Experiences with the identification of small rodents

Own experiences with the identification of mice and rats using ear tags, ear punching, toe clipping (for newborns only) and ear tattooing are given and compared with methods presented in literature. By far, ear tattooing by the newly developed pincers is the most suitable method for marking adult rodents for life. Tattooing is easy to apply, easy to read and not harmful to the animals. For newborn pups on day 4 p. p., the s. c. injection of India ink into the palm of paw is recommended.     

一种简易的啮齿动物切趾编码方法

提出一套改进的切趾标志编码系统。该编码系统具有趾号易于判读、切趾分布均匀的优点。本套编号系统具有一定的兼容性 ,可根据样地内动物个体的数量选择最小的切趾数 ,从而尽可能减少切趾对小哺乳动物行为与生存的影响     

Toe‐bud clipping of juvenile small marsupials for ecological field research: No detectable negative effects on growth or survival

Toe clipping is widely used to permanently mark many species of small vertebrates including marsupials, particularly didelphids and dasyurids. Small marsupials are marked as juveniles, by removing the tip of developing toe buds. It has recently been shown that survival and/or recapture probability decreases with increasing number of toes clipped in frogs. Because of this and other animal welfare concerns, toe clipping of adult vertebrates is increasingly being discouraged. The short‐ and long‐term effects of toe‐bud clipping have not been evaluated in marsupials. We used an experiment to test if marking more toes results in slower growth or higher mortality in the brown antechinus (Antechinus stuartii, Dasyuridae) in the short or long term. We found no harmful effects of toe‐bud clipping. There were no infections associated with clipping, marking more toes did not reduce growth in young or adults, and did not affect survival of young in captivity, survival of independent animals in the wild, or recapture probability. Toe‐bud clipping is done at an extremely immature stage, when the area cut is tiny and perception and memory of pain is unlikely to be a problem. We suggest that toe‐bud clipping is a humane and benign method of permanently marking antechinuses, and probably also the young of other morphologically similar small marsupials.     

Genomic DNA analysis from mouse toe lysates

The protocol described in this report provides a simple, accurate and efficient assay for detection of transgenes and mutations in large colonies of rodents, using crude lysates prepared from the digit cut from the animals for identification purposes. This can be done as early as 6 days of age, minimizing trauma to the mice and allowing assays to be completed long before weaning. Editor's note: The US Food and Drug Administration (FDA) had amended its regulations on good laboratory practice (GLP) for nonclinical laboratory studies regarding humane procedures for animal identification and expreimentation (Vol. 54 # 75). While discouraging use of toe clipping for animal identification, the FDA charged each facility's Institutional Animal Care and Use Committee (IACUC) with the responsibility for evaluation and final approval. C.A.P.     

The impact of tail tip amputation and ink tattoo on C57BL/6JBomTac mice

Genetic material for polymerase chain reaction (PCR) and Southern blot analysis on transgenic mice is normally obtained by tail biopsy. Additionally, it may be necessary to tattoo the mice, as it is essential to have a good and permanent identification. The aim of this study was to evaluate the effects of amputating the tip of the tail to obtain a biopsy for genetic analysis and of ink tattooing on welfare in C57BL/6J mice, a strain often used as genetic background for transgenes. The behaviour of the animals, fluctuating asymmetry (FA, a measure of developmental instability) and the level of restitution in the remaining part of the tail were evaluated and used for an assessment of the impact of these procedures on the welfare of the animals. One group of mice was marked by tail tattooing at various ages. Another group of mice were tail amputated at 12 or 20 days of age. Body weight and FA were followed, and at the end of the experiment, the level of fear/anxiety was assessed using a light-dark box. In the group of tail-amputated animals observation of climbing behaviour and a beam walking test for balance was performed. Seven weeks after tail amputation, the animals were euthanized. The remaining part of the tail was evaluated histopathologically. Body weight, behaviour in the light-dark box and balance test results were not influenced by tail amputation or tattooing. FA was only transiently increased by tattooing. Climbing behaviour was reduced just after tail amputation at 20 days of age. No signs of neuromas were found in the amputated tails, but seven weeks after amputation a significant number of mice did not have fully regenerated glandular tissue and hair follicles in the tail. It is concluded that both tail amputation and tail tattooing seem to have minor short-term negative effects on welfare and that the tissues on the tail probably do not regenerate fully after amputation.     

