasma cell tumor. Tumor cells were negative for CD204, IBA-1, E-cadherin, CD3, CD5, CD79a, CD20, and PAX5, and positive for MUM1, consistent with plasma cell origin. The presence of > 20% of circulating blastic plasma cells was consistent with primary plasma cell leukemia with plasmablastic morphology, a disease rarely described in veterinary medicine.

Thrombocytosis and central nervous system involvement in a case of canine acute megakaryoblastic leukemia.

This case report presents a 14-month-old female Poodle mix with acute megakaryoblastic leukemia based on a marked thrombocytosis, abnormal platelet morphology, circulating dwarf megakaryocytes, and blast cells in the blood. Bone marrow abnormalities included dysmegakaryopoiesis dygranulopoiesis, and an increased number of blast cells was observed in the blood. Extensive leukemic involvement was also found in the liver, spleen, lymph nodes, lungs, kidneys, and brain. The cytopathologic features of the abnormal circulating cells were highly suggestive of being megakaryocytic in origin, which was supported by negative myeloperoxidase staining and positive von Willebrand factor staining on immunocytochemistry (ICC). The neoplastic cells were also CD61 positive and had variable von Willebrand factor expression on ICC. Although there were only 25% blast cells in the bone marrow, which theoretically supported myelodysplastic syndrome, the hypothesis that this case represented acute myeloid leukemia of megakaryoblastic origin was confirmed by the continuous increase in circulating blast cell numbers during follow-up visits and the extensive leukemic involvement of parenchymal organs.

Insulin-like growth factor type 1 concentrations in hyperthyroid cats before and after treatment with thiamazole.

Objectives The aim of this study was to evaluate changes in circulating insulin-like growth factor type 1 (IGF-1) concentrations in hyperthyroid cats, before and after thiamazole treatment. Methods Thirty-four hyperthyroid cats were retrospectively included. Plasma free thyroxine (fT4) and IGF-1 concentrations were measured at diagnosis and 3 months after initiating antithyroid drug therapy. Results Median fT4 significantly decreased ( P <0.001) after treatment (from 78 pmol/l [range 43-122 pmol/l] to 31 pmol/l [range below assay limit of detection to 88 pmol/l]), whereas IGF-1 values significantly increased ( P <0.001) after treatment (from 117 ng/ml [33-341 ng/ml] to 221 ng/ml [36-865 ng/ml]). fT4 and IGF-1 concentrations were significantly negatively correlated both at diagnosis (r = -0.43, P = 0.01) and after treatment (r = -0.51, P = 0.002). Conclusions and relevance In cats, IGF-1 concentrations appear to be inversely proportional to the severity of hyperthyroidism and significantly increase after treatment with thiamazole.

Canine invasive mammary carcinomas as models of human breast cancer. Part 2: immunophenotypes and prognostic significance.

PURPOSE: Relevant animal models of human breast cancer are currently needed, especially for the aggressive triple-negative breast cancer subtype. Recent studies and our results (Part 1) indicate that spontaneous canine invasive mammary carcinomas (CMCs) resemble human breast cancer by clinics and pathology as well as behavior and prognostic indicators. We hypothesized that the current molecular classifications of human breast cancer, used for therapeutic decision, could be relevant to dogs. METHODS: Three hundred and fifty female dogs with spontaneous CMC and a 2-year follow-up were retrospectively included. By immunohistochemistry, CMCs were classified according to Nielsen (Clin Cancer Res 10:5367-5374, 2004) and Blows (PlosOne doi: 10.1371/journal.pmed.1000279, 2010) into the subtypes of human breast cancer. RESULTS: Four immunophenotypes were defined either according to Nielsen classification (luminal A 14.3%, luminal B 9.4%, triple-negative basal-like 58.6%, and triple-negative nonbasal-like 17.7% CMCs); or to Blows classification (luminal 1-: 11.4%, luminal 1+: 12.3%, Core basal phenotype: 58.6%, and five-negative phenotype: 17.7%). No HER2-overexpressing CMC as defined by a 3 + immunohistochemical score was observed in our cohort. By univariate and multivariate analyses, both immunophenotypical classifications applied to CMCs showed strong prognostic significance: luminal A or luminal 1+ CMCs showed a significantly longer disease-free interval (HR = 0.46), Overall (HR = 0.47), and Specific Survival (HR = 0.56) compared to triple-negative carcinomas, after adjustment for stage. CONCLUSIONS: In our cohort, triple-negative CMCs largely predominated (76%), were much more prevalent than in human beings, and showed an aggressive natural behavior after mastectomy. Dogs are thus potent valuable spontaneous models to test new therapeutic strategies for this particular subtype of breast cancer.

