Physiochemical and nutritional evaluation of whole kumquat fruits powder and its protective effect on thyroid hormones and blood sugar levels in diabetic rats / Avaliação físico-química e nutricional de frutas inteiras de kumquat em pó e seu efeito protetor sobre os hormônios da tireoide e os níveis de açúcar no sangue em ratos diabéticos

The present study was conducted to evaluate the chemical composition, antioxidant activity and hypoglycemic effects of whole kumquat (Ku) powder in diabetic rats fed a high-fat-high-cholesterol (HFHC) diet. The antioxidant activities were evaluated using stable 1,1-diphenyl 2-picrylhydrazyl (DPPH) free radical scavenging method, 2,2'-azinobis (3-ethyl benzo thiazoline-6-sulphonic acid) radical cation (ABTS) and Ferric reducing antioxidant power (FRAP). Total phenolic content was (51.85 mg GAE/g) and total flavonoid content was (0.24 mg Cateachin Equivalent, CE/g). DPPH and ABTS values were 3.32 and 3.98 mg Trolox equivalent (TE)/g where FRAP value was 3.00 mM Fe²+/kg dry material. A total of 90 albino rats were used in the present study. Rats group were as follows: normal diet; normal treated (2, 4, and 6% Ku.), diabetic rats (non-treated), diabetic + HFHC diet (non-treated), HFHC (non-treated), Diabetic (treated), HFHC (treated) and Diabetic + HFHC (treated). The diets were followed for 8 weeks. Blood samples were collected at the end of the experiment. Serum glucose was recorded and thyroid hormones (T4, Thyroxine and T3, Triiodothyronine) were conducted. Diet supplemented with Kumquat at different concentrations have a hypoglycemic effect and improve the thyroid hormones of both diabetic rats and HFHC diabetic rats.

O presente estudo foi conduzido para avaliar a composição química, a atividade antioxidante e os efeitos hipoglicêmicos do pó de kumquat (Ku) em ratos diabéticos alimentados com uma dieta rica em gordura e colesterol (HFHC). As atividades antioxidantes foram avaliadas usando o método de eliminação de radicais livres de 1,1-difenil 2-picrilhidrazil (DPPH), 2,2'-azinobis (ácido 3-etilbenzotiazolina-6-sulfônico) radical cátion (ABTS) e antioxidante redutor férrico potência (FRAP). O conteúdo fenólico total foi (51,85 mg GAE / g) e o conteúdo total de flavonoides foi (0,24 mg Cateachin Equivalent, CE / g). Os valores de DPPH e ABTS foram 3,32 e 3,98 mg equivalente de Trolox (TE) / g, em que o valor de FRAP foi de 3,00 mM Fe²+ / kg de material seco. Um total de 90 ratos albinos foi usado no presente estudo. O grupo dos ratos foi o seguinte: dieta normal: tratados normais (2, 4 e 6% Ku.), ratos diabéticos (não tratados), diabéticos + dieta HFHC (não tratados), HFHC (não tratados), diabéticos (tratados), HFHC (tratados) e diabéticos + HFHC (tratados). As dietas foram seguidas por 8 semanas. Amostras de sangue foram coletadas ao final do experimento. A glicose sérica foi registrada e os hormônios tireoidianos (T4, Tiroxina e T3, Triiodotironina) foram conduzidos. A dieta suplementada com kumquat em diferentes concentrações tem um efeito hipoglicêmico e melhora os hormônios tireoidianos tanto de ratos diabéticos quanto de ratos diabéticos com HFHC.

Pouteria ramiflora leaf extract on emulgel in wound healing activity in diabetic rats / Ação do extrato de folhas de Pouteria ramiflora em emulgel na atividade de cicatrização de feridas em ratos diabéticos

