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.     

Taking the stress out of blood collection: comparison of field blood‐sampling techniques for analysis of baseline corticosterone

Many ecological studies use stress hormones to assess the condition, health or disturbance levels of wild organisms. Common blood sampling protocols for this research involve trapping individuals and taking blood within three minutes to obtain a “baseline” for analysis of stress hormones (“conventional method”). In some situations it may be difficult to get an accurate measure of baseline values; therefore, alternative sampling techniques may be preferable. We compared corticosterone levels in samples taken via a newly developed, minimally invasive blood sampling technique with corticosterone levels in blood taken via the conventional method. We collected samples from incubating adult common terns Sterna hirundo via blood sucking bugs (Heteroptera, Triatominae) contained in “dummy eggs” (“bug method”) and compared measured corticosterone concentrations to concentrations in blood taken from the same birds using the conventional method. We found no significant differences in mean or variance of baseline corticosterone levels between samples collected via the different methods. This suggests that the bug method offers a viable alternative for hormone sampling.     

The effect of buprenorphine on the course of disease in laboratory rabbits infected with myxoma virus

The only method of assessing the virulence of myxoma virus is to record survival times of rabbits inoculated with the virus. This raises ethical concerns about using animals in experiments where death is the end point. We investigated whether or not the opioid analgesic buprenorphine could be used in rabbits without compromising the myxoma virus virulence assay and on the presumption that animals may suffer pain during the course of the disease. Thirty, 5-month-old New Zealand White rabbits were divided into two groups stratified for weight and gender, and inoculated intradermally with 100 pfu of the Standard Laboratory Strain (SLS) of myxoma virus. At day 6 post infection (p.i.), when eyelid swelling was first seen, each animal in one group was treated with 0.03 mg/kg buprenorphine, subcutaneously, morning and evening until death. Animals in the other group were untreated. Animals were weighed daily and rectal temperatures taken morning and evening. Intake of food and water was assessed as was general demeanor including respiratory effort. There was no significant difference in mean survival time, weight change, or demeanor between the two groups. Increased respiratory effort was seen from day 10 p.i. in animals surviving up to and beyond that time but again there was no difference between groups. Animals treated with buprenorphine refused food and water a day earlier than untreated animals, and had lower temperatures immediately prior to death. It was concluded that the opiate analgesic buprenorphine can be used without compromising the current virulence assay for the SLS of myxoma virus in New Zealand White rabbits but that the clinical signs of myxomatosis that could be attributed to pain were not abrogated.     

Immunization with a peptide of Sm B/B' results in limited epitope spreading but not autoimmune disease

An experimental model of systemic lupus erythematosus has recently been described in normal animals. We sought to confirm and extend this model, which involved immunization of normal rabbits and mice with a peptide of Sm B/B', PPPGMRPP. This peptide is an early target of the immune response in anti-Sm-positive patients with lupus. The peptide was used in a multiple Ag peptide format, with multiple copies of PPPGMRPP bound to an inert lysine backbone. New Zealand White rabbits and A/J and C57BL/10ScSn mouse strains were immunized with PPPGMRPP-MAP. Pepscan assays were used to determine the epitope spreading of the anti-PPPGMRPP-MAP response to other octamers of SmB/B' following immunization. We obtained high titer anti-PPPGMRPP-MAP IgG responses in the New Zealand White rabbits and A/J mice. The rabbits immunized with PPPGMRPP-MAP showed varying degrees of epitope spreading, while the A/J mice showed no spreading. We observed no autoantibodies to dsDNA or other anti-nuclear autoantibodies in our animals by ELISA or immunofluorescence, although anti-nuclear autoantibodies were found by Western blotting in some of the rabbits. No evidence of clinical disease was seen in our normal animals. These data underline the difficulties often associated with the reproduction of animal models in different laboratories.     

