Simple Cannulation Procedure for Serial Blood Sampling Through Cutaneous Ulnar Vein in Chickens

The objective of the study was to collect repeated, low-stress blood samples from the ulnar vein of chickens required for pharmacokinetic studies or hormonal assays. The study used 5 apparently healthy, unsexed, commercial broiler chickens about 6 weeks old and weighing 1.7–1.9 kg for serial sampling of blood. The study prepared the birds prior to cannulation and penetrated the catheter through the skin and into the lumen of the ulnar vein. The study successfully carried out serial blood samplings in 4 of 5 cannulated birds. Heparin (10%) solution maintained patency and prevented blood clot formation inside the cannula. However, the study found repeated clotting occurring in 1 bird. Cannula failed to maintain patency; the study could not carry out blood sampling properly, which was attributed to air embolism that might have occurred during catheter manipulation or repeated filling of cannula with heparin solution. The study observed no hematoma or inflammation at the site of cannulation. Owing to the advantages and to facilitate compliance with nonhuman animal welfare, this technique seems simple and efficient, allowing adoption for serial blood collection in chickens.     

Simple cannulation procedure for serial blood sampling through cutaneous ulnar vein in chickens

The objective of the study was to collect repeated, low-stress blood samples from the ulnar vein of chickens required for pharmacokinetic studies or hormonal assays. The study used 5 apparently healthy, unsexed, commercial broiler chickens about 6 weeks old and weighing 1.7-1.9 kg for serial sampling of blood. The study prepared the birds prior to cannulation and penetrated the catheter through the skin and into the lumen of the ulnar vein. The study successfully carried out serial blood samplings in 4 of 5 cannulated birds. Heparin (10%) solution maintained patency and prevented blood clot formation inside the cannula. However, the study found repeated clotting occurring in 1 bird. Cannula failed to maintain patency; the study could not carry out blood sampling properly, which was attributed to air embolism that might have occurred during catheter manipulation or repeated filling of cannula with heparin solution. The study observed no hematoma or inflammation at the site of cannulation. Owing to the advantages and to facilitate compliance with nonhuman animal welfare, this technique seems simple and efficient, allowing adoption for serial blood collection in chickens.     

Opioidergic control of luteinizing hormone release in the female rabbit: influence of ovariectomy and steroid replacement on pulsatile secretion

The influence of ovariectomy and steroid replacement on naloxone-induced changes in pulsatile secretion of luteinizing hormone (LH) in the female rabbit was examined. Blood samples were taken every 5 min through an indwelling catheter in the rabbit ear artery, and plasma was stored until assayed for LH by established radioimmunoassay procedures. In the intact animal, saline injection had no effect on LH secretion. Although naloxone (10 mg/kg) caused a 7-fold increase in mean LH pulse amplitude by 30 min after injection, this increase was not statistically significant because 5 of 11 animals did not respond. In animals ovariectomized 48 h previously, naloxone significantly increased LH concentration by 194% at 23 min after injection. When long-term ovariectomized rabbits were treated with estradiol benzoate and then were given naloxone, no significant increase in LH was observed, although many animals did respond. Treatment of long-term ovariectomized rabbits with 1 microgram estradiol benzoate and 100 micrograms progesterone or 1 mg testosterone propionate on Days 1 and 3 and naloxone on Day 4 resulted in a significant increase in LH 19-24 min later. Although there was an increase in pulse amplitude, no change was detected in pulse frequency after naloxone. These data suggest that the hypothesis of steroid-opioid coupling in the control of LH secretion is not applicable to the female rabbit.     

Femoral Arterial and Venous Catheterization for Blood Sampling,Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).     