The genus Pouteria has been studied because it presents various activities, among which is its anti-inflammatory potential. The effects of Pouteria ramiflora Carbopol gel on the healing of skin wounds in diabetic rats were evaluated by microscopic imaging. Streptozotocin was administered intraperitoneally in animals that had fasted for 12 hours, a situation confirmed by the glycemic index (> 240 mg dL-¹). An excision on the back of the animals was performed and three groups were formed: Control (Gel), Ethanolic extract (Ext) and Gel + extract 2% (Ext+gel); the histopathological evaluation occurred on the 7th, 14th, 21st and 30th days after the post-operative period. The results of the phytochemical prospecting of P. ramiflora extract demonstrated the major presence of phenolic compounds and flavonoids; the assessment of the inflammatory infiltrate on the 7th day was higher on group Ext and Ext+gel when compared to group Control; on the 14th day control and Ext (p<0.05). The quantification of fibroblasts was higher on the 7th day among the three treatments, control and Ext (p<0.05), on the 21st day. Angiogenesis showeda higher number of vessels in Ext+gel group (p<0.05) on the 7th day; in Control, Ext and Ext+gel (p<0.05) on the 14th day; and Control and Ext (p<0.05)on the 21st day. The histopathological results showed that the formulation Ext+gel was efficient in tissue reparation and decrease in inflammatory cells on the diabetic's animals.

O gênero Pouteria apresenta várias aplicações terapêuticas e, dentre elas, grande potencial antiflamatório. Os efeitos do gel de Pouteria ramiflora sobre a cicatrização de feridas na pele de ratos diabéticos foram avaliados pela histomorfometria. A estreptozotocina foi administrada por via intraperitoneal em animais após jejum de 12 horas, a confirmação de indução da diabetes foi confirmada pelo índice glicêmico (> 240 mg dL-1). Foi realizada uma incisão no dorso do animal e foram criados 3 grupos de tratamento: controle (gel carbopol), extrato etanólico (Ext) e Gel + extrato etanólico à 2% (Ext+gel); a avaliação histopatológica foi realizada no 7º, 14º, 21º e 30º dias após o período pós operatório. Os resultados da prospecção fitoquímica dos extratos de P. ramiflora demonstraram majoritariamente a presença de compostos fenólicos e flavonóides; o infiltrado inflamatório avaliado no 7º dia foi maior para animais do grupo controle em relação aos grupos Ext (p<0.05) e Ext+gel 2% (p<0.05); no 14º dia o controle e Exp (p<0.05) apresentaram aumento significativo dos infiltrados inflamatórios. A presença de fibroblastos foi elevada no 7º dia em todos os tratamentos. O processo da angiogênese mostrou um maior número de vasos sanguíneos entre os grupos Ext e Ext+gel (p<0.05) no 7º dia; no 14º dia o grupo controle, Ext (p<0.05), Control e Ext+gel (p<0.05) apresentaram aumento de vascularização, e no 21º dia apenas os grupos controle e Ext (p<0.05). Os resultados histopatológicos mostraram que a formulação gel carbopol + extrato etanólico a 2% foi eficiente na reparação de tecidos e na diminuição de células inflamatórias nos animais diabéticos.

Method for detecting hemodynamic alterations following a single gavage in rats.

It is known that administering a gavage to rodents evokes a cardiac reflex, due to gastrointestinal stimulation. Consequently, it is difficult to evaluate changes in hemodynamics after a single oral dose of a pungent or astringent, which alters the circulation by increasing sympathetic activity. In the present study, we developed a method for administering a gavage without significantly affecting hemodynamics measurements. We marked a gastric tube at 10 cm from the tip, to mark the distance from the oral cavity to the stomach body of Wistar male rats. Rats were intubated under urethane anesthesia.After 10-15 min of stabilization, we measured the mean blood pressure (MBP), heart rate (HR), and blood flow (BF) in the cremaster arteriole under two different conditions; condition 1: a pointed gastric tube, room temperature distilled water, and injected at normal speed (approximately 3 ml/min); condition 2: a rounded gastric tube, 37°C distilled water, and injection at 1.0 ml/min. Under condition 1, we observed striking hemodynamic alterations, due to the somatic afferent reflex. In contrast, under condition 2, these hemodynamic changes were nearly eliminated. In addition, we could clearly detect hemodynamic changes in rats after a single gavage treatment of pungent (capsaicin) or astringent (cinnamtannin A2). We observed transient increases in the HR and MBP soon after treatment with capsaicin. Moreover, cremasteric BF was elevated with cinnamtannin A2. These results confirmed the utility of the gavage method developed in this study.

Evaluation of copper metabolism in neonatal rats by speciation analysis using liquid chromatography hyphenated to ICP mass spectrometry.