Characterization of variations in rabbit hepatic progesterone 21-hydroxylase activity by serial biopsy

Earlier work has shown that the 21-hydroxylation of progesterone in the hepatic microsomal fraction of outbred New Zealand White rabbits varies over a 10-fold range. To determine whether the differences in 21-hydroxylase activity were due to a transient inductive effect, livers from a group of 28 rabbits were serially biopsied at least three times over a minimum period of two months. Both progesterone 21- and 16 alpha-hydroxylase activities were determined in the post-8700g supernatant of homogenates prepared from these biopsy samples. A substantial variability in both the 21- and 16 alpha-hydroxylase activity was observed for serial biopsy samples from individual rabbits. Each animal was found, however, to maintain relatively constant ratios of 21/16 alpha-hydroxylase activity throughout the course of the study. Previous studies have indicated that the 21-hydroxylase activity does not correlate with the 16 alpha-hydroxylase activity and that the 21-hydroxylase phenotype could be determined from the ratio of these activities. On the basis of this ratio, two groups of animals could be distinguished in the present study. Approximately 25% of the animals exhibited an elevated 21/16 alpha-hydroxylase ratio (greater than 1.5), the remainder were below this level. Furthermore, the expression of elevated levels of the 21-hydroxylase activity were found to be consistent within this subpopulation suggesting that a transient inductive effect is not responsible for the differences in 21-hydroxylase activity among populations of outbred New Zealand White rabbits. This study demonstrates the determination of the hepatic enzymatic phenotype while maintaining the animal for long periods of time and for subsequent investigations.     

不同品种实验兔血液流变学特性

目的分析比较市售中国白兔与我院自繁培育的封闭群新西兰白兔血液流变学特性的差异。方法采集新西兰白兔和中国白兔的血液样品,分别检测比较其WBV（高、中、低切变率）、PV、HCT、ESR等14项血液流变学指标。结果 （1）新西兰白兔与中国白兔不同性别间血液流变学特性没有显著性差异（P〉0.05）;（2）新西兰白兔和中国白兔血液流变学指标中,血浆粘度ηP,还原粘度低切Rls,还原粘度中切Rms,还原粘度高切Rhs及红细胞电泳指数RIE5个指标间有显著差异（P〈0.05）。其余9项指标差异不显著（P〉0.05）。结论实验兔的血液流变学特性与品种有一定的相关性。     

Lymph node topography of the head and neck in New Zealand White rabbits

Investigations of the lymphogenic metastatic spread of VX2 carcinomas in New Zealand White rabbits require an exact knowledge of the topography of cervical and facial lymph nodes. The topography of neck lymph nodes was evaluated from 16 rabbits macroscopically, histologically and by lymphographic investigations, and the possibility of their surgical removal (neck dissection) was examined. The upper aerodigestive tract and the ear of New Zealand White rabbits drain via four consistent groups of 12-18 lymph nodes. Except for the paratracheal lymph node, they are all easily accessible to surgery. The data presented in this study encourage the use of induced VX2 carcinomas in New Zealand White rabbits as an animal model to study the lymphogenic metastatic spread of squamous cell carcinomas of the head and neck. Such investigations could lead to an improvement of surgical and pharmaceutical treatment of this tumour entity.     

Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.     

A Diet High in Saturated Fat and Sucrose Alters Glucoregulation and Induces Aortic Fatty Streaks in New Zealand White Rabbits

A new and convenient animal model for studying peripheral vascular and coronary artery disease in diabetes was established in this study. Male New Zealand White rabbits weighing approximately 2 kg were divided into 2 groups: a normal control group fed standard laboratory chow and a diabetogenic diet–fed group received a high-fat/high-sucrose diet. The high-fat/high-sucrose diet (contained 10% lard and 37% sucrose) feeding was maintained for 6 months. Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, superoxide dismutase, nitric oxide, nitric oxide synthase, insulin, and glucose were quantitated at monthly or bimonthly intervals. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. The aortic samples were observed by electron microscopy. High plasma triglyceride and glucose concentrations were induced. At the end of 6 months, the aortic fatty streak lesions were present in the animals'' vascular specimens. As far as we know, this is the first report that demonstrates that New Zealand White rabbits can develop obvious aortic fatty streaks by feeding a high-fat/high-sucrose diet. Our results suggest that NewZealand White rabbits fed a high-fat/high-sucrose diet would provide a convenient model for studying peripheral vascular and coronary artery disease in diabetes.     

Nephrotoxicity of tiletamine in New Zealand white rabbits.

