Interaction of oxytetracycline and bacterial collangenase in the treatment of bovine retained placenta

Retained fetal membranes (RFM) is a postpartum disease that can negatively affect fertility and milk production in the dairy cow population. This disease predisposes the cow to postpartum metritis. Injections of bacterial (Clostridium histolyticum) collagenase have been shown to be highly effective in detaching RFM. Therefore, a combination therapy for RFM and prevention of metritis was hypothesized. This involved mixing collagenase with oxytetracycline, an antibiotic used frequently to treat metritis. However, it has been reported that tetracyclines inhibit collagenase activity. Inactivation of collagenase by oxytetracycline needed to be determined. To test for inactivation of collagenase, isolated bovine placentomes (metabolically active) were infused via umbilical vessels with a mixture of collagenase and oxytetracycline. Eight experimental groups were used: group 1, saline controls; group 2, collagenase 1200 units (U)/ml; groups 3, 4, and 5 contained 1200 U/ml of collagenase plus oxytetracycline (1 mg/ml, 0.1 mg/ml, 0.01 mg/ml, respectively); groups 6, 7, and 8 contained oxytetracycline at 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml, respectively. After 5 h of incubation in a warm (39°C) water bath, manometric pressure (pressure needed to separate cotyledon from caruncle), hydroxyproline (collagenolysis), and total protein (proteolysis) were determined. The antitumor antibiotic doxorubicin was used as a positive control for oxytetracycline. Doxorubicin has been reported to inhibit bacterial (C. histolyticum) collagenase. Experiments with doxorubicin were performed the same as experiments with oxytetracycline except that six groups were used instead of eight. Group 1, saline; group 2, collagenase 1200 U/ml; groups 3, 4, and 5 contained 1200 U/ml of collagenase plus doxorubicin (0.04 mg/ml, 0.02 mg/ml, and 0.01 mg/ml, respectively); group 6 contained doxorubicin at 0.02 mg/ml. It was also the purpose of this study to determine if oxytetracycline residues were present in milk and blood of cows given umbilical injections of collagenase (2 x 105 U/L saline) plus oxytetracycline (0.1 g). This combination therapy was administered to cows that had retained fetal membranes after natural delivery and to cows that had undergone cesarean sections. Samples of blood and milk (when available) were obtained and analyzed for oxytetracycline residues using Bacillus stearothermophilus disc assay. It was concluded that neither oxytetracycline nor doxorubicin inhibited collagenase in the bovine placentome. All milk and blood (plasma) samples tested negative for oxytetracycline residues