One step forward: contrasting the effects of Toe clipping and PIT tagging on frog survival and recapture probability

Amphibians have been declining worldwide and the comprehension of the threats that they face could be improved by using mark–recapture models to estimate vital rates of natural populations. Recently, the consequences of marking amphibians have been under discussion and the effects of toe clipping on survival are debatable, although it is still the most common technique for individually identifying amphibians. The passive integrated transponder (PIT tag) is an alternative technique, but comparisons among marking techniques in free‐ranging populations are still lacking. We compared these two marking techniques using mark–recapture models to estimate apparent survival and recapture probability of a neotropical population of the blacksmith tree frog, Hypsiboas faber. We tested the effects of marking technique and number of toe pads removed while controlling for sex. Survival was similar among groups, although slightly decreased from individuals with one toe pad removed, to individuals with two and three toe pads removed, and finally to PIT‐tagged individuals. No sex differences were detected. Recapture probability slightly increased with the number of toe pads removed and was the lowest for PIT‐tagged individuals. Sex was an important predictor for recapture probability, with males being nearly five times more likely to be recaptured. Potential negative effects of both techniques may include reduced locomotion and high stress levels. We recommend the use of covariates in models to better understand the effects of marking techniques on frogs. Accounting for the effect of the technique on the results should be considered, because most techniques may reduce survival. Based on our results, but also on logistical and cost issues associated with PIT tagging, we suggest the use of toe clipping with anurans like the blacksmith tree frog.     

Marking transgenic <Emphasis Type="Italic">Xenopus</Emphasis> froglets with passive micro transponders

Standard methods to mark Xenopus laevis individuals like tattooing or clipping toenails are inappropriate for the fast growing and regenerating small froglets and the previously used transponders are too large. In this study we successfully adapted micro transponders to tag these animals. Using these new transponders one can now tag small froglets directly after metamorphosis, which has not been possible previously. This new technique makes the breeding of transgenic frogs most efficient, because the frogs do not have to be kept separately and they grow much faster when kept together in large containers.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

吐鲁番沙虎头、体大小的两性异形及其食性的季节性变化

于2008年4月—2009年5月在新疆中国科学院吐鲁番沙漠植物园,捕获155号吐鲁番沙虎(Teratoscincus roborowskii)活体,对其两性异形和食性季节变化进行了研究,并收集单独暂养的吐鲁番沙虎粪便167份用于食性分析,同时测量各项形态学指标,切趾标记后放回原捕获地。结果表明:吐鲁番沙虎成体有两性异形,表现为雄性个体有较宽的头部,成体雌性具有较大的腋胯距;不同年龄组的两性个体头长和雄性个体的头宽随吻肛长都呈同速增长,只有雌性头宽的增长速率在个体发育过程中逐渐减小;吐鲁番沙虎摄入的动物性食物均为节肢动物,植物性食物为刺山柑果实。4—5月的生态位宽度为4.15,6—7月的生态位宽度为4.23,8—9月的生态位宽度为4.48。其中6—7月和8—9月的生态位重叠度最高。     

Evaluation of EMLA Cream for Preventing Pain during Tattooing of Rabbits: Changes in Physiological, Behavioural and Facial Expression Responses

Background

Ear tattooing is a routine procedure performed on laboratory, commercial and companion rabbits for the purpose of identification. Although this procedure is potentially painful, it is usually performed without the provision of analgesia, so compromising animal welfare. Furthermore, current means to assess pain in rabbits are poor and more reliable methods are required. The objectives of this study were to assess the physiological and behavioural effects of ear tattooing on rabbits, evaluate the analgesic efficacy of topical local anaesthetic cream application prior to this procedure, and to develop a scale to assess pain in rabbits based on changes in facial expression.

Methodology/Principal Findings

In a crossover study, eight New Zealand White rabbits each underwent four different treatments of actual or sham ear tattooing, with and without prior application of a topical local anaesthetic (lidocaine/prilocaine). Changes in immediate behaviour, heart rate, arterial blood pressure, serum corticosterone concentrations, facial expression and home pen behaviours were assessed. Changes in facial expression were examined to develop the Rabbit Grimace Scale in order to assess acute pain. Tattooing without EMLA cream resulted in significantly greater struggling behaviour and vocalisation, greater facial expression scores of pain, higher peak heart rate, as well as higher systolic and mean arterial blood pressure compared to all other treatments. Physiological and behavioural changes following tattooing with EMLA cream were similar to those in animals receiving sham tattoos with or without EMLA cream. Behavioural changes 1 hour post-treatment were minimal with no pain behaviours identifiable in any group. Serum corticosterone responses did not differ between sham and tattoo treatments.

