Characterization of bovine MHC class II DRB3 diversity in South American Holstein cattle populations.

Holstein cattle dominate the global milk production industry because of their outstanding milk production, however, this breed is susceptible to tropical endemic pathogens and suffers from heat stress and thus fewer Holstein populations are raised in tropical areas. The bovine major histocompatibility complex (BoLA)-DRB3 class II gene is used as a marker for disease and immunological traits, and its polymorphism has been studied extensively in Holstein cattle from temperate and cold regions. We studied the genetic diversity of the BoLA-DRB3 gene in South American Holstein populations to determine whether tropical populations have diverged from those bred in temperate and cold regions by selection and/or crossbreeding with local native breeds. We specifically studied Exon 2 of this gene from 855 South American Holstein individuals by a polymerase chain reaction (PCR) sequence-based typing method. We found a high degree of gene diversity at the allelic (Na > 20 and He > 0.87) and molecular (π > 0.080) levels, but a low degree of population structure (FST = 0.009215). A principal components analysis and tree showed that the Bolivian subtropical population had the largest genetic divergence compared with Holsteins bred in temperate or cold regions, and that this population was closely related to Bolivian Creole cattle. Our results suggest that Holstein genetic divergence can be explained by selection and/or gene introgression from local germplasms. This is the first examination of BoLA-DRB3 in Holsteins adapted to tropical environments, and contributes to an ongoing effort to catalog bovine MHC allele frequencies by breed and location.

Expression of Heparin-Binding EGF-Like Growth Factor (HB-EGF) in Bovine Endometrium: Effects of HB-EGF and Interferon-τ on Prostaglandin Production.

Heparin-binding EGF-like growth factor (HB-EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB-EGF, prostaglandins (PGs) and interferon-τ (IFN-τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB-EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB-EGF and IFN-τ on PGE2 and PGF2-α production by endometrial cells were investigated. RT-PCR analysis revealed that HB-EGF mRNA was greater at the mid-luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid- and late luteal stages than at the other luteal stages (p < 0.05). IFN-τ increased the expression of HB-EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB-EGF did not affect PGF2-α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2-α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN-τ significantly decreased HB-EGF-stimulated PGF2-α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB-EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN-τ increased their expression in cultured endometrial cells. HB-EGF and IFN-τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.

Insulin-like growth factor-1 regulates the expression of luteinizing hormone receptor and steroid production in bovine granulosa cells.

Luteinizing hormone LH plays important roles in follicular maturation and ovulation. The effects of LH are mediated by LH receptor (LHR) in the ovary. However, the factors that regulate the expression of LHR in bovine granulosa cells (GCs) are not well known. Insulin-like growth factor-1 (IGF-1) is known to play a key role in the acquisition and maintenance of functional dominance. To better understand the roles of LHR expression and IGF-1, we conducted three experiments to determine (i) mRNA expression of LHR in the GCs of developing follicles, (ii) the effects of IGF-1 on LHR mRNA expression in cultured GCs and (iii) the effects of IGF-1 on estradiol (E2), progesterone (P4) and androstenedione (A4) production by non-luteinized GCs. In experiment 1, small follicles (<6 mm Ø) expressed lower levels of LHR than mid-sized follicles (6-8 mm Ø) and large follicles (≥9 mm Ø) expressed the highest levels of LHR mRNA (p < 0.05). In experiment 2, IGF-1 (1 and 100 ng/ml) increased (p < 0.05) the expression of LHR mRNA in GCs from small and large follicles. In experiment 3, IGF-1 (0.1-100 ng/ml) increased A4 and E2 in GCs from both small and large follicles but increased P4 only in large follicles. IGF-1 in combination with LH (0.1 and 1 ng/ml) increased P4 and A4 in large follicles, and increased E2 and A4 in GCs of small follicles. These findings strongly support the concept that IGF-1 upregulates LHR mRNA expression as well as A4 and E2 production in GCs and that IGF-1 is required for determining which follicle becomes dominant and acquires ovulatory capacity.

Acute changes in the concentrations of prostaglandin F2α (PGF) and cortisol in uterine and ovarian venous blood during PGF-induced luteolysis in cows.