It is known that copper (Cu) is highly accumulated in several organs in the perinatal period, suggesting changes in Cu metabolism with development, although the precise mechanisms are still unclear. To elucidate the mechanisms underlying Cu accumulation in the organs of neonatal rats, we performed speciation analysis using high-performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry. In the neonatal rat liver immediately after birth, the Cu concentration was elevated 10-fold compared to that in the juvenile rat liver. Most of the accumulated Cu was bound to metallothionein, although Cu in Cu, zinc-superoxide dismutase (SOD) was reduced. Contrary to the hepatic Cu accumulation, the serum Cu concentrations in the neonatal rats were low due to the decreased amount of Cu bound to ceruloplasmin. The mRNA expression of antioxidant protein 1 (Atox1), a Cu chaperone that transports Cu to Atp7b, remained low up to two weeks after birth. These results suggest that Cu accumulation in the neonatal rat liver is caused by the low expression of Atox1, and the accumulation is useful to distribute Cu to Cu-containing anti-oxidative enzymes (e.g., SOD and Atox1) after respiration starts.

Purified Dietary Red and White Meat Proteins Show Beneficial Effects on Growth and Metabolism of Young Rats Compared to Casein and Soy Protein.

This study compared the effects of casein, soy protein (SP), red (RMP), and white meat (WMP) proteins on growth and metabolism of young rats. Compared to casein, the ratio of daily feed intake to daily body weight gain of rats was not changed by meat protein but reduced by SP by 93.3% ( P < 0.05). Feeding RMP and WMP reduced the liver total cholesterol (TC) contents by 24.3% and 17.8%, respectively ( P < 0.05). Only RMP increased plasma HDL-cholesterol concentrations (by 12.7%, P < 0.05), whereas SP increased plasma triacylglycerol, TC, and LDL-cholesterol concentrations by 23.7%, 19.5%, and 61.5%, respectively ( P < 0.05). Plasma essential and total amino acid concentrations were increased by WMP (by 18.8% and 12.4%, P < 0.05) but reduced by SP (by 28.3% and 37.7%, P < 0.05). Twenty-five liver proteins were differentially expressed in response to different protein sources. Therefore, meat proteins were beneficial for growth and metabolism of young rats compared to casein and SP.

Plasma Concentration of Meloxicam in Pediatric Rats.

In this study, we compared the plasma concentrations of meloxicam in pediatric rat pups (ages: 7, 14, 21, and 28 d) with those of young adult rats. Adult rats received 1.34 mg/kg SC meloxicam to determine the target peak plasma concentration (Cmax) for comparison with the pediatric animals. Pediatric rats received 1.34 mg/kg SC meloxicam, and in all age groups, Cmax met or exceeded that in adults (11.5 ±2.7 µg/mL). Plasma concentrations were similar between male and female pups within age groups, and peak plasma concentration was achieved more rapidly in rat pups than adults. The analgesic efficacy of this dose was not evaluated in this study.

Preanalytic processing of rat plasma influences thrombin generation and fibrinolysis assays.

BACKGROUND: Thrombin generation assays (TGA) have potential applications as measures of hemostatic balance in animal models. However, variations in plasma processing greatly influence human TGA, and may also impact on the translational value of TGA in animal studies. OBJECTIVES: The purpose of the study was to compare the performance characteristics of Sprague-Dawley rat plasma prepared by single vs double centrifugation protocols in TGA and fibrinolysis assays. METHODS: Platelet-poor plasma (PPP) from adult rats (n = 20 males; 20 females) was prepared by centrifugation at 1200g × 12 min, or 2 sequential centrifugations of 2500g × 15 min. Plasma aliquots were assayed fresh and after freeze-thaw in a commercial fluorogenic TGA (Technothrombin TGA, Technoclone) using 2 different trigger reagents containing approximately 7 pM human tissue factor. In addition to TGA variables (lag time, peak thrombin, endogenous thrombin potential), we compared clotting time test and fibrinogen concentration, residual platelet and platelet-derived microparticle (PMP) counts measured by flow cytometry, and variables of fibrin clot formation and lysis measured in turbidimetric assays. RESULTS: Single-centrifugation PPP demonstrated significantly greater thrombin-generating potential regardless of trigger reagent, yielded higher residual platelet and procoagulant PMP counts, and more stable fibrin clot profiles. The influence of a freeze-thaw cycle on TGA varied depending on trigger reagent, and male sex was associated with an overall "procoagulant" phenotype. CONCLUSIONS: Preanalytic processing and sex have significant effects on many functional measures of hemostasis in rats. A standardized double centrifugation protocol to prepare PPP is recommended for future studies.