Conclusions

Ear tattooing causes transient and potentially severe pain in rabbits, which is almost completely prevented by prior application of local anaesthetic cream. The Rabbit Grimace Scale developed appears to be a reliable and accurate way to assess acute pain in rabbits.     

Genetic evidence of <Emphasis Type="Italic">Ranavirus</Emphasis> in toe clips: an alternative to lethal sampling methods

Amphibian populations have been undergoing declines on a global scale. Among the many threats to these populations are emergent infectious diseases (EIDs). The Ranavirus in particular has been found within many declining amphibian populations. Although non-lethal sampling methods exist for some amphibian groups, such as salamanders, the anurans are traditionally tested using a lethal method. By comparing traditional liver samples and a new non-lethal method of toe clipping we prove that the Ranavirus can also be determined in frogs using a non-lethal method, a much needed tool in threatened populations. This method will allow for ranaviral detection without further impacting declining populations, and can further be used for other research questions.     

Evaluation of marking and tagging methods for genetic studies in carp

A variety of marking and tagging methods were tested on common carp (Cyprinus carpio L.) with the aim of identifying suitable methods for genetic studies in this and other species of carp. Elastomer and Alcian blue dye marking; Cold and Silver nitrate branding; Floy, Fingerling, Carlin disc and visible implant tags; and fin clipping were all tested on a range of sizes of common carp (from mean weights of 10–25 g up to 600–800 g). The branding and tagging methods tested did not give satisfactory retention rates. A combination of elastomer marking and fin clipping was then tested as a method for strain identification in a growth comparison trial on catla (Catla catla Hamilton) and found to be satisfactory for this purpose. Passive integrated transponders (PIT) tags were used to individually identify catla of wild or hatchery origin being grown for use as broodstock. These had almost 100% (98.8%) retention rates, but are expensive compared to most other tagging methods.     

Effects of simulated winter browsing on mountain birch foliar chemistry and on the performance of insect herbivores

Winter browsing by mammalian herbivores is known to induce a variety of morphological and physiological changes in plants. Browsing has been suggested to decrease the carbohydrate reserves in woody plants, which might lead to reduced tannin production in leaves during the following summer, and consequently, to increased herbivore damage on leaves. We conducted a clipping experiment with mature mountain birch trees and measured the effects of clipping on birch growth, leaf chemistry and toughness, as well as on the performance of insect herbivores. Leaves grew larger and heavier per unit area in the clipped ramets and had a higher content of proteins than leaves in the control trees. Clipping treatment did not affect the total content of sugars in the leaves (mg g^?1), suggesting that a moderate level of clipping did not significantly reduce the carbohydrate pools of fully‐grown mountain birch trees. Furthermore, the contents of proanthocyanidins (condensed tannins) and gallotannins were slightly higher in the leaves of clipped ramets, contrary to the hypothesis of reduced tannin production. The effects of clipping treatment on leaf and shoot growth and on foliar chemistry were mainly restricted to the clipped ramets, without spreading to untreated ramets within the same tree individual. The effects of clipping on leaf characters varied during the growing season; for instance, leaf toughness in clipped ramets was higher than toughness in control trees and ramets only when leaves were mature. Accordingly, clipping had inconsistent effects on insect herbivores feeding at different times of the growing season. The generally small impact of clipping on herbivore performance suggests that the low intensity of natural browsing at the study area, simulated by our clipping treatment, does not have strong consequences for the population dynamics of insect herbivores on mountain birch via enhanced population growth caused by browsing‐induced changes in food quality.     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

Tattooing in reconstruction of the nipple and areola: a new method

In order to produce the characteristic color of the nipple-areola complex and in unilateral reconstructions to match the other side, a modified tattooing method has been introduced. The quite different requirements between reconstruction and ornamental studio tattooing can only be satisfied by different techniques. For the first time, pigment particles have been suspended in a gel rather than adsorbed on an opaque mineral. Our porcine study has shown how the pigment is distributed in the dermis without penetration of the gel particles. The first 16 of the authors' 29 patients have been reviewed with an analysis of their appearance. Using this new method, a third achieved a completely natural appearance and two-thirds were judged to be intermediate, compared with an unnatural, although sometimes acceptable, result in all the earlier conventional tattoos.     