Prostaglandin F2α (PGF) is considered to be the main luteolysin in cattle. We have previously demonstrated that cortisol (Cr) suppresses PGF production in non-pregnant bovine endometrium. This study was carried out to test whether exogenous PGF increases ovarian and/or uterine PGF production and to determine the temporal relationship between PGF and Cr in ovarian and uterine circulations during PGF-induced luteolysis in cows. Catheters were inserted into the ovarian vein (OV), uterine vein (UV) and jugular vein (JV) of 10 cows on Day 9 of the oestrous cycle (Ovulation = Day 0) for frequent blood collection. On Day 10, the cows were divided randomly into two groups and treated with a luteolytic dose of a PGF analogue (cloprostenol) or saline solution. Blood samples were collected at -0.25, 0, 0.25, 0.5, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). The basal concentrations of PGF and Cr in OV and UV plasma were not significantly different. Injection of a PGF analogue induced more than twofold increases in the levels of PGF between 0.25 and 1 h in UV plasma, but not in OV plasma. PGF increased (p < 0.05) the concentrations of Cr in OV, UV and JV plasma between 0.5 and 1 h. The Cr levels in OV, UV and JV plasma were similar. The PGF levels in UV plasma decreased after Cr reached its highest levels. The overall results suggest that the uterus rather than the ovary increases PGF production in response to PGF injection. Based on the temporal changes of PGF and Cr in the ovarian and uterine circulations, Cr may act to reduce uterine PGF production in non-pregnant cows in vivo.

Conversion of cortisone to cortisol and prostaglandin F2α production by the reproductive tract of cows at the late luteal stage in vivo.

Previous in vitro studies demonstrated that bovine endometrium has the capacity to convert inactive cortisone to biologically active cortisol (Cr) and that Cr inhibits cytokine-stimulated prostaglandin F(2α) (PGF) production. This study was carried out to test the hypothesis that bovine reproductive tract has the capacity to convert cortisone to Cr in vivo and to evaluate the effects of intravaginal application of exogenous cortisone on uterine PGF secretion during the late luteal stage. The temporal relationships between PGF and Cr levels in uterine plasma were also determined. Catheters were inserted into jugular vein (JV), uterine vein (UV), vena cava caudalis (VCC) and aorta abdominalis (AA) of six cows on Day 15 of the oestrous cycle (ovulation = Day 0) for frequent blood collection. On Day 16, the cows were divided randomly into two groups and infused intravaginally with vaseline gel (10 ml; control; n = 3) or cortisone dissolved in vaseline gel (100 mg; n = 3). Blood samples were collected at -2, -1, -0.5, 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal application of cortisone increased plasma concentrations of Cr between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and at 1.5 h in AA. The plasma concentrations of PGF in UV and of PGF metabolite in JV were greater at 0.5 and 1 h in the cortisone-treated animals than in control animals. The levels of PGF in UV blood plasma decreased after Cr reached its highest levels. The overall findings suggest that the female reproductive tract has the capacity to convert cortisone to Cr in vivo. Based on the temporal changes of PGF and Cr levels in the uterine plasma, a biphasic response in PGF secretion was found to be associated to the Cr increase induced by the cortisone treatment at the late luteal stage in non-pregnant cows.

Take action to prevent diabetes--the IMAGE toolkit for the prevention of type 2 diabetes in Europe.