Eradication of ear mites from naturally infested conventional research rabbits using ivermectin

Rabbits naturally infested with ear mites were treated with ivermectin injection for cattle, subcutaneously at the rate of 400 mcg/kg; which was repeated in 15 to 17 days. Rabbits treated as described and housed in a conventional vivarium environment were found to be free of mites during a subsequent 33 to 139 day observation period. Side effects were minimal and associated with occasional transient discomfort at the injection site. Ivermectin appears to be safe and effective for treating rabbits with ear mites. The prospects of eradicating mites from infested rabbit colonies using this method of treatment is promising.     

眼镜蛇伤模型造模探讨及多项指标的动态观察

目的 探讨眼镜蛇伤模型的造模方法并观察模型多项指标的动态变化。方法 用中华眼镜蛇毒于家兔左小腿作皮下注射制作眼镜蛇伤模型,并分别于注蛇毒前1天,注蛇毒后6h,24h和7天从家兔耳缘静脉采血作血液和生化等多项指标的检查。结果 与生理盐水组相比,眼镜蛇毒组的家兔在注毒后数小时至24h可出现炎症反应和急性弥漫性血管内凝血(DIC),心功能及水盐代谢亦受到一些影响。结论 利用家兔可制作出眼镜蛇伤模型。     

Comparison of clinical pathology parameters with two different blood sampling techniques in rats: retrobulbar plexus versus sublingual vein

Blood samples were taken from the retrobulbar venous plexus or the sublingual vein of male HamIbm:Wist rats to compare clinical pathology parameters between the two sampling techniques. By analogy with a pharmacokinetic study, blood was sampled six times during one day from unfasted animals. After 3 weeks of recovery, blood was taken from fasted animals on a single occasion. In addition, prolactin and corticosterone levels were determined to compare stress-related effects between the two sampling methods. Body weight development and food consumption were similar after single as well as after repeated blood sampling for the two blood sampling techniques. Haemotological evaluation showed a gradual decrease in erythrocyte count, haemoglobin concentration and haematocrit after repeated blood sampling. Repeated withdrawal of blood samples over 24 h corresponding to approximately 22% of the total blood volume resulted in a decrease in red blood cell parameters by up to 30%. The withdrawal of approximately 10% of the total blood volume was associated with a decrease in these parameters by up to 10% and should not be exceeded for animal welfare reasons and to allow a reliable evaluation of data in a study. Repeated blood sampling was associated with an initial decrease in the number of white blood cells, mainly due to a reduction in lymphocytes; white blood cell counts were slightly increased one day after. The decrease in lymphocytes and the increase in neutrophils after repeated sampling were generally slightly more pronounced in the blood from the retrobulbar plexus than from the sublingual vein. Comparison of serum clinical chemistry data showed significantly higher activities of creatine kinase and aspartate aminotransferase in samples from the retrobulbar plexus. These findings suggest a higher degree of tissue damage with blood sampling from the retrobulbar plexus than from the sublingual vein. Despite a large inter-individual variability, higher mean values of prolactin on each occasion and corticosterone after a single sample in fasted animals indicate a higher stress associated with blood sampling from the retrobulbar plexus.     

Arterial baroreflex modulation of heat-induced vasodilation in the rabbit ear

Ryan, Kathy L., W. Fred Taylor, and Vernon S. Bishop.Arterial baroreflex modulation of heat-induced vasodilation in therabbit ear. J. Appl. Physiol. 83(6):2091-2097, 1997.The purpose of this study was to determinewhether nonthermal baroreflexes arising from cardiopulmonaryand/or arterial baroreceptors modulate rabbit ear blood flow(EBF) during hyperthermia. Intact and sinoaortic-denervated (SAD)rabbits were chronically instrumented with a Doppler ultrasonic flowprobe for measurement of EBF velocity (kHz). During whole body heatingin conscious rabbits, EBF and ear vascular conductance (EVC) increasedas core temperature increased until maximal plateau levels of EBF andEVC were reached. The maximal plateau level of EVC attained during heatstress was lower in SAD than in intact rabbits. Subsequentintrapericardial administration of procaine at maximal EBF blockedcardiac afferents but did not alter EVC in either animal group. In asecond experiment, ramp decreases in mean arterial pressure wereproduced by vena caval occlusion at maximal EBF. In intact rabbits, EBFand EVC decreased linearly as mean arterial pressure fell, but EBF andEVC did not decrease during vena caval occlusion in SAD rabbits. Thusneither pharmacological nor mechanical unloading of cardiacbaroreceptors results in reflex vasoconstriction in the heat-stressedrabbit ear. However, baroreflexes arising from arterial baroreceptorsmay modulate EBF in heat-stressed rabbits.