Determination of toe-off event time during treadmill locomotion using kinematic data

Researchers collecting gait kinematic data during treadmill locomotion are often interested in determining the times of toe off and heel strike for each stride. In the absence of additional hardware, only position data collected with motion-capture equipment may be available. Others have published methods for using kinematic data for detecting overground gait events. However, during treadmill locomotion, especially running, overground methods may not possess sufficient accuracy. The purpose of this paper is to describe a method for using kinematic data to determine the time of toe off during treadmill locomotion. Ten subjects walked and ran on a treadmill while a motion-capture system collected positional data from heel and toe markers. The treadmill was equipped with force platforms that allowed an accurate determination of foot-ground contact. The time of toe off was determined using the vertical component of the toe marker, and this method was found to have greater accuracy for event detection than other published methods. Researchers can use the described method to determine times of heel strike and toe off during treadmill locomotion using only kinematic data.     

Movements and mortality of common goldeneye Bucephala clangula broods in a patchy environment

We tested the effect of timing and intensity of deer browsing on compensatory response by willow grown for bioenergy. Browsing was simulated several times during summer and once during winter. The clipping was done at different intensities during the first year after establishment in a new willow plantation, and during the first year after harvest in an older willow coppice. Total aboveground biomass and biomass available for deer browsing were recorded both at the end of the first and the second growing periods after clipping. Both in the new plantation and in the older coppice, the willows fully compensated for biomass losses after winter clipping, irrespective of clipping intensity. On the other hand, total biomass production usually decreased after high-intensity summer clipping. Such a seasonal difference may be explained by the nutrition of remaining buds after clipping. In the new willow plantation, total produced biomass after summer clipping still bottomed at about 50% of the level of control stools at the end of the experiment. In the older coppice, usual browsing levels by deer in summer, roughly corresponding to the moderate clipping intensities used in this experiment, did not influence total produced biomass. The newly established willow stools reacted to summer clipping by undercompensating in terms of biomass available for deer browsing, while older stools could overcompensate. Compared to stools clipped in late summer, willow stools clipped during early summer were able to compensate earlier and stronger. The higher the proportion of twig biomass removed by summer clipping, the lower the compensatory growth by willow. Depending on the circumstances, the fast-growing willows responded within the whole continuum from under- to overcompensation, and some implications for the management of willow plantations and large herbivores are discussed.     

Complications of tattooing

Pathologic consequence of tattooing is relatively rare, but in light of the great number of persons in military service, where tattooing seems to be popular, consideration of the complications seems worth while.Four unusual cases of patients with cutaneous lesions in tattoos are reported. One patient had Boeck's sarcoid, in which the cutaneous eruption was limited solely to the tattooed areas on the body and involved all the various colors of the tattoo. The cutaneous eruption was only a single manifestation of generalized sarcoidosis. Another patient had secondary syphilis with the cutaneous lesions involving multi-colored tattoos, including the red areas. These tattoos were applied in Hawaii, and presumably the red dye was not the usual cinnabar (mercuric sulfide). As a general rule, the cutaneous lesions of secondary syphilis do not involve the red areas because of the treponomicidal action of mercuric sulfide.In the third case the characteristic eruption of discoid lupus erythematosus was present. It began over the red areas of a tattoo on the patient's forearm. It has been stated that mercuric sulfide is a photo-sensitizing agent, and it is believed that this explains the localization of the eruption in this case. The fourth patient had eruption, caused by mercury sensitivity, in an area of tattoo. The eruption was noted soon after the tattooing was done, and it persisted for seven years.     

COMPLICATIONS OF TATTOOING

Pathologic consequence of tattooing is relatively rare, but in light of the great number of persons in military service, where tattooing seems to be popular, consideration of the complications seems worth while.Four unusual cases of patients with cutaneous lesions in tattoos are reported. One patient had Boeck''s sarcoid, in which the cutaneous eruption was limited solely to the tattooed areas on the body and involved all the various colors of the tattoo. The cutaneous eruption was only a single manifestation of generalized sarcoidosis.Another patient had secondary syphilis with the cutaneous lesions involving multi-colored tattoos, including the red areas. These tattoos were applied in Hawaii, and presumably the red dye was not the usual cinnabar (mercuric sulfide). As a general rule, the cutaneous lesions of secondary syphilis do not involve the red areas because of the treponomicidal action of mercuric sulfide.In the third case the characteristic eruption of discoid lupus erythematosus was present. It began over the red areas of a tattoo on the patient''s forearm. It has been stated that mercuric sulfide is a photo-sensitizing agent, and it is believed that this explains the localization of the eruption in this case.The fourth patient had eruption, caused by mercury sensitivity, in an area of tattoo. The eruption was noted soon after the tattooing was done, and it persisted for seven years.