When we ask people what they value most, health is usually top of the list. While effective care is available for many chronic diseases, the fact remains that for the patient, the tax payer and the whole of society: prevention is better than cure. Diabetes and its complications are a serious threat to the survival and well-being of an increasing number of people. It is predicted that one in ten Europeans aged 20-79 will have developed diabetes by 2030. Once a disease of old age, diabetes is now common among adults of all ages and is beginning to affect adolescents and even children. Diabetes accounts for up to 18 % of total healthcare expenditure in Europe. The good news is that diabetes is preventable. Compelling evidence shows that the onset of diabetes can be prevented or delayed greatly in individuals at high risk (people with impaired glucose regulation). Clinical research has shown a reduction in risk of developing diabetes of over 50 % following relatively modest changes in lifestyle that include adopting a healthy diet, increasing physical activity, and maintaining a healthy body weight. These results have since been reproduced in real-world prevention programmes. Even a delay of a few years in the progression to diabetes is expected to reduce diabetes-related complications, such as heart, kidney and eye disease and, consequently, to reduce the cost to society. A comprehensive approach to diabetes prevention should combine population based primary prevention with programmes targeted at those who are at high risk. This approach should take account of the local circumstances and diversity within modern society (e.g. social inequalities). The challenge goes beyond the healthcare system. We need to encourage collaboration across many different sectors: education providers, non-governmental organisations, the food industry, the media, urban planners and politicians all have a very important role to play. Small changes in lifestyle will bring big changes in health. Through joint efforts, more people will be reached. The time to act is now.

A European evidence-based guideline for the prevention of type 2 diabetes.

BACKGROUND: The prevalence and socioeconomic burden of type 2 diabetes (T2DM) and associated co-morbidities are rising worldwide. AIMS: This guideline provides evidence-based recommendations for preventing T2DM. METHODS: A European multidisciplinary consortium systematically reviewed the evidence on the effectiveness of screening and interventions for T2DM prevention using SIGN criteria. RESULTS: Obesity and sedentary lifestyle are the main modifiable risk factors. Age and ethnicity are non-modifiable risk factors. Case-finding should follow a step-wise procedure using risk questionnaires and oral glucose tolerance testing. Persons with impaired glucose tolerance and/or fasting glucose are at high-risk and should be prioritized for intensive intervention. Interventions supporting lifestyle changes delay the onset of T2DM in high-risk adults (number-needed-to-treat: 6.4 over 1.8-4.6 years). These should be supported by inter-sectoral strategies that create health promoting environments. Sustained body weight reduction by >or= 5 % lowers risk. Currently metformin, acarbose and orlistat can be considered as second-line prevention options. The population approach should use organized measures to raise awareness and change lifestyle with specific approaches for adolescents, minorities and disadvantaged people. Interventions promoting lifestyle changes are more effective if they target both diet and physical activity, mobilize social support, involve the planned use of established behaviour change techniques, and provide frequent contacts. Cost-effectiveness analysis should take a societal perspective. CONCLUSIONS: Prevention using lifestyle modifications in high-risk individuals is cost-effective and should be embedded in evaluated models of care. Effective prevention plans are predicated upon sustained government initiatives comprising advocacy, community support, fiscal and legislative changes, private sector engagement and continuous media communication.

Quality indicators for the prevention of type 2 diabetes in Europe--IMAGE.

BACKGROUND: The marked increase of type 2 diabetes necessitates active development and implementation of efficient prevention programs. A European level action has been taken by launching the IMAGE project to unify and improve the various prevention management concepts, which currently exist within the EU. This report describes the background and the methods used in the development of the IMAGE project quality indicators for diabetes primary prevention programs. It is targeted to the persons responsible for diabetes prevention at different levels of the health care systems. METHODS: Development of the quality indicators was conducted by a group of specialists representing different professional groups from several European countries. Indicators and measurement recommendations were produced by the expert group in consensus meetings and further developed by combining evidence and expert opinion. RESULTS: The quality indicators were developed for different prevention strategies: population level prevention strategy, screening for high risk, and high risk prevention strategy. Totally, 22 quality indicators were generated. They constitute the minimum level of quality assurance recommended for diabetes prevention programs. In addition, 20 scientific evaluation indicators with measurement standards were produced. These micro level indicators describe measurements, which should be used if evaluation, reporting, and scientific analysis are planned. CONCLUSIONS: We hope that these quality tools together with the IMAGE guidelines will provide a useful tool for improving the quality of diabetes prevention in Europe and make different prevention approaches comparable.

Leukotrienes affect secretory function of ovarian cells in vitro.

