Comparison of sublingual, facial and retro-bulbar blood sampling in mice in relation to animal welfare and blood quality.

INTRODUCTION: Repeated blood sampling is a common procedure in laboratory mice, but at present it is unknown which technique has the least impact on the animals when large or repeated blood samples are required. Retro-bulbar sinus puncture is a reliable technique but has been shown to cause many changes in the animals, why sublingual and facial vein puncture have been suggested as suitable alternatives. This study investigated 1) which of the three blood sampling techniques had the least impact on nest building activity, level of faecal corticosterone metabolites, body weight, fur status, and macroscopic changes, 2) whether the blood sampling techniques gave rise to variation in blood quality between blood samples, and 3) whether sublingual and facial vein puncture should be performed with or without anaesthesia in female C57BL/6 mice. METHOD: Three hundred and sixty C57BL/6 female mice divided into five batches were included in the study and randomized to a short (blood sampling on Day 8, 9 and 10) or a long protocol (blood sampling on Day 8, 15 and 22). Each protocol consisted of six identical groups: sublingual vein puncture (SVP), sublingual vein puncture in isoflurane (SVPiso), facial vein puncture (FVP), facial vein puncture in isoflurane (FVPiso), retro-bulbar sinus puncture (RBP), and a control group (CONTROL) with only scruffing being performed. At baseline (Day 2) nest building activity (NBA) was assessed and faecal pellets collected for evaluation of faecal corticosterone metabolites (FCM). The day after each blood sampling day NBA and FCM were reassessed. RESULTS AND CONCLUSION: None of the blood sampling techniques proved to be superior to the others in any of the measured parameters. Finally, sublingual and facial vein puncture performed under anaesthesia gave rise to variation in the quality of the blood. A refinement of all three techniques are therefore warranted.

Fasting of male mice - Effects of time point of initiation and duration on clinical chemistry parameters and animal welfare.

Fasting of mice is a common procedure, which can affect the outcome of the study as well as animal welfare. In this study, we assess the effects of fasting, fasting duration and fasting initiation time in relation to light schedule and present suggestions for optimization of fasting. Male C57BL/6NCrl mice were fasted for 0, 3, 6, 12, 18 and 24 hours initiated either in the light period (photophase) or the dark period (scotophase). Body weight, gastric content, body temperature, corticosterone and 19 routine clinical chemistry parameters were evaluated. Fasting caused significant changes in most of the measured parameters. Increasing duration of fasting resulted in increasing physiological changes. Fasting initiated in the scotophase caused more significant changes than fasting initiated in the photophase. To cause the least physiological changes in mice and increase animal welfare, mice should preferably be fasted in the photophase and for the shortest possible period allowed by the experimental purpose of fasting.

Endocrine disrupting effects in rats perinatally exposed to a dietary relevant mixture of phytoestrogens.

Dietary phytoestrogens may prevent certain human diseases, but endocrine activity has been reported in animal studies. Sprague-Dawley rats were exposed perinatally to a 1-, 10- or 100-fold "high human dietary intake" mixture of 12 phytoestrogens consisting of mainly the lignan secoisolarici resinol and the isoflavones genistein and daidzein. This mixture induced persistent adverse effects, as adult male mammary glands showed hypertrophic growth. A reduced anogenital distance in newborn males indicated an anti-androgenic mode of action. Testosterone levels, testis and prostate weights, and expression of selected genes in testis and prostate were unaffected. Decreased serum estradiol was seen in genistein-exposed dams. This study indicated adverse effects at high intake levels in rats, but does not provide evidence for risk of phytoestrogen-mediated endocrine disruption at normal human dietary consumption levels. Further studies are warranted to increase the knowledge upon which risk assessment on dietary phytoestrogen exposure during pregnancy and infancy is based.

Impact of blood sampling technique on blood quality and animal welfare in haemophilic mice.

Blood collection in mice can be a challenge, in particular for samples used for coagulation analysis as initiation of coagulation during the procedures can influence the results. Blood collection from the retrobulbar venous plexus is commonly used but the method remains controversial. Several alternatives exist but not all are applicable to mice with a compromised coagulation system because of subsequently excessive bleeding. We therefore wanted to explore whether blood collection by puncture of the submandibular vein could replace blood collected from the retrobulbar venous plexus during pharmacokinetic and pharmacodynamic studies in mice lacking coagulation factor VIII (FVIII). The plasma concentrations of recombinant activated factor VII were independent of the blood collection method in a pharmacokinetic study. The same applied to the thromboelastographic profile of mice with normal coagulation in a pharmacodynamic study. However, excessive haemorrhages were observed in all FVIII knockout mice after a single puncture of the submandibular vein and 60% of the mice were euthanized 2-4 h after the blood collection. In contrast, no or only slight haemorrhage was observed in animals subjected to blood collection from the retrobulbar venous plexus. No signs of distress determined by blood glucose level or clinical abnormalities of the eye were observed after puncture of the retrobulbar venous plexus. In conclusion, blood collected by puncture of the submandibular vein and retrobulbar venous plexus has a quality which allows it to be used in coagulation assays. However, because of excessive bleedings, puncture of the submandibular vein is not recommended in mice lacking FVIII.