The role of fetal serotonin (5-HT) in bovine placenta detachment

The enzyme collagenase is involved in degrading uterine collagen during postpartum involution. It has been reported that cultures of rat and human myometrial smooth muscle cells produce collagenase when grown in medium containing fetal bovine serum (FBS) but not in medium containing newborn bovine serum (NBS). The substance in FBS that induces the cells to produce collagenase is 5-hydroxytryptamine (5-HT, serotonin). The concentration of 5-HT in FBS is between 30 µM-SG µM. In contrast, NBS has a concentration of approximately 1 µM or less. It has been proposed that 5- HT serves as a signal to initiate uterine collagen hydrolysis. Furthermore, a 5-HT transporter exists in the mouse and human placenta. The function of the transporter is not known, however it is speculated that it may act in growth and development of the fetus. Five-HT is known to act as a growth factor for many cell types yet its effect on placental cells is not known. In addition, the source of 5-HT found in placental tissue is unclear. It may be that the source for placental 5-HT is the fetal intestine since 95% of the body\u27s 5-HT is located in the gastrointestinal tract and intestinal resection causes a drop in blood concentration of 5-HT. The working hypothesis of this research was that 5-HT from the fetal intestine is a proliferation factor that supports placental cell growth and inhibits activity of matrix metalloproteinases (MMPs) during pregnancy. It is suspected that withdrawal of fetal 5- HT during partum causes placenta separation by cessation of cell proliferation and stimulation of MMP secretion. The objectives of this research were to: determine 5-HT concentrations in blood and tissues of bovine fetuses and neonates; determine whether or not 5-HT acts as a proliferation factor for cultured placental cells; determine whether 5- HT stimulates or inhibits placental MMP activity in cultured cells, isolated placentomes, and pregnant cows; and immunolocalize 5-HT and collagenase in placental tissue. Blood, intestine, cotyledon, caruncle, and muscle were collected from mid-term and full-term gestation fetuses at an abattoir and from 24 hour and 48-72 hour old calves obtained from The University of Tennessee Dairy Farm. Blood was collected from the umbilical cord of cesarean section fetuses and from pregnant and non-pregnant cows. Samples were analyzed using an enzyme immunoassay procedure. Fetal blood 5-HT concentrations remained elevated from mid (54,111 nM) through full (51,640 nM) pregnancy, declined (P %le; 0.05) during delivery (13,625 nM), and stayed low in 24 hour old calves (24,460 nM), 48-72 hour old calves (18,400 nM), and in cows (8,004 nM). Five-HT concentration in intestine of mid (5,321 ng/g) and full (7,059 ng/g) gestation fetuses and 48-72 hour old calves (6,618 ng/g) was significantly higher (P ≤ 0.05) than 24 hour old calves (1,410 ng/g) and cows (3,049 ng/g). Concentration of 5-HT in placenta of mid (4,570 ng/g) and full (5,788 ng/g) gestation fetuses was significantly higher (P ≤ 0.05) than postpartum placenta (1,176 ng/g). Concentration of 5-HT in muscle of mid gestation fetuses (1,412 ng/g) and 24 hour old calf (1,759 ng/g) were significantly lower (P ≤ 0.05) than in full gestation fetuses (4,941 ng/g). Cotyledon and caruncle cells were cultured from primary explants of cow placenta. Ten thousand cells were plated in each well of a 96-well plate. Cells were either used as control or treated with 5-HT ranging in concentration from 2.5 µM to 10 µM. The proliferative effect of 5-HT was determined by incorporation of 3H-thymidine into DNA of cells, a tetrazolium-based colorimetric proliferation assay, and cell count. Increasing concentrations of 5-HT were shown to stimulate incorporation of ^H-thymidine into DNA of cells; however, 5-HT was inhibitory of the reaction in the colorimetric proliferation assay. Five-HT had no effect on cell number when counted on a hemacytometer or by a Coulter counter. Cultured cotyledon and caruncle cells were grown in serum-free medium, and medium supplemented with either FES or NBS and stimulated with 5 µM 5-HT. Media samples were analyzed for MMP activity using a fluorometric technique. Five-HT did not stimulate MMP activity; however, when all 5-HT samples were pooled (2.05 nM), there was significant (P ≤ 0.05) inhibition of MMP activity compared to control (3.81 nM). Isolated placentomes were: (1) perfused for 4 hours with blood containing 50 ≤M 5-HT then incubated for 4 hours with 5-HT, and (2) infused with 5 µM 5-HT and incubated for 4 hours to 11 hours. Matrix metalloproteinase activity was determined using a manometric technique and hydroxyproline and total protein analysis. There were no differences (P ≥ 0.05) in manometric pressure (force needed to separate cotyledon from caruncle) between control (118 mm Hg) and 5-HT (116 mm Hg) treated placentomes nor in the amount of total protein released in control (1.70 mg/dL) and 5- HT (1.61 mg/dL) treated placentomes. However, amount of hydroxyproline released was discretely higher (P ≤ 0.05) from 5-HT (2.06 µg/mL) treated placentomes than from controls (1.57 µg/mL). Fourteen near-term cows were induced to deliver with injections of dexamethasone and PGF2α. These injections also cause approximately 70% of cows to retain their placenta. Twenty-four hours later, 7 cows were injected with 50 µg/kg of 5-HT and 7 cows were injected with saline every 12 hours until delivery or for a total of 3 days. In each group, all but 1 cow retained placenta. Results of these experiments indicated that 5-HT does not stimulate MMP activity in the induced model. Cotyledons and caruncles from mid and full gestation and naturally delivered placentae were fixed for light microscopy and electron microscopy study. Using light microscopy, 5-HT was localized to the surface membrane of epithelial cells and connective tissue of prepartum cotyledons and caruncles and postpartum cotyledons. Using electron microscopy, 5-HT was localized in close proximity to collagen fibers of postpartum cotyledons. We were unable to localize collagenase in the bovine placenta. The results of this research support the following; (1) a pattern of 5-HT concentration change during pregnancy and parturition; (2) 5-HT may be a proliferation factor for placenta; (3) 5-HT did not stimulate MMP activity in placenta; (4) 5-HT is present in the bovine placenta during pregnancy and postpartum