The aim of this study was to determine which cells are the source of production and target for leukotriene (LTs) action within the bovine ovary. Luteal (CL, days 14-16 of the oestrous cycle), steroidogenic cells (LSC) and endothelial cells (LEC) of the bovine corpus luteum (CL), and granulosa cells (GC) were isolated enzymatically, cultured in a monolayer and incubated with LTC(4), LTB(4), Azelastine (an antagonist of LTC(4)) or Dapsone (an antagonist of LTB(4)). Then cells were collected for determination of mRNA expression for LT receptors (LTRs) and 5-lipoxygenase (5-LO) by real time RT-PCR, and media were collected for determination of prostaglandin (PG)E(2), F(2α), progesterone (P4; LSC only), endothelin-1 (ET-1; LEC only) and 17-ß oestradiol (E2; GC only). The greatest mRNA expression for LTR-II and 5-LO were found in LEC, whereas LTR-I mRNA expression did not differ among cell types. The level of PGE(2) increased after LTs treatment in each type of ovarian cell, excluding LTC(4) treatment in LEC. The secretion of PGF(2α) was also increased by LTs, but decreased after LTB(4) treatment of LSC. In GC cultures, both LTs stimulated E2 secretion; in LEC cultures, LTB(4) stimulated whereas LTC(4) inhibited P4 secretion; in LEC cultures, LTC(4) stimulated but LTB(4) inhibited ET-1 secretion. The results show that LTs are produced locally and are involved in PGs production/secretion in all examined cells (LSC, LEC and GC) of bovine ovary. Leukotriene treatment modulate secretion of E2, by GC, P4 by LSC and ET-1 by LEC, which indicates that LTs are involved in regulation of ovarian secretory functions.

Acute effects of prostaglandin F(2alpha) on systemic oxytocin and progesterone concentrations during the mid- or late-luteal phase in mares.

The acute effects of prostaglandin F(2alpha) (PGF) on circulating oxytocin and progesterone concentrations were characterized in mares during the mid- or late-luteal phase. Pony mares were randomly assigned to the following experimental groups based on treatment with PGF (2.5mg) or saline on Day 8 or Day 13 (Day 0=ovulation): PGF-8, PGF-13, saline-8, or saline-13 (n=7/group). Mares were fitted with indwelling, jugular vein catheters and two blood samples (-5 and 0 min) were collected prior to treatment. Treatments were administered into the jugular vein (0 min) and blood collection continued thereafter at 1 min intervals until 5 min and then at 5 min intervals until 60 min. Based on the combined data of -5 and 0 min samples, mares on Day 8 had greater (P<0.05) oxytocin concentrations than mares on Day 13. On Day 8, PGF treatment resulted in a biphasic pattern of oxytocin release. Oxytocin concentrations increased (P<0.05) 1 min after PGF treatment, decreased (P<0.05) from 1 to 10 min, and increased (P<0.05) from 10 to 30 min. Oxytocin concentrations were greater (P<0.05) from 1 to 3 min in PGF-treated than saline-treated mares and at most sample times from 15 to 60 min. On Day 13, oxytocin concentrations were greater (P<0.05) in PGF-treated than in saline-treated mares for most sample times. Mares treated with PGF on Day 8 had greater (P<0.05) oxytocin concentrations at 25, 30, and 40 min than mares on Day 13. Progesterone concentrations on Day 8 also increased by 1 min after PGF, decreased toward basal concentrations by 2-3 min, and then increased to a maximum 10 min after treatment. Subsequently, circulating progesterone decreased (P<0.05) below pretreatment concentrations by 40-50 min after PGF. In conclusion, treatment with PGF resulted in an immediate and biphasic increase in progesterone concentrations prior to the expected decrease. Treatment of mares with PGF on Day 8 resulted in an overall greater increase in systemic oxytocin concentrations compared to treatment on Day 13, and the increase on Day 8 was biphasic.

Temporal relationships among LH, estradiol, and follicle vascularization preceding the first compared with later ovulations during the year in mares.