Impact of Diet Containing Grape Pomace on Growth Performance and Blood Lipid Profile of Young Rats.

Grape pomace (GP), the residue of grapes after wine making, is rich in dietary polyphenols and fiber, and it has potential to serve as a functional food ingredient to improve health. However, high polyphenol diets have also been reported to inhibit the growth of young animals and cause liver necrosis. This study investigated the effect of diets containing different amounts of GP on the growth performance and blood lipid profile by using a young rat model. Twenty female Sprague-Dawley rats of age 7 weeks were randomly divided into four groups that were fed AIN-93G diets that were modified by substituting 0%, 10%, 20%, and 30% of carbohydrate with GP for 10 weeks (the diets, thus, obtained contained 0%, 6.9%, 13.8%, and 20.7% of GP). The group fed original AIN-93G (0% GP) was used as control. Feed consumption, body weight, length, and height were recorded weekly. Blood samples were taken biweekly to analyze plasma lipid profile. At the end of the feeding period, the rats were fasted overnight and euthanized by exsanguination under anesthesia. Livers, hearts, and kidneys were collected, and their weights were recorded. Results show that the diet containing a maximum of 20.7% of GP did not influence the body weights, lengths, and heights of rats. As the GP content increased, the blood triglyceride and very low-density lipoproteins (VLDL) decreased, the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) increased slightly but were statistically significant, and total cholesterol remained constant. In conclusion, GP in the AIN-93G diet did not influence the growth performance of young rats, but it exhibited both positive and negative effects on the blood lipid profile.

Assessment of endogenous 25-hydroxyvitamin D serum concentrations by liquid chromatography-tandem mass spectrometry in various animal species.

BACKGROUND: Mass spectrometry (MS) has become the preferential method for the analysis of vitamin D in the clinic, yet no single platform is utilized for preclinical species in drug development studies. For vitamin D, the MS platform can provide certain benefits such as applicability of a single assay for multiple species, low cost, and high specificity. OBJECTIVES: A quantitative liquid chromatography-tandem MS (LC-MS/MS) assay for 25-hydroxyvitamin D3 (25OHD3 ) and D2 (25OHD2 ) was validated for rat, dog, mouse, and monkey, and suitability for drug development studies was assessed. METHODS: Standards were used to determine intra- and inter-assay accuracy and precision for LC-MS/MS. Extraction recovery and carryover due to instrumentation were determined. Repeat analyses of pooled serum samples from rat, dog, mouse, and monkey were assessed for precision, and other serum samples were used to determine the normal range in each species and detect biologically relevant changes. RESULTS: For both 25OHD3 and 25OHD2 , inaccuracy was ≤ 6%, and imprecision was ≤ 13%. Extraction recovery was 75% for 25OHD3 and 72% for 25OHD2 , and carryover was ≤ 0.1%. Measurable concentrations of 25OHD3 were recorded in serum samples from all species tested, but no 25OHD2 as diets were only fortified with 25OHD3 . This dataset provides preliminary information for the determination of RIs for 25OHD3 in rat, dog, mouse, and monkey with the LC-MS/MS platform. CONCLUSIONS: The LC-MS/MS assay was accurate and precise for determination of endogenous concentrations of 25OHD3 in serum samples from drug development studies in rat, dog, mouse, and monkey.

Reference intervals for biochemical blood variables, packed cell volume, and body temperature in pet rats (Rattus norvegicus) using point-of-care testing.