     

An alternative, less invasive blood sample collection technique for serologic studies utilizing triatomine bugs (Heteroptera; Insecta)

The collection of blood samples for serological studies is often stressful for the focus animal. Recently, the use of bloodsucking bugs, such as Dipetalogaster maximus or Triatoma infestans (Reduviidae; Triatominae; Heteroptera), has been suggested as a new and less invasive method for blood collection. To evaluate this technique, we collected paired blood samples from 20 domestic rabbits (Oryctolagus cuniculus) during a study of rabbit hemorrhagic disease virus (RHDV). For each rabbit, blood samples were collected by the conventional method (needle and syringe from the vena auricularis) and through feeding by D. maximus. Samples were tested for RHDV antibodies using standard test kits at three different dilutions. Antibody titers were identical for 56 paired samples and differed in only four cases. The simple matching indices were 1 for the 1:10 dilution and 0.9 for the 1:100 and 1:1000 dilutions. The major advantages of the new technique are 1) the possibility to obtain blood from animals where veins are inaccessible and 2) the fact that anesthesia of focus animals may not be necessary.     

Evolution of glycogen and blood metabolites after controlled suckling at two periods of lactation in young rabbits

In natural conditions young rabbit nurses once a day and therefore ingests the whole of his daily caloric intake during a single meal. The present work investigates glucose homeostasis during perinatal period in young rabbit by assessing blood glucose and glycogen stores before and after one single meal. Ponderal data, glycogen and blood metabolites were determined in 1-4 day- and 17-21 day-old rabbits before suckling and at different times (1, 3, 6, 9, 24, 48, 72 h) after controlled suckling. In "young" and "old" rabbits hepatic glycogen stores were exhausted after 48 and 72 h of fast. Within the first hours following milk ingestion, muscle and carcass glycogen did not vary until 9 h in the "young" and until 24 h in the "old" without notable variation of glycemia. From 24 to 72 h young rabbits were in a fasting period with low hepatic glycogen and a decrease of muscle and carcass glycogen, but glycaemia decreased only slightly at 48 and 72 h in "young" and at 72 h in "old" As blood alanine was decreased, it appears that gluconeogenesis was effective and that alanine-glucose and Cori cycles were operating in these conditions.     

In vivo and in vitro CO2 titration curves in the rabbit: adaptation to hypercapnic conditions

Arterial blood acid-base status of unanesthetized, unrestrained rabbits was studied during 6-12 hr of exposure to 7, 10 and 14.5% CO2. Most of the changes in blood acid-base status occurred during the first 20-60 min of exposure to hypercapnia and only minor changes occurred during the remaining exposure period (up to 12 hr). Blood buffer values obtained were not different from those reported for other terrestrial mammals. The whole body buffer values obtained here for the rabbit (0.58 nM H+/mmHg PCO2) is higher than that reported previously for man and dog. This relatively high whole body buffer value complies well with the high tolerance to CO2 reported for the rabbit.     

Experimental Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Background

A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.

Methods

Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.

Findings

Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.

Conclusions

These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.     

A simplified technique for producing an ischemic wound model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 °C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.     