The dog as a naturally-occurring model for insulin-like growth factor type 1 receptor-overexpressing breast cancer: an observational cohort study.

BACKGROUND: Dogs spontaneously develop invasive mammary carcinoma with a high prevalence of the triple-negative (TN) subtype (lack of ER-Estrogen Receptor and PR-Progesterone Receptor expression, lack of HER2-Human Epidermal Growth Factor Receptor 2 overexpression), making this animal model relevant for investigating new therapeutic pathways. Insulin-like growth factor Type-1 receptor (IGF1R) is frequently overexpressed in primary human breast cancers, with a growing role in the TN phenotype. The purpose of this study was to investigate the Dog as a candidate model for IGF1R-overexpressing mammary carcinoma. METHODS: 150 bitches with canine mammary carcinoma (CMC) and a known 2-year follow-up were retrospectively included. IGF1R expression was assessed by immunohistochemistry (IHC) using a similar scoring system as for HER2 in breast cancer. The prognostic value of the IGF1R expression was assessed in terms of overall and specific survival as well as disease-free interval (DFI). RESULTS: 47 CMC (31 %) were classified as luminal and 103 (69 %) as triple-negative (TN-CMC). 41 % of CMC overexpressed IGF1R (IHC score 3+) of which 76 % were TN-CMC and 62 % grade III. IGF1R overexpression was associated with aggressive features including lymphovascular invasion, histological grade III, low ER expression and the TN phenotype. Univariate and multivariate analyses revealed that IGF1R overexpression was associated with shorter overall and specific survivals and shorter DFI in TN-CMC. CONCLUSIONS: IGF1R overexpression is common and related to a poor outcome in canine invasive mammary carcinoma, particularly in the triple negative subtype, as in human breast cancer. Preclinical studies using the Dog as a spontaneous animal model could be considered to investigate new therapies targeting IGF1R in triple-negative breast cancer.

Shunting between the CVC and both the azygos vein and thoracic duct in a dog with CTD.

A 5 mo old female rottweiler was referred for evaluation of a suspected congenital heart disease. Clinical signs included anorexia, exercise intolerance, and severe loss of body condition. Clinical examination revealed dyspnea, pale mucous membranes, and weak femoral pulses. Pleural and abdominal effusions and iron deficiency anemia were identified. A distended intrathoracic caudal vena cava (CVC) visible on thoracic radiographs suggested that the modified transudate abdominal effusion was the result of improper venous return to the right side of the heart. Cor triatriatum dexter (CTD) was diagnosed via echocardiography but did not explain all the anomalies detected during a contrast echocardiography. Abnormal communications between the CVC and azygos vein and the CVC and thoracic duct were subsequently identified by abdominal ultrasonography and angiography. Medical management with diuretics, iron supplements, and surgical treatment of CTD resulted in normalization of the respiratory rate, the exercise intolerance, and the anemia. To the authors' knowledge, this is the first reported case of CTD associated with shunts between the CTV and both the azygos vein and thoracic duct in dogs. This report emphasizes the importance of presurgical assessment of concurrent thoracic and abdominal congenital vascular abnormalities.

Mammary gland carcinoma in a dog with peripheral blood and bone marrow involvement associated with disseminated intravascular coagulation.

A 7-year-old female Leonberger dog was referred to the National Veterinary School of Lyon Teaching Hospital with a 2-day history of anorexia and bleeding. A mammary mass had been removed 7 months earlier, but histologic examination was not performed. On physical examination, the dog was depressed and had pale mucous membranes and numerous petechiae and hematomas. Significant laboratory findings were moderate thrombocytopenia, prolonged prothrombin, activated partial thromboplastin, and thrombin times, hypofibrinogenemia, and increased concentration of fibrin(ogen) degradation products. A peripheral blood smear, buffy coat preparation, and bone marrow aspirate contained low numbers of large atypical cells that had moderate nuclear:cytoplasmic ratios, oval nuclei with multiple prominent nuclei, and basophilic cytoplasm with villous projections. A small nodule was found in the left inguinal mammary gland, and a fine-needle aspirate contained cells similar to those in blood and bone marrow. In samples of blood, bone marrow, and the mammary mass, the neoplastic cells were immunoreactive for cytokeratin. The diagnosis was mammary carcinoma with secondary disseminated intravascular coagulation (DIC) and disseminated tumor cells in bone marrow and circulating tumor cells in blood; this diagnosis was not confirmed by histopathologic examination. Owing to clinical deterioration and the poor prognosis, the dog was euthanized and a necropsy was not performed. This is the first report of a canine mammary carcinoma with circulating tumor cells and secondary DIC.