Diameter of the preovulatory follicle, plasma concentrations of LH and estradiol, and vascularization of the follicle wall, based on color-Doppler signals, were characterized in 40 pony mares for 6 days preceding ovulation (Days -6 to -1; preovulatory period). Comparisons between the preovulatory periods preceding the first compared with a later ovulation during the year were used to study the relationships between LH and estradiol and between vascularization and estradiol. Diameter of the preovulatory follicle was greater (P<0.02) and concentration of LH was less (P<0.02) during the first preovulatory period, whereas concentration of estradiol was not different between the first and second preovulatory periods. Vascularized area (cm(2)) of the follicle wall increased at a reduced rate during the first preovulatory period, as indicated by an interaction (P<0.03) between day and group. Vascularized area was similar between the preovulatory groups on Day -6, and a reduced rate of increase resulted in a lesser (P<0.001) area on Day -1 before the first ovulation (1.4+/-0.1cm(2)) than before a later ovulation (2.2+/-0.2 cm(2)). Results demonstrated that follicle vascularization and the LH surge were attenuated preceding the first ovulation of the year with no indication that estradiol was involved in the differences between the first and later ovulations.

Phytoestrogen metabolites are much more active than phytoestrogens themselves in increasing prostaglandin F(2alpha) synthesis via prostaglanin F(2alpha) synthase-like 2 stimulation in bovine endometrium.

Phytoestrogens have recently been suggested to be the cause of infertility by stimulating luteolytic prostaglandin (PG) F(2alpha) secretion from endometrium in cattle. The purpose of this study was to examine the enzymatic and molecular mechanisms involved in the preferential induction of PGF(2alpha) synthesis by phytoestrogens, and whether phytoestrogens influence endometrial cell viability. Cultured bovine endometrial epithelial and stromal cells were exposed to phytoestrogens (daidzein and genistein) and their metabolites (equol and p-ethyl phenol) for 24h. Prostaglandin F(2alpha) and PGE2 were stimulated by phytoestrogens in both stromal and epithelial cells, with a preference for PGF(2alpha) synthesis in epithelial cells (P<0.001). Although RT-PCR and Western Blot analyses did not reveal the influence of phytoestrogens on either gene expression or protein level of cyclooxygenase-2 (COX-2) and PGE2 synthase (PGES) in stromal and epithelial cells (P>0.05), the stimulative effects of equol and p-ethyl phenol on PGF(2alpha) synthase-like 2 (PGFSL2) gene expression and protein level were observed only in epithelial cells (P<0.05). The same compounds did not affect PGFSL2 gene expression and protein in stromal cells (P>0.05). Exposure to phytoestrogens and their metabolites decreased cell viability in both stromal and epithelial cells. Stromal cell viability decreased to 50% of the control and was more evident than that in epithelial cells (P<0.001). The overall results suggest that infertility in cattle, caused by phytoestrogen-dependent preferential stimulation of luteolytic PGF(2alpha) synthesis, is caused by increasing PGFSL2 in epithelial cells, and by decreasing stromal cell viability, which are the main source of luteotropic PGE2 production.

Regulation of prostaglandin synthesis by interleukin-1alpha in bovine endometrium during the estrous cycle.

Interleukin (IL)-1 has been suggested to participate in regulation of many reproductive functions. To investigate the possible role of IL-1alpha as a local regulator in bovine endometrium, we determined the effects of IL-1alpha on prostaglandin (PG) E2 and PGF(2alpha) output by the bovine endometrium at different stages of the estrous cycle. The expressions of IL-1alpha and IL-1 receptor type 1 (IL-1RT1) mRNA in bovine endometrium were also studied. Bovine uteri were classified into six stages (estrus: day 0; early luteal: days 2-3; developing luteal: days 5-6; mid luteal: days 8-12; late luteal: days 15-17; and follicular: days 19-21). After 1h of pre-incubation, endometrial tissues (20-30mg) were exposed to 0 or 10ng/ml IL-1alpha for 4h. IL-1alpha significantly stimulated PGE2 output throughout the luteal stages, with the highest response during the mid luteal stage, while it did not stimulate PGE2 output during the estrus and the follicular stage. On the other hand, IL-1alpha significantly enhanced PGF(2alpha) output throughout the estrous cycle except in the endometrium from the mid luteal stage, with the highest response at the follicular stage (P<0.001). The treatment of endometrial tissue with IL-1alpha resulted in an increase of the PGE2:PGF(2alpha) ratio at the mid luteal stage, and in a decrease during the late luteal and follicular stages of the estrous cycle. A semiquantitative reverse transcription-polymerase chain reaction revealed that IL-1alpha and IL-1RT1 mRNA are expressed in the endometrium throughout the estrous cycle. IL-1alpha mRNA expression was greater in the early luteal stage than in the estrus, late luteal, and follicular stages (P<0.05). IL-1RT1 mRNA was greater in the late luteal stage than in the other stages (P<0.05). The overall results suggest that IL-1alpha is produced in bovine endometrium throughout the estrous cycle, and plays some roles not only in maintenance of CL, but also in luteolysis by regulating the local PGE2:PGF(2alpha) ratio in bovine endometrium during the estrous cycle.