Chronic central vein catheterization for intraoperative and long-term venous access in swine

Chronic venous access and repeated blood sampling for research purposes in large swine ideally should be possible without sedation, restraint or direct venipuncture of deep vessels. An operative technique of cranial vena cava catheterization and chronic catheter maintenance methods are described which were used successfully in the placement of 11 silicone rubber catheters in 10 animals. All were used for repeated blood sampling, as well as intraoperative infusion of medications and large fluid volumes. Long term patency was excellent with 10 catheters patent at the end of the study interval, up to 14 weeks after insertion. Serial blood sampling was accomplished easily without restraint. Catheter damage, infection or malfunction was rare. Proper maintenance and careful aseptic blood sampling render the cranial vena cava catheter a safe and reliable alternative to direct venipuncture in swine.     

Sampling of cardiovascular data; how often and how much?

Long-term measurement of cardiovascular variables by telemetry in laboratory animals has become indispensable in recent years. However, limited battery life and management of large volumes of recorded data are major drawbacks. These limitations can often be overcome by intermittent sampling of data. The question is, how much data does one need to collect to accurately reflect the underlying average value? To investigate this, 24-h continuous recordings of rabbit heart rate, arterial pressure, and integrated renal sympathetic nerve activity (RSNA) were resampled using a variety of protocols that differed with respect to the number of individual sampling periods used and the total amount of time that was sampled. The absolute percentage errors of estimates of the daily mean, standard deviation, and interquartile range were calculated for each sampling protocol. A similar analysis was repeated using arterial pressure data from rats. The results show that the number of sampling periods spread throughout the day had more effect than the total amount of data recorded. For example, just 2 h of total sampling time spread over 12 evenly spaced 10-min periods gave estimates of the daily mean of blood pressure and heart rate with <1% error and RSNA with <3% error. We show that accurate estimates of the daily mean of arterial pressure, heart rate, and RSNA can all be made using scheduled recording, and we recommend recording a minimum of 2 h/day spread over a number of periods throughout the day.     

Chronic silastic central venous catheterization for induction, maintenance and support of persistent granulocytopenia in rabbits

In order to investigate new approaches in diagnosis, prevention and treatment of infectious complicating chemotherapy-induced granulocytopenia, we developed and prospectively evaluated a method of chronic central venous catheterization for the induction, maintenance and support of persistent granulocytopenia in rabbits. The method entails a central venous silastic catheter with a subcutaneous tunnel and a heparin lock device for repeated non-traumatic sampling of blood and administration of medications. During the course of 10 months, 226 rabbits were studied. Mean duration of catheter placement was 27 days, 17 of which were spent in granulocytopenia. Two-way flow was sustained throughout the duration of placement in 205 rabbits (91%) and for 5,845 (95%) of a total 6,163 catheter-days. All but two catheters could be flushed throughout the duration of their placement. Postoperative infectious complications related to catheter insertion developed in less than 1% of the rabbits. This method of chronic catheterization safely provides long-term venous access for studies requiring frequent venous access, including the painless induction, maintenance, and support of chronic granulocytopenia in rabbits.     

Thromboxane-B(2) levels in serum of rabbits receiving a single intravenous dose of aqueous extract of garlic and onion

We have shown previously that fresh garlic extract is effective in reducing thromboxane formation by platelets both in vivo and in vitro animal models of thrombosis. In the present study, the effect of different concentrations of a single dose of aqueous extracts of garlic and onion were evaluated on serum thromboxane-B(2)synthesis in rabbits. Different concentrations of garlic and onion were administered as single doses in the ear vein of rabbits. Rabbits were bled before and at different intervals after the infusion of garlic or onion extracts. Venous blood was collected and allowed to clot at 37 degrees C for 1 h. Thromboxane-B(2)level was measured in the serum by radioimmunoassay. It was observed that garlic inhibits the thrombin-induced platelet synthesis of TXB(2)in a dose-and time-dependent manner. Maximum inhibition of TXB(2)occurred between 0.5 h and 6 h at 25 and 100 mg kg(-1)garlic. At 24 h post-garlic infusion TXB(2)inhibition was reduced to 15% of the control and TXB(2)levels were comparable to that of the control values at 72 h pots-garlic infusion. Infusion of 100 mg kg(-1)onion extract did not elicit any inhibitory effect on TXB(2)synthesis in the serum of rabbit during the treatment period. The rapid recovery of platelet cyclooxygenase activity after infusion of a single dose of garlic suggests that garlic should be taken more frequently in order to achieve beneficial effects in the prevention of thrombosis.     

