Miscellaneous Techniques Tail clipping is frequently used to obtain tissue for DNA analysis of transgenic mice.  Less than 10 mm of the tail tip should be removed.  The tail ossifies between 2 - 4 weeks of age, therefore, mice older than 3 weeks of age must be anesthetized for the procedure.  Measures should be employed to stop bleeding at the site of amputation.  Put pressure on the cut end of the tail with a piece of gauze until bleeding stops.  A product called “Quick Stop” powder also works well to assist with hemostasis and is used by dipping the cut surface of the tail into the powder after the major bleeding has been arrested by pressure. Oral gavage is commonly used in drug and toxicology studies to deliver a precise amount of test material, and can also be used to administer medications.  A special gavage needle with a ball at the end is used to deliver materials directly into the stomach. The ball on the needle prevents entry into the trachea.  The length of the gavage tube required is determined by measuring the distance from the mouth to the last rib.  The mouse should be held with the head and neck extended.  If the gavage tube does not easily pass into the esophagus, remove it and try again. Recognition of Pain and Distress in Mice Because animals cannot volunteer to participate in medical research, we are ethically constrained to provide humane care, and to alleviate as much pain and distress as is possible in such animals.   This is mandated by the PHS Policy on Humane Care and Use of Laboratory Animals.  The U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (upon which PHS Policy is based), states that “Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative.  Unless the contrary is established, investigators should consider that procedures that cause pain and distress in human beings may cause pain or distress in other animals.”  The proper use of anesthetic and analgesic drugs helps to alleviate pain and distress during procedures.  It is imperative that researchers learn to recognize the signs of pain and distress in mice.  Inconvenient or not, the benefit of the doubt must always go to the animal.  The most common signs of pain and distress in mice listed in order of increasing severity, include: (1) ruffled or “spikey” fur (mouse looks unkempt);  (2) weight loss which may be mild to severe, anorexia, dehydration;  (3) ocular discharge;  ( 4) lethargy, depression, or reluctance to move;  (5) sitting with the back in a hunched position;  (6) ataxia (uncoordinated muscle movements), regional or generalized weakness;  (7) tremors, which may be intermittent to persistent depending on the condition of the animal;  (8) hypothermia;  (9) labored respiration; and (10) cyanosis, or a blue tinge to the mucous membranes (Table 1).  Any animals exhibiting combinations of 2 to 3 minor signs, or a single major sign should be euthanized immediately. Animals in pain and distress may not interact with their cage mates, or may interact with them in a more aggressive manner.  They may also become more aggressive towards human handling. Female mice may cannibalize litters in response to pain and distressing situations.  Animals may squeal when picked up or when an affected area is touched.  Persistent vocalization and crying indicates substantial pain or distress that should be relieved immediately.  Moribund animals should be euthanized immediately. Euthanasia Mice may be euthanized by placing them into a CO2 chamber until they have expired.   Please note that a compressed gas cylinder is the only source of CO2 that may be used for euthanasia purposes at LSSU.  According to the most recent Report of the American Veterinary Medical Association Panel on Euthanasia, CO2 for euthanasia may not be generated by the use of dry ice, fire extinguishers, or antacids as these methods are unreliable in producing the required concentration of CO2 and insuring a fast and painless death.  Mice may not be crowded into cages before euthanasia by this method as it induces stress and may result in inefficient euthanasia.  No more than the maximum allowable number of mice housed in a cage may be euthanized together. Cervical dislocation and decapitation are considered to be physical methods of euthanasia and may be used at LSSU.  Unless the IACUC has granted an exception based on scientific justification, the mouse must be anesthetized prior to euthanasia by these methods.  If cervical dislocation is used as a means of euthanasia, investigators must be responsible for ensuring that all personnel have been properly trained and consistently apply the technique humanely and effectively.  If decapitation is used as a method of euthanasia, the guillotine used must be maintained in good working order and serviced on a regular basis to ensure sharpness of blades.  The blade must be dropped quickly and forcefully so that a clean cut and not a slow crush is obtained.  The use of plastic restraint cones appears to reduce distress from handling, minimizes the chance of injury to personnel, and improves positioning of the animal in the guillotine.   Euthanasia of neonates requires special considerations.  Neonatal rodents are extremely resistant to hypoxia induced by CO2, therefore, this is not considered to be a valid method of euthanasia during the first postnatal week.  Pups up to one week of age may be anesthetized using isoflurane or halothane and then decapitated, or may be euthanized with an overdose of the indicated inhalant anesthetics.  As with adult mice, pups greater than one week of age may be euthanized with a gas inhalant anesthetic, CO2, or pentobarbital alone. Investigators should consult the AVMA classification of "Methods of Euthanasia by Species" from the "Report of the AVMA Panel on Euthanasia". Methods are summarized in Table 2. It is always the responsibility of the investigator to ensure that the animal is dead before disposal of the carcass.  Cessation of the heartbeat by palpation of the thoracic cavity is used to determine that the animal is no longer alive.  Alternatively, the thoracic cavity may be opened to obtain a terminal blood sample or to perform perfusion.  Cessation of respiration alone is not a reliable indicator of death and may only indicate an extremely deep state of anesthesia. Occupational Health Concerns Development of allergies to species of animals used in research, especially rodents and rabbits, is one of the most common problems encountered by both animal care workers and investigators. While the most common manifestations of this sensitivity are nasal symptoms, itchy eyes, and rashes, it is estimated that up to 10% of chronically exposed individuals will develop asthma which can be life-threatening.  The majority of allergies induced by mice are due to a protein found in the urine.  This protein can become airborne, and individuals that are extremely sensitive can be adversely affected by simply walking into a room where mice are housed.  The use of gloves, laboratory coats, and other protective clothing helps to minimize exposure and prevent the development of allergies.  The use of filter top cages also helps to minimize the amount of protein that can be aerosolized.   Wash well with soap after working with the mice.  Anaphylaxis may occur in extremely allergic individuals if they are bitten by a mouse or receive a puncture wound from a needle that has mouse proteins on it.  Development of allergies should be reported to the LSSU Health Center.   Anyone bitten by a mouse should report the injury immediately to the LSSU Health Center (635-2110).  A bite from a mouse may result in a puncture wound, and any bite wound should be cleaned thoroughly to prevent bacterial infections.  A current tetanus immunization is recommended for anyone working with mice, as such injuries may provide entry for the tetanus bacterium.  Transmission of infectious diseases from mice to humans is rare today because of the care used in rearing and housing laboratory mice, however, the introduction of wild-caught rodents may cause diseases to occur.  Diseases which may be readily transmitted from infected mice to humans include lymphocytic choriomeningitis, salmonellosis, and leptospirosis.  Of special concern to those handling wild mice in the southwest are Hantavirus infection (pulmonary form in the U.S.) and bubonic plague.  Wild rodents should never be handled without gloves and protective clothing. Comments & suggestions  should be directed to jroese@lssu.edu Visit my website Step 2 - Insert the needle with gentle, steady pressure.  Administer the substance slowly.  No resistance should be felt. Step 1 - A gavage needle is used to deliver materials directly into the stomach.  The mouse is held with head and neck extended. The length of the gavage tube is determined by measuring from the mouth to the last rib.