Blood flow: a key regulatory component of corpus luteum function in the cow.

Prostaglandin F2alpha (PGF2alpha) is the primary luteolysin in the cow. During the early luteal phase, the corpus luteum (CL) is resistant to the luteolytic effect of PGF2alpha. Once mature, the CL becomes responsive to PGF2alpha and undergoes luteal regression. These actions of PGF2alpha coincide with changes in luteal blood flow (BF): PGF2alpha has no effect on BF in the early CL, but acutely increases BF in the peripheral vasculature of the mature CL within 30 min of PGF2alpha injection. During spontaneous luteolysis, luteal BF increases on Days 17-18 of the estrous cycle, prior to any decrease in plasma progesterone (P). The increase in luteal BF is synchronous with an increase in plasma PGFM levels, suggesting that pulsatile release of PGF2alpha from uterus stimulates the increase in luteal BF. Serial biopsies of these CL showed that mRNA expression for endothelial nitric oxide synthase (eNOS) together with endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) increases on Days 17-18 when the luteal BF is elevated. On Day 19 when plasma P level firstly decreases, eNOS mRNA returns to the basal level whereas ET-1 and ACE mRNA remains elevated. Cyclooxygenase-2 (COX-2) mRNA expression increases on Day 19. In support of these data, an in vivo microdialysis study revealed that luteal ET-1 and angiotensin II (Ang II) secretion increases and precedes PGF2alpha secretion during spontaneous luteolysis. In conclusion, we show for the first time that an acute increase of BF occurs in the peripheral vasculature of the mature CL together with increases in eNOS expression and ET-1 and Ang II secretion in the CL during the early stages of luteolysis in the cow. We propose that the increase in luteal BF may be induced by NO from large arterioles surrounding the CL, and simultaneously uterine or exogenous PGF2alpha directly increases ET-1 and Ang II secretion from endothelial cells of microcapillary vessels within the CL, thereby suppressing P secretion by luteal cells. Taken together, our results indicate that an acute increase in luteal BF occurs as a first step of luteolysis in response to PGF2alpha. Therefore, local BF plays a key role to initiate luteal regression in the cow.

Human exposure to selamectin from dogs treated with revolution: methodological consideration for selamectin isolation.

This study was undertaken to determine selamectin residue in dog's blood and in gloves worn while petting dogs after Revolution application. Revolution contains the active ingredient selamectin (a semisynthetic avermectin), which controls endoparasites and ectoparasites, including adult fleas, flea eggs, ticks, heartworms, ear mites, and sarcoptic mange in dogs, for 30 days. Revolution was applied topically on a group of six adult house hold dogs (240 mg selamectin/dog). The gloves worn for 5 min while petting the dogs were collected in glass jars and the blood samples (5 mL/dog) were collected in EDTA tubes at 0 h, 24 h, and 72 h, and at 1, 2, 3, 4, and 5 weeks post-Revolution application for selamectin residue determination. At no time during the study did the dogs show any signs of toxicity, weight loss, or change in body temperature. Extracts of the blood and the gloves were analyzed for selamectin residue using RP-HPLC coupled with a UV detector (246 nm). Selamectin standard used for peak identification and quantitation was purified from Revolution. Selamectin residue was detected in the blood (10.26 +/- 1.06 ng/mL) only at 72 h post-Revolution application, probably due to its poor dermal absorption and rapid elimination from the circulation. In the glove extracts, the highest concentration of selamectin (518.90 +/- 66.80 ppm) was detected 24 h after Revolution application. Transferable residue of selamectin in gloves from dog's coat was detected at a lesser magnitude after 1 week of Revolution application, and that was followed by a further descending trend during the second, third, and fourth weeks. No selamectin residue was detected in the glove extracts after the fifth week. In spite of selamectin's binding to the sebaceous glands of the skin, gloves contained significant transferable residue. Thus, these findings suggest that repeated exposure to selamectin can pose potential health risks, especially to veterinarians, veterinary technologists, dog trainers/handlers, and pet owners.