Tiletamine and zolazepam, the two constituents of Telazol, were evaluated independently to determine which agent was responsible for the nephrotoxicity caused by Telazol in New Zealand White rabbits. Five rabbits were injected i.m. with 32 mg/kg of tiletamine, four animals received 7.5 mg/kg of tiletamine, and five rabbits received 32 mg/kg of zolazepam. Urinalysis was performed and blood urea nitrogen and serum creatinine were monitored for 7 days postinjection. In all five rabbits injected with the high dose of tiletamine, blood urea nitrogen and creatinine rose by 3 days postinjection and increased steadily throughout the week. By 4 days postinjection, urine protein and glucose were elevated and cellular and protein casts were present. No serum chemistry or urine abnormalities were detected in rabbits receiving low doses of tiletamine, zolazepam, or in the four control rabbits. All animals were euthanized and necropsied at 7 days postinjection. Histopathology showed severe renal tubular necrosis in all five rabbits injected with 32 mg/kg tiletamine. Mild nephrosis was present in three of four rabbits injected with 7.5 mg/kg of tiletamine. No lesions were present in the zolazepam-injected or control rabbits. The results of this study show that tiletamine is the constituent responsible for the nephrotoxicity of Telazol in rabbits. They further demonstrate that doses commonly used for anesthetic induction or restraint can produce renal lesions in rabbits.     

Pair housing of rabbits reduces variances in growth rates and serum alkaline phosphatase levels

New Council of Europe regulations mandate housing of two rabbits in the same cage space currently used to house one, provided the animals are socially compatible. This study was designed to assess changes in growth and selected serum chemistry parameters due to pair housing or single housing of rabbits. Six sets of four female siblings of Crl:KBL(NZW)BR rabbits were used. The animals were seven weeks old on arrival. Two siblings of each set were allocated to pair housing, two to single housing. The animals were housed in stainless steel cages (120 cm x 60 cm x 60 cm) with a perforated floor, including a shelf (60 cm x 30 cm) at 30 cm height from the floor. The rabbits were provided with an aspen cube (5 cm x 5 cm x 5 cm), one item per animal. The rabbits were weighed and blood samples were taken from the auricular central artery at four different times during the study. Blood sera were assayed for a set of routinely assayed clinical chemistry parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (APHOS), blood urea nitrogen (BUN), cholesterol (CHOL) and protein (PROT). Mean and variance profiles over the study period were statistically analysed by multivariate analysis of variance. No differences in mean profiles were detected; however, weight (P = 0.0002) and APHOS (P = 0.017) variances were significantly lower in pair-housed animals. The reduction in variance on growth and APHOS attributable to pair housing appears to be rather large. During the 21-week study, occasional fighting was seen between the pair-housed rabbits. After sexual maturity, further major fighting bouts resulted in significant trauma that necessitated the cessation of the study. In conclusion, pair housing appears to have a decreasing effect on growth and APHOS variance, but antisocial behaviour such as fighting remains a serious problem.     

A method of endoscopic endotracheal intubation in rabbits.

Endotracheal intubation in rabbits is a formidable task and has led to the devising of a new endoscopic method. Sixty New Zealand White rabbits (2.0-3.5 kg) underwent experimental left thoracotomy requiring mechanical ventilation. After anaesthetic induction was achieved, the animal was placed in a supine position. A rigid 30 degrees endoscope was passed through a 4.5 mm endotracheal tube (ET) and then used to advance, under direct vision, through the oropharynx and vocal cords. Once passing the vocal cords, the ET was advanced into the trachea as the endoscope was withdrawn. All animals were successfully intubated within 30 s to 2 min and then ventilated. No complications or deaths occurred during or after intubation, or postoperatively. No instances of oesophageal intubation occurred. All animals survived long-term. Thirty animals underwent necropsy at 10 days and, 30 at 30 days, with no postoperative evidence of orotracheal injury. We have established a new method of endotracheal intubation in rabbits. It is reliable, effective, non-traumatic, safe, and expeditious.     

