The research presented here was supported by Charles River.

<ol>
	<li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> JAALAS. 50, 600-613 (2011).</li><li>Diehl, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J. Appl. Toxicol. 21, 15-23 (2001).</li><li>Hurst, J. L., West, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+anxiety+in+laboratory+mice.">Taming anxiety in laboratory mice.</a> Nat. Methods. 7, 825-826 (2010).</li><li>Maurer, B. M., D&#246;ring, D., Scheipl, F., K&#252;chenhoff, H., Erhard, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+gentling+programme+on+the+behaviour+of+laboratory+rats+towards+humans.">Effects of a gentling programme on the behaviour of laboratory rats towards humans.</a> Appl. Anim. Behav. Sci. 111, 329-341 (2008).</li><li>Cloutier, S., Newberry, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+conditioning+technique+to+reduce+stress+associated+with+repeated+intra-peritoneal+injections+in+laboratory+rats.">Use of a conditioning technique to reduce stress associated with repeated intra-peritoneal injections in laboratory rats.</a> Appl. Anim. Behav. Sci. 112, 158-173 (2008).</li><li>Romanovsky, A. A., Kulchitsky, V. A., Simons, C. T., Sugimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+of+fever+research:+why+are+polyphasic+fevers+often+thought+to+be+biphasic.">Methodology of fever research: why are polyphasic fevers often thought to be biphasic.</a> Am. J. Physiol. 275, 332-338 (1998).</li><li>Turner, P. V., Pekow, C., Vasbinder, M. A., Brabb, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+equipment+considerations,+vehicle+selection,+and+solute+preparation.">Administration of substances to laboratory animals: equipment considerations, vehicle selection, and solute preparation.</a> JAALAS. 50, 614-627 (2011).</li><li>Lukas, G., Brindle, S. D., Greengard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+route+of+absorption+of+intraperitoneally+administered+compounds.">The route of absorption of intraperitoneally administered compounds.</a> J. Pharmacol. Exp. Ther. 178, 562-564 (1971).</li><li>AALAS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Mouse Handbook. , (2009).</li><li>AALAS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> LAT Training Manual. , (2009).</li><li>AALAS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> LATg Training Manual. , (2009).</li><li>Barnett, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Animal Technology. , 440 (2007).</li><li>Baumans, V., Pekow, C. A., Hau, J., Schapiro, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Laboratory Animal Science. 1, 401-446 (2010).</li><li>Bogdanske, J. J., Hubbard-Van Stelle, S., Riley, M. R., Schiffman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Mouse Procedural Techniques. , (2011).</li><li>Danneman, P., Suckow, M. A., Brayton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Mouse. , (2000).</li><li>Sharp, P. E., La Regina, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Rat. , (1998).</li></ol>

The authors are employees of Charles River.