BACKGROUND: Rats (Rattus norvegicus) are increasingly kept as pets, thus more and more requiring veterinary care. Important differences exist between pet and laboratory rats, but little information is available on pet rats. Diagnosis and treatment of pet rat diseases would benefit from RIs specific for this animal population. OBJECTIVES: The aim was to establish RIs for biochemical blood variables, PCV, and body temperature in pet rats. Additionally, influences of sex and feed rationing method (ad libitum, restricted) on these variables were determined. METHODS: American Society for Veterinary Clinical Pathology (ASVCP) recommendations were followed. Biochemical variables were measured using an automated chemistry analyzer, the VetScan VS2. Nonparametric statistical methods were used to determine RIs and effects of sex and feed rationing method. RESULTS: Reference intervals were established using values of 123 clinically healthy pet rats (except for globulins and albumin/globulin ratio, n = 120) and were: glucose 6.6-13.7 mmol/L, total proteins 66-88 g/L, albumin ≥ 38 g/L, globulins 12-35 g/L, albumin/globulin ratio 1.0-4.7, urea nitrogen 2.5-6.6 mmol/L, creatinine ≤ 53 µmol/L, total bilirubin 4-7 µmol/L, ALP 40-442 IU/L, ALT 22-137 IU/L, amylase 502-1113 IU/L, sodium 133-144 mmol/L, potassium 3.6-5.3 mmol/L, calcium 9.5-10.9 mg/dL, phosphorus 2.3-7.0 mg/dL, PCV 40-50%, and body temperature 35.8-39.3°C. Sex significantly affected 10 variables. No significant influence was found with feed limitation CONCLUSIONS: Reference intervals reported in this study will be useful for interpretation of biochemistry analysis in pet rats and therefore improve pet rat medicine.

Hematologic and Biochemical Reference Values of the Australian Bush Rat (Rattus fuscipes).

We provide hematologic (n = 34) and biochemical (n = 30) blood values for wild-caught Australian bush rats (Rattus fuscipes). Hematology values have similar range limits compared with other rat species. Biochemistry values for glucose, alanine transaminase, aspartate aminotransferase, and creatine kinase have higher maximum ranges compared with other rats.

Plasma amino acid profiles at various reproductive stages in female rats.

We measured the plasma levels of amino acids at various reproductive stages in female rats, including the estrous cycle, pregnancy and lactation, and compared the resulting amino acid profiles using two- or three-dimensional figures. These figures revealed that the amino acid profiles of pregnant and lactating dams differed considerably from those during the estrous cycle or in male rats. The plasma levels of individual amino acids were almost the same between proestrus, estrus, metestrus and diestrus, and their profiles did not differ significantly. However, the amino acid profiles changed during pregnancy and lactation in dams. The plasma Ser level decreased significantly in mid and late pregnancy, whereas Tyr, Gly and His decreased significantly in the late and end stages of pregnancy, and Trp and Lys significantly decreased and increased at the end of pregnancy, respectively. Much larger changes in amino acid profiles were observed during lactation, when the levels of many amino acids increased significantly, and none showed a significant decrease. Plasma Pro, Ser and Gly levels increased continuously from day 1 until day 15 of lactation, whereas Asn and Met increased significantly from days 1 and 5 respectively until the end of lactation. These results suggest that the profiles of plasma amino acids show characteristic changes according to reproductive stage and that it may be necessary to consider such differences when performing amino acid-based diagnosis.

Plasma cholinesterase activity of rats, western grey kangaroos, alpacas, sheep, cattle, and horses.

INTRODUCTION: Plasma cholinesterase activity levels of various species may be of interest to toxicologists or pathologists working with chemicals that interfere with the activity of plasma cholinesterase. METHODS: We used a pH titration method to measure the plasma cholinesterase activity of six mammalian species. RESULTS: Plasma cholinesterase activity varied up to 50-fold between species: sheep (88 ± 45 nM acetylcholine degraded per ml of test plasma per minute), cattle (94 ± 35), western grey kangaroos (126 ± 92), alpaca (364 ± 70), rats (390 ± 118) and horses (4539 ± 721). DISCUSSION: We present a simple, effective technique for the assay of plasma cholinesterase activity levels from a range of species. Although labour-intensive, it requires only basic laboratory equipment.

Overview of pharmacokinetics.