Herpesvirus saimiri strains from three DNA subgroups have different oncogenic potentials in New Zealand white rabbits

Herpesvirus saimiri is a primate tumor virus that induces acute T-cell lymphomas in New World monkeys. Strains of this virus have been previously classified into three groups on the basis of extreme DNA variability of the rightmost region of unique L-DNA. To compare the oncogenic potentials of various strains, we inoculated New Zealand White rabbits with viruses representing groups A, B, and C of herpesvirus saimiri. The results showed that a group C strain were highly oncogenic in New Zealand White rabbits; however, group A or B viruses were not oncogenic in these rabbits. Analysis of DNAs of tumor tissues and lymphoid cell lines established from tumors showed that the viral genome exists in circular episomal form. To identify which part of the genome of the group C strain is responsible for the highly oncogenic phenotype, group B-C recombinant strains were constructed by an efficient drug selection technique. Two group B recombinant strains in which the right-end 9.2 kilobase pairs of unique DNA is replaced by group C virus DNA were oncogenic in rabbits, indicating that the rightmost sequences contribute to the oncogenic properties of the group C strain. Oncogenicity of herpesvirus saimiri has been traditionally evaluated in New World monkeys; infection of rabbits with group C strain 484-77 offers a much more accessible animal model to study the mechanism of oncogenicity of this virus.     

An evaluation of analgesia associated with the immobility response in laboratory rabbits

The immobility response (IR) was studied in rabbits to evaluate its analgesic properties and reliability as a method of restraint. The participation of the endogenous opioid system in IR was studied indirectly by evaluating the effects of the narcotic antagonist naloxone on this phenomenon. Twenty-four adult New Zealand White rabbits were subjected to six noxious stimuli while restrained by IR and while restrained under control conditions. Testing on each animal was repeated under both conditions following the administration of naloxone. The noxious stimuli consisted of three levels of electric shock (10 volts, 30 volts, and 50 volts) applied to the shaved forearm, and mechanical pressure applied to the pinna, front toe, and hind toe. Withdrawal and changes in blood pressure, heart rate, and respiration were used as indicators of pain perception. Distress associated with noxious electrical and pressure stimulation was significantly reduced by IR, which suggested that the phenomenon does have a significant analgesic component. However, the rabbits showed wide variability in their susceptibility to IR induction, and even animals which did not withdraw in response to noxious stimulation under IR sometimes exhibited physiological changes suggestive of distress. Therefore, IR should not be considered as a reliable or humane alternative to analgesic/anesthetic drugs for laboratory rabbits. Naloxone had little effect on IR or IR-associated analgesia.     

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.     

Formation of 15-HETE as a major hydroxyeicosatetraenoic acid in the atherosclerotic vessel wall

Atherosclerosis was induced in New Zealand White rabbits through cholesterol feeding. Aortae were taken out from treated animals and incubated with arachidonic acid. Aortae from cholesterol-fed animals converted arachidonic acid into 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). This conversion was not seen in aortae from control animals. The immediate precursor of 15-HETE, 15-HPETE, is an inhibitor of prostacyclin synthetase and might hamper prostacyclin production.     

Blood-sucking bugs as a gentle method for blood-collection in water budget studies using doubly labelled water

During doubly-labelled water (DLW) experiments, blood collection by venous puncture may traumatize animals and consequently affect the animals' behaviour and energy budget. Recent studies have shown that blood-sucking bugs (Triatominae; Heteroptera) can be used instead of conventional needles to obtain blood from animals. In this paper, we validate the bug method in captive nectar-feeding bats, Glossophaga soricina, for water budget analysis by comparing the daily water flux estimated with the DLW method with values measured by an energy balance method. As the mean daily water flux of the DLW method was not significantly deviating from the expected value, blood-sucking bugs may substitute more invasive methods of blood collection in DLW experiments. Based on the DLW estimates, daily energy and water intake rates were calculated and compared to values measured with the energy balance method. The DLW method and the energy balance method yielded on average similar results regarding the daily energy intake (DLW method: 48.8+/-14.2 kJ d(-1) versus energy balance method: 48.1+/-9.9 kJ d(-1)) and daily water intake (DLW method: 13.7+/-2.4 mL d(-1) versus energy balance method: 14.7+/-3.0 mL d(-1)). Based on the calculated water and sugar intake per day, we estimated the sugar concentration of ingested nectar to equal on average 16.2+/-2.4% (mass/mass), which fell close to the measured sugar concentration of 17% (mass/mass) bats fed on during the experiment. We conclude that it is possible to extrapolate mean daily energy and water intake for animal groups, populations and species based on DLW estimates, but due to the large variance of results (low accuracy), it seems inadequate to calculate values for single individuals.     