This protocol should be viewed as an introduction to animal handling and substance administration meant to supplement hands-on training provided at the researcher's facility. The means of restraint that will be used and the routes of substance administration should be considered in the experimental design and when the research protocol or ethics committee protocol is written.
Training in animal-related procedures is vital to the success of research. To perform most experiments, animals must be handled by research staff, and the better the animal handling, the less stressed the animal 3. Accustoming animals to gentle human contact can reduce stress and make animals more tractable research subjects 4,5. Handling stress has been shown to affect some types of research 6 and it is possible it can affect others as well. Restraint of rodents should be accomplished with careful, but firm handling (a tentative grip is likely to result in injury to rodent and handler) and should be for the shortest duration practical. Restraint methods are usually chosen based on the size of the animal or the access sought. For example, handling adult rats by the scruff, although possible, is often met with strong resistance by the rat, especially if the handler is inexperienced. Holding a mouse or rat by hand can make access to the tail veins difficult and a restraint device is often chosen to keep the animal as still as possible.
When researchers handle animals, they are often seeking to administer a compound or biologic for further study. The route of administration of substances can affect absorption, bioavailability, and suitability for a particular experiment. Familiarity with various routes should provide researchers with the ability to administer their substance in the best way possible for their research. For example, a route that promotes rapid absorption of a substance, such as intravenous or intraperitoneal, should not be used if the researcher wants to administer the substance in a slower-acting manner. Recent reviews of some of these techniques and considerations for volume, equipment, and solute may be found in two papers by Turner et al. 1,7
Whenever substances are to be administered to laboratory rodents, consideration should be given to the proper size of equipment and volume of substance (outlined in Table 1). Improperly sized equipment or large volumes may result in discomfort, injury, or death of the animal. Generally, substances administered parenterally are sterile, except where the research goals would make this impossible (i.e., bacterial studies). Compounds and biologicals should be in a solute or vehicle that will have the least effect on the animal. A physiologic pH (7.3 -7.4) is generally well-accepted, especially for subcutaneous, intramuscular, and intraperitoneal routes. Non-physiologic pH levels in substances administered by these routes may result in pain or necrosis and tissue damage. Wider ranges of pH are tolerated with intragastric and intravenous routes 7. In small rodents, another important consideration is the possibility of chilling if large volumes of room-temperature fluids are given. If fluids are being administered intravenously or intraperitoneally, especially in support of an ill animal, they should be warmed to body temperature (37 &deg;C).
The routes of administration discussed in this protocol are ones commonly used in many research programs, are simple to master, and generally do not require anesthesia. An almost infinite variety of routes of administration are possible, however, including intracranial, intrathecal, epidural, intratracheal, intraosseous, and intraarticular to name but a few. Training in these specialized routes of administration should be sought from people who have extensive experience with the route and good outcomes.
In rodents, the intranasal route is typically used to study substances introduced to the lungs via a more "natural"; method than intratracheal instillation. Mice and rats are obligate nose-breathers, so inducing them to inhale very small amounts of fluid is not difficult, even in conscious animals. Since the nasal mucosa is well-supplied with blood vessels, intranasal administration of some substances can be similar to intravenous administration. This route is not recommended in animals with rhinitis, however, as that may compromise absorption. Attempts to administer large volumes by the intranasal route may result in dyspnea or drowning of the animal.
Intramuscular injections provide rapid absorption of substances. Intramuscular injections can be challenging in rats and mice due to their small size and correspondingly small muscles. They are performed in the hind legs. Due to the potential for damage of the sciatic nerve, the quadriceps femoris is the muscle of choice.
Although both the subcutaneous and intradermal routes involve the skin, there are differences between the bioavailability of substances placed in the skin vs the subcutis. Subcutaneous administration is often considered a "deposition" route, with slower absorption than other routes, such as intravenous or intraperitoneal. Intradermal administration is used for very small volumes of substances, typically immunostimulatory substances such as adjuvant-antigen mixtures. In both cases, the substances administered should be of physiologic pH and non-irritating. Intradermal or subcutaneous injections should not be performed in the scruff, since this is a commonly used site for restraining the rodent.
Intravenous and intraperitoneal administration are often considered equivalent in rodents. Intravenous routes of dosing provide more rapid uptake of compounds, however, while intraperitoneal administration should be considered roughly equivalent to oral administration 8. Care should be taken with compounds administered intraperitoneally as they may cause pain if improperly buffered. The common route of intravenous bolus administration in rodents is via the tail veins. If chronic intravenous administration of a substance is desired, implantation of venous or arterial cannulae should be considered. Substances administered intravenously should be delivered aseptically and should be shown to be safe to administer intravenously. For example, substances that may induce hemolysis ,thrombosis, or vasculitis are not appropriate for intravenous administration.
The intragastric or oral gavage route is often used to mimic a common dosing route in humans. It also allows for precise dosing of substances when compared to oral administration through food or water. Bioavailability of compounds administered via gavage will vary based on the fed/fasted state of the animal, as well as the solute or vehicle of the compound or biological. Gavage or feeding needles should be of the appropriate size for the animal being used, and should be cleaned between animals, if disposable gavage needles are not practical. Injuries caused by gavage are not uncommon and include deposition of the substance into the lung or rupture of the stomach or esophagus. Training should be supervised by an experienced party and undertaken on euthanized animals first, then anesthetized animals (that will be euthanized) before gavage on awake animals is attempted. First gavage attempts on awake animals should involve average-sized animals and small volumes of a substance, such as saline, that will not cause injury if the procedure goes awry. Animals should be closely assessed for signs of distress, such as gasping, turning blue, bleeding, or excess salivation ,after gavage and euthanized if necessary. If euthanasia is required, the animal should be necropsied to determine why the gavage procedure failed.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Needles</td>
 <td>Various</td>
 <td>Various</td>
 <td>Needles are sold by both gauge and length. Check both before ordering.</td>
 </tr>
 <tr>
 <td>Syringes</td>
 <td>Various</td>
 <td>Various</td>
 <td>Always choose an appropriate size for the volume to be administered. </td>
 </tr>
 <tr>
 <td>DecapiCones</td>
 <td>Braintree Scientific</td>
 <td>DC-200, DCL-120, MDC-200</td>
 <td>Available in mouse and rat sizes.</td>
 </tr>
 <tr>
 <td>Rodent restrainer</td>
 <td>Harvard Apparatus, Braintree Scientific, Plas-Labs, others</td>
 <td>&nbsp;</td>
 <td>Available in clear Plexiglas, adjustable plastic, and sized for mice and rats.</td>
 </tr>
 <tr>
 <td>50 ml conical tube</td>
 <td>Various</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Feeding needles</td>
 <td>VWR, Popper and Sons</td>
 <td>Various</td>
 <td>Fit the needle gauge and length to the animals as described above. Both disposable and reusable feeding needles are available.</td>
 </tr>
</tbody>
</table>