Hematology, plasma biochemistry, and hormonal analysis of captive Louisiana pine snakes (Pituophis ruthveni): effects of intrinsic factors and analytical methodology

Blood analyte data are useful in health assessments and management of reptiles. There is a knowledge gap for blood analyte data of the endangered Louisiana pine snake (LPS; Pituophis ruthveni). The objectives of this study were to provide baseline hematology, plasma biochemical, and hormone data of captive LPS, to compare the data in juvenile and adult snakes and in adult snakes by sex, and to investigate methodological differences in hormone (serum vs. plasma) and protein analyses (total solids versus total protein). Blood samples from apparently healthy captive LPS were analyzed for hematology and plasma biochemistry (n = 11) and plasma and serum hormone analyses (n = 9). Packed cell volume (PCV) and absolute heterophils were significantly higher in adult compared with juvenile LPS, while PCV, white blood cell count, and absolute lymphocytes were higher in adult males compared with adult females. Significantly higher plasma concentrations were found in adults compared with juveniles for calcium, total protein, total solids, albumin, globulins, and bile acids. No significant differences were observed in 17β-estradiol measured in serum and plasma when comparing adults and juveniles and for 17β-estradiol in adult males and females. Plasma concentrations of 17β-estradiol were significantly lower than in serum. Serum testosterone in two adult males was 8.33 and 35.53 nmol/L, respectively, while it was undetectable in females and juveniles (n = 5). This study is the first to provide baseline information on blood analytes in endangered LPS, which will be useful for individual animals in managed care and as baseline for future population-level assessments

Cortisol, progesterone, 17α-hydroxyprogesterone, and TSH responses in dogs injected with low-dose lipopolysaccharide

Background. Stress and diseases such as endotoxemia induce cortisol synthesis through a complex biosynthetic pathway involving intermediates (progesterone, and 17αhydroxyprogesterone (17α-OHP)) and suppression of the hypothalamus-pituitarythyroid axis. Objective. To measure plasma concentrations of cortisol, progesterone, 17α-OHP, and thyroid stimulating hormone (TSH) in dogs experimentally injected with intravenous low-dose lipopolysaccharide (LPS). Our hypothesis was that LPS treatment would elicit a significant increase in cortisol and its precursors, and a significant decrease in TSH concentration. Methods. Hormone measurements were performed on blood samples left over from a previous investigation (2011) on the effect of low-dose LPS on hematological measurands. Five sexually intact female dogs, none in estrous at the time of the study, were administered saline treatment two weeks prior to LPS treatment. LPS was administered intravenously at a dose of 0.1 µg/kg. Blood was collected before (baseline, time -24 hours) and 3-, 6- and 24-hours post-injection. Mixed model analysis for repeated measures was used, with both treatment and time as the repeated factors. Ranked transformation were applied when diagnostic analysis exhibited violation of normality and equal variance assumptions. Post hoc multiple comparisons were performed with Tukey’s adjustment. Statistical significance was defined as p \u3c 0.05. Results. Significant differences relative to baseline values were detected following both treatments. Compared to baseline, dogs had significantly higher cortisol and 17α-OHP at 3-hours, and significantly lower TSH at 3- and 6-hours following LPS treatment. Dogs had significantly lower TSH at 6- and 24- following saline treatment. Though not statistically significant, the trend in progesterone concentrations was similar to cortisol and 17α-OHP, with an increase at 3-hours post-injection followed by a decrease close to baseline following both LPS and saline. Cortisol and 17α-OHP concentrations were higher after LPS treatment than after saline treatment at 3- and 6-hours post-injection, but differences were not statistically significant, and no significant differences between treatments were detected for any other hormone or timepoint

Adrenocortical Challenge Response and Genomic Analyses in Scottish Terriers With Increased Alkaline Phosphate Activity