In addition to having selective potency against the molecular target(s), a compound must be able to reach its intended site of action in vivo in sufficient quantity and for the appropriate duration of time to exert a biological effect. The fate of a compound after in vivo administration depends upon its absorption, distribution, metabolism, and excretion (ADME), as well as its target residence time. The concentration of the compound in the blood, plasma, and other tissues represents the sum of all of these processes. Described in this unit are protocols for administering a compound by various routes to rats and for collecting the appropriate samples to determine the pharmacokinetics profile. The basic terms used in pharmacokinetics studies are defined, and representative examples are given to illustrate important variables to consider.

Development and validation of a sensitive and selective LC-MS/MS method for the determination of trans δ-veniferin, a resveratrol dehydrodimer, in rat plasma and its application to pharmacokinetics and bioavailability studies.

In this study, an accurate and reliable LC-MS/MS assay was firstly developed and validated for the quantitative determination of trans δ-veniferin (TVN) in rat plasma. Chlorzoxazone was used as the internal standard (IS). After one-step protein precipitation with methanol, the analyte and IS were separated on an ODS column by gradient elution with mobile phase of acetonitrile-0.2% formic acid at a flow rate of 0.3mL/min. Negative electrospray ionization was performed using multiple reactions monitoring (MRM) mode with transitions of m/z 453.0>410.9 for TVN, and m/z 168.0>132.0 for IS. Good linearity (R≥0.996) was observed over the concentration range of 5-5000ng/mL for TVN with a lower limit of quantification (LLOQ) of 5ng/mL. The mean recoveries for TVN and IS were 91.05% and 96.68%, respectively. The intra- and inter-day precisions (RSD) were no more than 10.5% and accuracies (RE) were within the range of -6.3% to 2.1%. The validated method was suitable for quantification of TVN and successfully applied to the pharmacokinetic study of TVN after oral and intravenous administration to rats. The oral absolute bioavailability of TVN was 14.2% in rat.

Determination of mesoridazine by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study in rats.

The object of the present study was to develop and validate an assay method of mesoridazine in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples from rats were prepared by simple protein precipitation and injected onto the LC-MS/MS system for quantification. Mesoridazine and chlorpromazine as an internal standard (IS) were separated by a reversed phase C18 column. A mobile phase was composed of 10mM ammonium formate in water and acetonitrile (ACN) (v/v) by a linear gradient system, increasing the percentage of ACN from 2% at 0.4min to 98% at 2.5min with 4min total run time. The ion transitions monitored in positive-ion mode [M+H](+) of multiple-reaction monitoring (MRM) were m/z 387>126 for mesoridazine and m/z 319>86 for IS. The detector response was specific and linear for mesoridazine at concentrations within the range 0.001-4µg/ml and the correlation coefficient (R(2)) was greater than 0.999 and the signal-to-noise ratios for the samples were ≥10. The intra- and inter-day precision and accuracy of the method were determined to be within the acceptance criteria for assay validation guidelines. The matrix effects were approximately 101 and 99.5% from rat plasma for mesoridazine and chlorpromazine, respectively. Mesoridazine was stable under various processing and/or handling conditions. Mesoridazine concentrations were readily measured in rat plasma samples after intravenous and oral administration. This assay method can be practically useful to the pharmacokinetic and/or toxicokinetic studies of mesoridazine.

Absorption and metabolism of piceatannol in rats.

Piceatannol (trans-3,3',4,5'-tetrahydroxystilbene), a natural analogue of resveratrol, has multiple biological functions. Nevertheless, piceatannol's biological fate is yet to be determined. In this study, we evaluated the absorption and metabolism of piceatannol in rats. Furthermore, the area under the plasma concentration curves (AUC) and metabolic pathway of piceatannol were compared with those of resveratrol. We determined the plasma concentrations of piceatannol, resveratrol, and their respective metabolites following their intragastric administration. Resveratrol metabolites were only conjugates, whereas piceatannol metabolites were piceatannol conjugates, O-methyl piceatannol, and its conjugates. The AUC for piceatannol, resveratrol, and their metabolites increased in a dose-dependent manner (90-360 µmol/kg). The AUC for total piceatannol was less than that for total resveratrol, whereas the AUC for piceatannol (8.6 µmol·h/L) after piceatannol and resveratrol coadministration was 2.1 times greater than that for resveratrol (4.1 µmol·h/L). The greater AUC for piceatannol was a result of its higher metabolic stability.