manual restraint and common compound administration routes in mice and rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Watch this Scientific Journal Video about Manual Restraint and Common Compound Administration Routes in Mice and Rats at JoVE.com

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

manual-restraint-common-compound-administration-routes-mice

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Biology

Unit 1

General Pharmacological Principles

SCIENTISTS IN ACTION

286.0K ビュー.  Charles River. 研究用げっ歯類で安全かつ人道的に働くことはハンドリングと拘束方法のコアコンピテンシーを必要とします。この記事では、安全に処理するために必要な基本的な原則を提示し、効果的にマウスやラットに化合物を投与するであろう。

ビデオ: Manual Restraint and Common Compound Administration Routes in Mice and Rats

Working safely and humanely with research rodents requires a core competency in handling and restraint methods.  This article will present the basic principles required to safely handle and effectively administer compounds to mice and...

immunostimulatory agent evaluation: lymphoid tissue extraction and injection route-dependent dendritic cell activation

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating...

evaluation of blood-brain barrier breakdown in a mouse model of mild traumatic brain injury

Author Spotlight: Advancing Traumatic Brain Injury Research by Mapping Blood-Brain Barrier Breakdown Using Dual-Dye Fluorescence

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same...

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

utilizing an orally dissolving strip for pharmacological and toxicological studies: a simple and humane alternative to oral gavage for animals

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral...

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

https://cloudfront.jove.com/CDNSource/teasers/66653_b.jpg

imaging denatured collagen strands in vivo and ex vivo via photo-triggered hybridization of caged collagen mimetic peptides

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

the dimethylnitrosamine induced liver fibrosis model in the rat

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic...

induction and scoring of graft-versus-host disease in a xenogeneic murine model and quantification of human t cells in mouse tissues using digital pcr

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify...

lung microrna profiling across the estrous cycle in ozone-exposed mice

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous...

corneal and limbal alkali injury induction using a punch-trephine technique in a mouse model

Author Spotlight: Advancing Research in Corneal Opacity Treatment and Regeneration 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and...

A Modified Biopsy Punch Method for Precise Alkali Burns in Mice

https://cloudfront.jove.com/CDNSource/teasers/65609_b.jpg

assessing changes in volatile general anesthetic sensitivity of mice after local or systemic pharmacological intervention

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals.  Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled...

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

the bm12 inducible model of systemic lupus erythematosus (sle) in c57bl/6 mice

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

an efficient and high yield method for isolation of mouse dendritic cell subsets

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic...

studying murine small bowel mechanosensing of luminal particulates

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel...

the bioconjugation and radiosynthesis of 89zr-dfo-labeled antibodies

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

neurodevelopmental reflex testing in neonatal rat pups

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

quantitative measurement of intrathecally synthesized proteins in mice

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized...

models of murine vaginal colonization by anaerobically grown bacteria

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown...

an epithelial abrasion model for studying corneal wound healing

An Epithelial Abrasion Model for Studying Corneal Wound Healing

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and ...

https://cloudfront.jove.com/CDNSource/teasers/65056.jpg

Developing Gene Transformation and Editing Methods in Bamboo

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

Author Spotlight: Advancing Gene Editing in Bamboo Leaves for Sustainable Plastic Alternatives 

In this study, a novel in planta gene expression and gene editing method mediated by Agrobacterium was developed in bamboo. This method greatly improved the efficiency of gene function validation in bamboo, which has significant implications for accelerating the process of bamboo breeding.

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...