Scottish terriers (ST) frequently have increased serum alkaline phosphatase (ALP) of the steroid isoform. Many of these also have high serum concentrations of adrenal sex steroids. The study's objective was to determine the cause of increased sex steroids in ST with increased ALP. Adrenal gland suppression and stimulation were compared by low dose dexamethasone (LDDS), human chorionic gonadotropin (HCG) and adrenocorticotropic hormone (ACTH) response tests. Resting plasma pituitary hormones were measured. Steroidogenesis-related mRNA expression was evaluated in six ST with increased ALP, eight dogs of other breeds with pituitary-dependent hyperadrenocorticism (HAC), and seven normal dogs. The genome-wide association of single nucleotide polymorphisms (SNP) with ALP activity was evaluated in 168 ST. ALP (reference interval 8–70 U/L) was high in all ST (1,054 U/L) and HAC (985 U/L) dogs. All HAC dogs and 2/8 ST had increased cortisol post-ACTH administration. All ST and 2/7 Normal dogs had increased sex steroids post-ACTH. ST and Normal dogs had similar post-challenge adrenal steroid profiles following LDDS and HCG. Surprisingly, mRNA of hydroxysteroid 17-beta dehydrogenase 2 (HSD17B2) was lower in ST and Normal dogs than HAC. HSD17B2 facilities metabolism of sex steroids. A SNP region was identified on chromosome 5 in proximity to HSD17B2 that correlated with increased serum ALP. ST in this study with increased ALP had a normal pituitary-adrenal axis in relationship to glucocorticoids and luteinizing hormone. We speculate the identified SNP and HSD17B2 gene may have a role in the pathogenesis of elevated sex steroids and ALP in ST

Elevated Testosterone and Progestin Concentrations in a Spayed Female Rabbit with an Adrenal Cortical Adenoma

This case was described briefly in a recent book chapter (Lennox AM, Fecteau KA: 2014, Endocrine disease. In: BSAVA Manual of Rabbit Medicine, eds. Meredith A, Lord B, pp 274–276. British Small Animal Veterinary Association, Gloucester, UK). In the previous description, the tumor was described as a pheochromocytoma; however, further evaluation suggested that it more closely resembled an adrenal cortical adenoma. A 10-year-old, spayed female rabbit was presented for a behavior change of 8 months’ duration. The rabbit was inappropriately urinating and defecating, as well as demonstrating aggressive behaviors such as chasing, biting, and mounting various objects. The rabbit had elevated progesterone, 17-hydroxyprogesterone, and testosterone concentrations, and ultrasound examination of the abdomen showed a round, homogenous nodule measuring 1.1 × 0.8 × 0.9 cm in the region of the left adrenal gland. Necropsy revealed a unilateral adrenal cortical adenoma. To the authors’ knowledge, this is the first complete description of a female rabbit with an adrenal cortical adenoma documented in the literature

Variability of serum aldosterone concentrations in pet ferrets (Mustela putorius furo)

OBJECTIVE To explore sources of serum aldosterone concentration variability in a population of healthy and diseased ferrets, determine a preliminary 1-sided reference interval for serum aldosterone concentration in healthy ferrets, and identify a decision limit to differentiate healthy from diseased ferrets on the basis of serum aldosterone concentration. DESIGN Prospective threshold definition and diagnostic accuracy study. ANIMALS 78 healthy (n = 56) and diseased (22) ferrets. PROCEDURES Serum aldosterone concentrations were measured on consecutively admitted ferrets, and an upper reference limit for aldosterone concentrations was established. Sensitivity and specificity of aldosterone concentration cutoffs to differentiate healthy from diseased ferrets were estimated with receiver operating characteristic curve analysis. RESULTS Measurements of serum aldosterone concentrations in the ferrets showed wide variability, with a median concentration of 4.75 pg/mL (interquartile range, 0.55 to 17.9 pg/mL; range, 0.02 to 283.9 pg/mL) and 76% (59/78) of samples having concentrations < 18 pg/mL. Ferrets that were healthy, older, or sexually inactive had significantly lower aldosterone concentrations. The upper limit of the reference interval for healthy ferrets was 13.3 pg/mL (90% confidence interval, 9.9 to 16.9 pg/mL). Analysis of receiver operating characteristic curves indicated that an aldosterone concentration cutoff value of 7.6 pg/mL differentiated healthy ferrets from diseased ferrets with a sensitivity of 72.7% and specificity of 73.2% (area under the curve, 0.79; 95% confidence interval, 0.67 to 0.91). CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that high aldosterone concentrations should not be considered diagnostic of primary hyperaldosteronism in ferrets. A need exists to develop better tests to identify primary hyperaldosteronism