The effect of unilateral partial facial paralysis on nasofrontal sutural growth: an experimental study in the rabbit.

In a previous study in the rabbit, it was demonstrated that paralysis of the midfacial musculature results in decreased anteroposterior growth of the snout. At the end of growth, these animals showed macroscopically striking similarities to animals with unilateral fusion of the nasofrontal suture. In this study, whether nasofrontal sutural growth is unilaterally restricted in animals with unilateral partial facial paralysis was investigated. A left-sided partial facial paralysis was induced in sixteen 12-day-old New Zealand White rabbits. At the ages of 5, 9, 12, and 17 weeks, four animals were randomly assigned to be killed for analysis of nasofrontal sutural growth. In each animal, the left experimental side was compared with the right control side. By means of histomorphometric measurements, it was shown that diminished sutural growth activity was present on the left paralyzed side in periods of rapid growth. On the other hand, no significant alterations in sutural width were found. These findings seem to explain some of the macroscopic growth alterations (i.e., diminished anterior maxillary length) observed in rabbits with unilateral partial facial paralysis.     

The effect of different sample collection methods on rabbit blood parameters

Nowadays great deal of research is physiological field is conducted on experimental animals and there is a lot of criticism from the wide public on methods used. Therefore, recently there is a lot of effort focused on the welfare of the animals. Main aim of this study is to determine the effect of experimental sample collection method on the selected parameters of stress. In the experiment two sample collections of rabbit blood from marginal ear vein were realized – first using standard method with one person fixing the animal and other collecting the blood using gently fixating the animal. In the second groups experimental method of inserting the experimental animal into a sack and further collection in dark was realized. During the experiment the levels of cortisol – main stress indicator in organism and other health parameters of animals including mineral profile and haematological parameters were observed. Our results show no significant changes in levels of cortisol but also a decreasing tendency in the sample from the second (dark) collection. Haematological parameters were generally in the reference values and any significant changes except levels of lymphocytes and percent of lymphocytes which shown significant increase in the second collection period were found. Also the levels of mean corpuscular haemoglobin and percent of neutrophils unveiled a significant decrease in values. Values of mineral profile parameters have indicated no significant changes except the levels of phosphorus. Based on the result we can state that the experimental sample collection has no effect on blood parameters of the animals but we spectated a statistically insignificant decrease in the levels of cortisol which can suggest that the dark collection is possibly less stressful to the animals.     

Endometrial venous aneurysms in three New Zealand white rabbits.

Hematuria in rabbits has been associated with uterine adenocarcinoma, uterine polyps, renal infarction, urolithiasis, cystitis, bladder polyps, and pyelonephritis. Three adult female New Zealand White rabbits (Oryctolagus cuniculus) developed apparent hematuria, as suggested by blood in their excreta pans. They had been immunized with antigen-adjuvant emulsions, but had uneventful clinical histories. Physical examination disclosed no abnormalities, and laboratory tests, including hematology, serum chemistries, urinalyses, urine cultures, ultrasonography, and intravenous pyelography disclosed mild anemia, hematuria, and proteinuria in two of the rabbits. Antibiotic therapy failed to alleviate clinical signs. Two rabbits were euthanized because of persistent urogenital bleeding and the third rabbit underwent exploratory laparotomy and ovariohysterectomy. Multiple endometrial venous aneurysms were present in the uteri of all rabbits and urogenital bleeding was attributed to episodic bleeding from these lesions. Varices and aneurysms of uterine subserosal and myometrial venous plexuses, but not of endometrial vessels in women have been reported. To our knowledge, endometrial venous aneurysms have not been reported in animals previously. Our findings indicate that the differential diagnoses for sporadic apparent hematuria in female rabbits should include endometrial aneurysms.