Expression of 11beta-hydroxysteroid dehydrogenases in bovine follicle and corpus luteum.

In glucocorticoid target organs, local concentrations of active glucocorticoid are determined by the relative expression of two 11beta-hydroxysteroid dehydrogenases (HSDs): bi-directional 11beta-HSD type1 (11HSD1) that mainly activates cortisone to cortisol, and dehydrogenase 11beta-HSD type2 (11HSD2) that inactivates cortisol to cortisone. In this study, we examined the expression of mRNA encoding these two 11beta-HSDs in bovine granulosa cells harvested from preovulatory follicles and corpora lutea (CL). Ovaries were obtained from Holstein cows at a local slaughterhouse. Follicles larger than 10 mm in diameter and CL were dissected and follicular fluid and granulosa cells were taken. Corpora lutea were weighed and their stages were morphologically assessed (stage I, days 1-4; stage II, days 5-10; stage III, days 11-17; stage IV, days 8-20). Follicles were classified into four groups according to their hormonal status (oestradiol (E(2)): progesterone (P(4))>1: oestrogen active; E(2):P(4)<1: oestrogen inactive) and stage of the oestrous cycle (luteal or follicular phase). Total RNA was extracted with phenol-chloroform and subjected to a semi-quantitative RT-PCR for 11HSD1, 11HSD2 and beta-actin. Concentrations of steroids in follicular fluid were determined by an enzyme immunoassay. In granulosa cells, only 11HSD1 mRNA was detected. There was a negative correlation between the expression of 11HSD1 and the concentration of cortisol in follicular fluid (P<0.05), indicating 11HSD1 may act as a dehydrogenase in the bovine follicle. Both types of 11beta-HSDs were expressed in CL. The levels of mRNA for both isozymes were high in stage I and II, and were decreased in stage III CL. In stage IV CL, the expression of 11HSD2 but not 11HSD1 mRNA increased. These results indicate that the bovine granulosa cells and CL express 11HSD1 and 11HSD2, and they may play an important physiological role in the bovine ovary through modulating the local glucocorticoid environment.

In vitro regulation of local secretion and contraction of the bovine oviduct: stimulation by luteinizing hormone, endothelin-1 and prostaglandins, and inhibition by oxytocin.

The precise regulatory mechanisms of cyclic oviductal contraction in the cow are unclear. The purpose of this study was to evaluate the effect of luteinizing hormone (LH), steroids, prostaglandins (PGs) and peptides on the oviductal contraction and secretion of PGs and endothelin (ET-1). In addition, the cyclic expression of mRNA for ET-1 and its receptors (ET-R) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). In the in vitro microdialysis study, an infusion of LH alone or in combination with progesterone (P(4)), estradiol-17beta (E(2)) and/or ET-1 stimulated pronounced release of PGE(2), PGF(2alpha) and ET-1 in the oviducts from cows in the follicular and postovulatory phases. The addition of LH, LH+P(4)+E(2) and/or ET-1 to the medium increased the amplitude of oviductal contraction. However, oxytocin (OT) completely blocked the responses of oviductal secretion and contraction. In contrast, these substances did not show any effect in the oviducts from cows in the mid luteal phase. Similar expression patterns of mRNA encoding for ET-R type A and type B were found, which were highest during the postovulatory phase, lower during the luteal phase, with the lowest expression during the follicular phase. We suggest that the preovulatory LH surge, together with increasing E(2) levels from the Graafian follicle and a basal P(4) from regressing corpora lutea (CL), stimulates maximum oviductal production of PG and ET-1, resulting in oviductal contraction for a rapid transport of gametes. OT released from the newly-formed CL may block these mechanisms, and slow contractions for transport of the embryo to the uterus.