Variability of serum aldosterone concentrations in pet ferrets (Mustela putorius furo)

OBJECTIVE To explore sources of serum aldosterone concentration variability in a population of healthy and diseased ferrets, determine a preliminary 1-sided reference interval for serum aldosterone concentration in healthy ferrets, and identify a decision limit to differentiate healthy from diseased ferrets on the basis of serum aldosterone concentration. DESIGN Prospective threshold definition and diagnostic accuracy study. ANIMALS 78 healthy (n = 56) and diseased (22) ferrets. PROCEDURES Serum aldosterone concentrations were measured on consecutively admitted ferrets, and an upper reference limit for aldosterone concentrations was established. Sensitivity and specificity of aldosterone concentration cutoffs to differentiate healthy from diseased ferrets were estimated with receiver operating characteristic curve analysis. RESULTS Measurements of serum aldosterone concentrations in the ferrets showed wide variability, with a median concentration of 4.75 pg/mL (interquartile range, 0.55 to 17.9 pg/mL; range, 0.02 to 283.9 pg/mL) and 76% (59/78) of samples having concentrations < 18 pg/mL. Ferrets that were healthy, older, or sexually inactive had significantly lower aldosterone concentrations. The upper limit of the reference interval for healthy ferrets was 13.3 pg/mL (90% confidence interval, 9.9 to 16.9 pg/mL). Analysis of receiver operating characteristic curves indicated that an aldosterone concentration cutoff value of 7.6 pg/mL differentiated healthy ferrets from diseased ferrets with a sensitivity of 72.7% and specificity of 73.2% (area under the curve, 0.79; 95% confidence interval, 0.67 to 0.91). CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that high aldosterone concentrations should not be considered diagnostic of primary hyperaldosteronism in ferrets. A need exists to develop better tests to identify primary hyperaldosteronism

Comparison of testis structure, function and thyroid hormone levels in control C57BL/6 mice and anti-mullerian hormone over expressing mice

Anti-Mullerian hormone (AMH) is considered as a negative regulator of postnatal Leydig cell (LC) differentiation, because AMH over expressing mice (Mt-hAMH mice) testes are deficient in LC. Therefore, in the present study Mt-hAMH mice was used as a model to examine the process of postnatal LC differentiation. Testis structure-function studies were performed in age-matching Mt-hAMH and C57BL/6 (controls) mice; testicular components were quantified and circulating testosterone and thyroid hormone levels (thyroxine/T4 and triiodothyronine/T3; necessary for postnatal LC differentiation) were determined. Results revealed that Mt-hAMH mice were heavier and their testis weights were smaller compared to controls. Mast cells were present in Mt-AMH testis interstitium, but absent in controls. The absolute volumes of seminiferous tubules (ST), testis interstitium, LC and blood vessels per testis were lower and lymphatic space was higher in Mt-hAMH mice than in controls (p<0.05). The average cell LC volume and their number per testis, ST length, plasma testosterone, luteinizing hormone-stimulated testosterone secretion per testis and per LC in vitro, plasma T4 and T3 were significantly lower in Mt-hAMH mice compared to controls (p<0.05). Increased body weight in Mt-hAMH mice could be attributed to the reduced T4 and T3. Reduced testis weight in Mt-AMH mice is explained by the reduced ST volume in them. Reduced plasma testosterone, testicular and LC testosterone secretion in vitro in Mt-hAMH mice can be explained by the reduced number, size and steroidogenic potential of LC in Mt-hAMH mice. Study revealed several structure-function deficiencies in Mt-AMH mouse compared to controls, which were not documented in previous investigations. As hypothyroidism causes arrest in postnatal LC differentiation, it is suggested that the reduced LC number in Mt-hAMH testes could be at least in part due to their reduced thyroid hormone levels. However, latter concept needs to be further tested in future investigations