Utilization of dried blood spots within drug discovery: modification of a standard DiLab(®) AccuSampler(®) to facilitate automatic dried blood spot sampling

The use of dried blood spots (DBS) in preclinical studies has seen an enormous increase over the past two years. Despite its positive impact on the 3Rs (reduce, replace and refine), its uptake in exploratory drug discovery has been limited due mainly to protracted method development time in bioanalysis but also the need for small volumes (<20 μL) to be sampled manually. Automatic blood sampling technology such as the DiLab(?) AccuSampler(?) is widely used in drug discovery to facilitate exploratory rodent-based pharmacokinetic and pharmacokinetic/pharmacodynamic studies with minimal animal handling. Propranolol was orally administered to a Han-Wistar rat attached to either a standard DiLab(?) AccuSampler(?) or a retrofitted unit designed to directly collect the DBS samples. In all, 50 or 20 μL blood samples were then collected via the standard or retrofitted unit, respectively, at six timepoints over a 7 h period. After drying and storage the DBS samples were analysed for propranolol via liquid chromatography-mass spectrometry. In this report we demonstrate that a standard DiLab(?) AccuSampler(?) can be easily retrofitted to facilitate automatic dried blood spot sampling and that time-concentration data generated from these samples are equivalent to that from manually spotted samples.     

Less invasive blood sampling in the animal laboratory: clinical chemistry and haematology of blood obtained by the Triatominae bug Dipetalogaster maximus

Dipetalogaster maximus (Dipmax), a blood-sucking bug belonging to the family Reduviidae, has been used to obtain blood samples, for example for clinical chemistry and haematology, in a variety of zoo animals and wildlife. Using this bug allows stress-free blood sampling as the bug is able to draw blood without the mammal noticing the bug. In laboratory animal science, the need for blood samples from unstressed animals may arise, especially in animal behaviour research. The use of Dipmax bugs may prove a valuable tool for this purpose. To validate the method, we compared an array of standard blood parameters sampled from New Zealand White rabbits, sampled either by the use of bugs or by the conventional method; puncture of vena auricularis caudalis. The overall hypothesis was that there was no significant difference in clinical chemistry and haematological parameters between the bug method and the conventional method. A total of 17 clinical parameters as well as 12 haematological parameters were measured and compared in New Zealand White rabbits. The results showed that for 13 of these 29 analysed parameters, the bug method and the conventional method did not give significantly different results, and the obtained results were thus directly comparable. For the remaining parameters the obtained results were significantly different. However, all parameters were measurable in the bug samples. The influences of the bug metabolism on these parameters are discussed.     

A rapid and non-surgical procedure for jugular catheterization of pigs

A rapid and non-surgical method for jugular catheterization in pigs was set up in 30 piglets of 6.2 kg, 23 pigs of 46 kg and 84 kg and two lactating multiparous sows. The animal was restrained on a V-shaped table (piglets) or with a rope around the mandible (slaughter pigs and sows). The vein was located with the Vacutainer system and a wire guide was inserted into the Vacutainer needle up to the vein lumen. When the needle was removed, the catheter was inserted over the wire guide and advanced until it penetrated the skin and thereafter, the vein wall. The catheter was fixed outside by a large tape and coiled inside a patch just behind the ears. The technique utilizes readily available material and is no more risky for the animal than a single blood sampling. Moreover, it can be performed within 15 to 20 min (including animal restraint) within pens. This new approach might have important implications not only for research purposes by facilitating repeated blood samplings but also for projects which require a rapid and easy method for testing of any kind of pharmaceutical or other type of products under husbandry conditions.