Resistance tests and field trials with bromadiolone for the control of Norway rats (Rattus norvegicus) on farms in Westphalia, Germany.

BACKGROUND: The aim of this study was to determine the incidence and the level of resistance to bromadiolone among rats on farms suspected of being foci of resistance by using the international normalised ratio (INR)-based blood clotting response (BCR) test. Whether the level of reduced susceptibility constitutes 'practical resistance' was subsequently determined in field trials. RESULTS: The 2.5 multiple of the ED(50) baseline was used to test for the incidence of resistance, and higher multiples in the range of the suspected resistance factor were used to investigate the degree of resistance. The ED(50) values of bromadiolone in resistant rats were confirmed in the range 4.70-7.05 mg kg(-1) for males and 4.62-6.61 mg kg(-1) for females. Variations within these ranges appeared between farms. According to the BCR resistance tests, 50-100% of rats were classified as resistant prior to the field trials; 29-100% of rats survived the treatments. CONCLUSION: BCR tests based on the use of the INR and baselines are suitable for determining the incidence and for assessing the level of resistance in populations of Norway rats. The majority of rats of the Westphalian resistant strain, characterised by the Y139C marker in VKOR, are resistant to bromadiolone under practical control conditions.

Vitamin K requirement and reproduction in bromadiolone-resistant Norway rats.

BACKGROUND: Nucleotide polymorphisms in the VKORC1 gene can be linked to anticoagulant rodenticide resistance in Norway rats (Rattus norvegicus Berkenhout). This provides a fitness advantage to rats exposed to anticoagulant actives, but may also cause fitness costs. The vitamin K requirement and reproductive parameters of bromadiolone-resistant rats (Westphalian resistant strain; VKOR variant Tyr139Cys) and bromadiolone-susceptible Norway rats were compared. RESULTS: At vitamin K deficiency, blood clotting times increased in all homozygous resistant males within 8 days and in 80% of homozygous resistant females within 15 days. There was little effect on blood clotting in heterozygous males and no effect in heterozygous females and VKOR wild-type individuals. Litter size was about 20% higher in sensitive pairs compared with resistant pairs. Testes growth, male gonad weight, sperm motility and testis cell concentration were unaffected by the mutation. CONCLUSIONS: The VKOR variant Tyr139Cys causes considerable physiological cost in Norway rats in terms of vitamin K requirement and reproduction. This may affect the distribution and spread of resistant individuals in the wild. Decreased litter size of resistant parents seems to be due to lowered female reproductive performance, as there was no significant effect of the mutation on any aspects of male reproduction considered, but this requires further study.

Stability of hematologic analytes in monkey, rabbit, rat, and mouse blood stored at 4°C in EDTA using the ADVIA 120 hematology analyzer.

BACKGROUND: The time from sampling to analysis can be delayed when blood samples are shipped to distant reference laboratories or when analysis cannot be readily performed. OBJECTIVE: The objective of this study was to evaluate the stability of hematologic analytes in blood samples from monkeys, rabbits, rats, and mice when samples were stored for up to 72 hours at 4°C. METHODS: Blood samples from 30 monkeys, 15 rabbits, 20 rats, and 30 mice were collected into EDTA-containing tubes and were initially analyzed within 1 hour of collection using the ADVIA 120 analyzer. The samples were then stored at 4°C and reanalyzed at 24, 48, and 72 hours after collection. RESULTS: Significant (P<.0003) changes in hematologic analytes and calculations included increased HCT and MCV and decreased MCHC and cell hemoglobin concentration mean (CHCM) at 72 hours and increased MPV at 24 hours in monkeys; increased MCV at 72 hours and MPV at 48 hours and decreased monocyte count at 24 hours in rabbits; increased MCV and decreased MCHC, CHCM, and monocyte count at 24 hours in rats; increased MCV, red cell distribution width, and MPV and decreased MCHC, CHCM, and monocyte count at 24 hours in mice. CONCLUSIONS: Although most of the changes in the hematologic analytes in blood from monkeys, rabbits, rats, and mice when samples were stored at 4°C were analytically acceptable and clinically negligible, the best practice in measuring hematologic analytes in these animals is timely processing of blood samples, preferably within 1 hour after collection.