Bovine retained placenta: hormonal concentrations in fetal and maternal placenta.

The aim of this study was to evaluate the relationship between the occurrence of retention of the fetal membranes (RFM) and the hormonal concentrations of progesterone, estradiol-17beta, prostaglandin E(2) (PGE(2)), prostaglandin F(2alpha) (PGF(2alpha)), oxytocin (OT), oxytocin receptor (OT-R), endothelin-1 and angiotensin II (Ang II) in the placental tissues of cattle. Parturition was induced in nine Holstein cows by a single injection of PGF(2alpha) on Day 274 of gestation. Six out of nine cows in the induced group did not release the fetal membranes within 12 h after parturition and served as the RFM group, and the remaining three cows in that group, which released their fetal membranes within 12 h, served as the non-RFM group. Five other cows calved spontaneously and served as controls. The placental tissues were collected immediately (0 h) and at 6 h after parturition. The hormonal concentrations were measured by enzyme immunoassay in maternal and fetal placental tissues from RFM, non-RFM and control cows. There were no differences in P4 and E2 concentrations among the RFM, non-RFM and control groups. The mean PGF(2alpha) concentration of the RFM group was lower than those of the non-RFM and control groups in the maternal part of the placenta. In maternal tissues, the OT and OT-R concentrations in the RFM group were lower than those at 0 and 6 h after parturition in the non-RFM group. Additionally, the Ang II concentration of the RFM group in both the maternal and fetal parts of placental tissues tended to be higher than those of the other groups. In conclusion, the present results suggest that ET-1 and Ang II may play differential tissue-specific roles in the placental unit that may amplify the local endocrinological cascade involving OT, OT-R and PGF(2alpha) interactions which are necessary for normal placental separation in the cow.

Effectiveness of propranolol for cocaine dependence treatment may depend on cocaine withdrawal symptom severity.

Propranolol may reduce symptoms of autonomic arousal associated with early cocaine abstinence and improve treatment outcome. This trial was an 8-week, double-blind, placebo-controlled trial of propranolol in 108 cocaine dependent subjects. The primary outcome measure was quantitative urinary benzoylecgonine levels. Secondary outcome measures included treatment retention, addiction severity index results, cocaine craving, mood and anxiety symptoms, cocaine withdrawal symptoms, and adverse events. Propranolol treated subjects had lower cocaine withdrawal symptom severity but otherwise did not differ from placebo treated subjects in any outcome measure. However, in a secondary, exploratory analysis, subjects with more severe cocaine withdrawal symptoms responded better to propranolol in comparison to placebo. In these subjects, propranolol treatment was associated with better treatment retention and lower urinary benzoylecgonine levels as compared with the placebo treatment. Propranolol may be useful only for the treatment of cocaine dependent patients with severe cocaine withdrawal symptoms.

Vascular control of ovarian function: ovulation, corpus luteum formation and regression.

Hemodynamic changes are involved in the cyclic remodeling of ovarian structures. A transrectal color Doppler ultrasonography was used to assess the blood flow and changes in the vasculature that take place in the follicle wall and within the corpus luteum (CL) during specific physiological events such as ovulation, CL development, and CL regression in cows. To investigate the local release of vasoactive peptides, steroid hormones, and prostaglandins (PGs) in the ovarian microenvironment, the capillary membranes (0.2mm diameter and 5-10mm length) of a microdialysis system (MDS) were implanted into the follicle wall and the CL in vitro. Furthermore, in vivo experiments were conducted with the same MDS membranes surgically implanted in follicle wall or on CL along with ovarian venous and jugular catheters to collect simultaneous, real-time information on the ovarian and systemic changes in the secretion of factors regulating vascular function. Based on the results obtained from the series of in vitro and in vivo experiments, we propose that a functional "cross-talk" occurs between the vascular components (endothelial cells) and steroidogenic cells to control follicular and luteal functions in the bovine ovary.