Exposure to the widely used fungicide mancozeb causes thyroid hormone disruption in rat dams but no behavioral effects in the offspring.

The widely used fungicide mancozeb has been shown to cause hypothyroxinemia and other adverse effects on the thyroid hormone system in adult experimental animals. In humans, hypothyroxinemia early in pregnancy is associated with adverse effects on the developing nervous system and can lead to impaired cognitive function and motor development in children. The aim of the present study was therefore to assess whether perinatal mancozeb exposure would cause developmental neurotoxicity in rats. Groups of 9-21 time-mated Wistar rats were dosed with 0, 50, 100, or 150 mg mancozeb/kg body weight (bw)/day by gavage from gestation day (GD) 7 to postnatal day (PND) 16, and total thyroxine (T(4)) levels were measured in dams during gestation. On PND 16, hormone levels and several organ weights were measured in the offspring, whereas motor activity, startle response, and cognitive function were assessed in the adult offspring. The dose of 150 mg/kg/day caused neurotoxicity in the pregnant dams and was therefore reduced to 100 mg/kg bw/day in mid study. T(4) levels showed a dose-dependent and significant decrease in dams from all three dose groups on GD 15, whereas offspring T(4) levels, thyroid weights, and histology were unaffected on PND 16. No effects on reproductive organ weights were seen, and no behavioral changes were observed. Taken together, these results indicate that in rats, moderate maternal hypothyroxinemia during gestation does not necessarily lead to hyperactivity or reduced special learning abilities in the offspring. Mancozeb exposure did, however, reduce T(4) levels in dams and may therefore still be a potential contributor to thyroid disruption in humans and in result adversely affects the developing brain.

Evaluation of endocrine disrupting effects of nitrate after in utero exposure in rats and of nitrate and nitrite in the H295R and T-screen assay.

Animal studies have shown that nitrate acts as an endocrine disrupter affecting the androgen production in adult males. This raises a concern for more severe endocrine disrupting effects after exposure during the sensitive period of prenatal male sexual development. As there are no existing studies of effects of nitrate on male sexual development, the aim of the study was to examine how in utero exposure to nitrate would affect male rat fetuses. Pregnant dams were dosed with nitrate in the drinking water from gestational day (GD) 7 to GD21 at the following dose levels 17.5, 50, 150, 450, and 900 mg/l. At GD21, fetuses were examined for anogenital distance, plasma thyroxine levels, testicular and plasma levels of testosterone and progesterone, and testicular testosterone production and histopathology. In addition, endocrine disrupting activity of nitrate and nitrite were studied in two in vitro assays, the H295R assay and T-screen. There were no consistent indications that nitrate induces anti-androgenic effects in male fetuses or that prenatal nitrate exposure affected the thyroid axis. However, a more comprehensive study with long-term exposure before and during pre- and postnatal development would be relevant to sufficiently address the concerns based on the indications for endocrine disrupting effects in adult animals.

Dysgenesis and histological changes of genitals and perturbations of gene expression in male rats after in utero exposure to antiandrogen mixtures.

We investigated the ability of a mixture of three androgen receptor antagonists to induce disruption of male sexual differentiation after perinatal exposure. The aim was to assess whether the joint effects of vinclozolin, flutamide, and procymidone can be predicted based on dose-response data of the individual chemicals. Chemicals were administered orally to pregnant Wistar rats from gestational day 7 to postnatal day 16. Changes in reproductive organ weights and of androgen-regulated gene expression in prostates from male rat pups were chosen as end points for extensive dose-response studies. With all end points, the joint effects of the three antiandrogens were dose additive. Histological evaluations showed that dysgenesis and hypoplasia of prostates, seminal vesicles, and epididymis were seen with the highest mixture doses. No changes were observed in any single-compound low-dose group for these lesions, nor were there histopathological changes in the testes. Pronounced dysgenesis of external genitals was observed with all doses of the mixture, and severe dysgenesis was seen with a mixture for which the individual compounds caused no effects. A combination of doses of each chemical that on its own did not produce significant reductions in the weights of seminal vesicles and PBP C3 expression induced a marked mixture effect. Thus, antiandrogens cause additive effects on end points of various molecular complexities such as alterations at the morphological and the molecular level. Exposure to antiandrogens, which appears to exert only small effects when judged on a chemical-by-chemical basis, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.