Waldenström's macroglobulinemia in a dog with a bleeding diathesis.

An 11-year-old female spayed Whippet dog was referred to the Veterinary Hospital of the National Veterinary School of Lyon with a 3-month history of chronic bleeding, cutaneous masses suggestive of hematomas, and hemorrhagic diarrhea. Laboratory abnormalities included leukopenia with neutropenia and lymphopenia, rouleaux formation, marked hyperproteinemia with hyperglobulinemia and normoalbuminemia, hypercalcemia, markedly increased serum urea concentration, mildly increased creatinine concentration, and proteinuria. Hemostatic screening tests showed marked prolongation of thrombin time, increased concentrations of fibrin(ogen) degradation products and D-dimers, and slightly prolonged activated partial thromboplastin time. Serum and urine protein electrophoresis revealed a monoclonal peak identified as IgM by immunoelectrophoresis and an M-spike, respectively. Cytologic evaluation of bone marrow revealed a hypocellular sample with lymphoplasmacytic cells comprising approximately 50% of all nucleated cells. These findings were consistent with a diagnosis of Waldenström's macroglobulinemia with secondary bleeding diathesis. Treatment with prednisone and chlorambucil was initiated, and the dog had a good response.

Serum insulin-like growth factor type 1 concentrations in healthy dogs and dogs with spontaneous primary hypothyroidism.

Circulating insulin-like growth factor type 1 (IGF-1) concentrations in dogs have been correlated with standard breed bodyweight (SBBW or breed size). Thyroid and somatotropic functions, which have common effects and regulatory mechanisms, were investigated in hypothyroid dogs. IGF-1 was measured in 495 adult healthy dogs (N) and in 220 primary hypothyroid dogs (HOT) with clinical and biological signs of primary hypothyroidism. IGF-1 was determined as a function of SBBW (kg): ≤15 (group A); 1540 (group D). In HOT dogs, median fT4 and c-TSH values were 9pmol/L and 1.5ng/mL, respectively. A significant correlation between bodyweight (BW) and IGF-1 was observed in both HOT and N dogs. The median IGF-1 value (ng/mL) was significantly higher (P<0.01) in HOT dogs compared to N in groups B, C and D (230 vs. 182; 316 vs. 230; 606 vs. 306 respectively). In conclusion, IGF-1 concentration should be interpreted in the context of SBBW in dogs and increases in spontaneous primary hypothyroidism. However, it remains unclear if this association is directly due to hypothyroidism or is the result of the weight gain accompanying hypothyroidism.

Castration of horses under total intravenous anaesthesia: analgesic effects of lidocaine.

OBJECTIVE: To evaluate the effects of local anaesthesia with lidocaine for castration of horses under intravenous anaesthesia. STUDY DESIGN: Prospective, randomized, blinded clinical trial. ANIMALS: Fifteen equidae, scheduled to undergo castration under total intravenous anaesthesia, were randomly distributed in two groups. One group received lidocaine injections (group L: two ponies, four horses, two donkeys) and the other received saline (group S: two ponies, three horses, two donkeys). METHODS: Behaviour, heart rate (HR) and respiratory rate (f(R)) were evaluated prior to anaesthesia. Body mass was measured using an electronic scale and testicular volumes were estimated. The animals were anaesthetized with acepromazine intramuscularly and romifidine intravenously followed 10 minutes later by ketamine. Following romifidine administration lidocaine or saline was administered subcutaneously along the incision line and by intratesticular and intrafunicular injection. Based on clinical observations (movement, f(R), and cranial nerve reflexes) incremental intravenous doses of ketamine and romifidine were administered. HR, f(R), oscillometric mean arterial blood pressure (MAP), duration of surgery, movement and additional doses were recorded. Surgical conditions were assessed using a visual analogue scale (VAS) and a simple descriptive scale (SDS). Recovery was assessed by two assistants, unaware of treatment, acting separately using a VAS and a SDS. Group means were compared using Mann-Whitney and Wilcoxon tests and the Kruskal-Wallis signed rank test for matched pairs used to compare groups at different points (p < 0.05). RESULTS: The number (median, range) of incremental doses (4 [1-5] compared to 1.5 [1-4]) and movements (1 [1-5] compared to 0 [0-1]) were higher (p = 0.01 for both) in the control group than in the lidocaine group. Groups were similar for other recorded variables. CONCLUSIONS AND CLINICAL RELEVANCE: These results show the effectiveness of lidocaine used as a local anaesthetic adjunct to intravenous anaesthesia in horses undergoing castration.