Comments & suggestions  should be directed to jroese@lssu.edu Visit my website Recognition of Pain and Distress in Rats 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 rats listed in order of increasing severity, include: (1) ruffled or “spikey” fur (rat looks unkempt);  (2) weight loss which may be mild to severe, anorexia, dehydration;  (3) ocular discharge which may include porphyrin staining around the eyes;  ( 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 characterized by cool extremities;  (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 Rats 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 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.   Rats may not be crowded into cages before euthanasia by this method as it induces stress and may result in inefficient euthanasia.  Only the original occupants of the cage may be euthanized at one time.  Do not introduce strange animals into the cage prior to euthanasia. A chamber pre-filled with a volatile inhalation anesthetic such as isoflurane or halothane may be used in lieu of CO2.  Because the liquid forms of volatile anesthetics are an irritant to the skin and mucous membranes, care must be taken to ensure that the rat does not come into direct contact with the agent.  An overdose of the injectable anesthetic pentobarbital may also be used at a dosage of 100 mg/kg body weight intraperitoneally. 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 rat must be anesthetized prior to euthanasia by these methods.  Cervical dislocation may be used only for rats weighing less than 200 grams.  If cervical dislocation is used as a means of euthanasia. investigators are responsible for ensuring that personnel have been properly trained and consistently apply the technique humanely and effectively.  If decapitation is used as a method of euthanasia the guillotine must be maintained in good working order and serviced on a regular basis to ensure sharpness of blades.  When decapitation is used as the means of euthanasia the blade must be dropped quickly and forcefully so that a clean cut and not a slow crush is obtained.  Correct use of the guillotine on un-anesthetized rats requires considerable training in order to restrain the animal with as little distress as possible, as well as to avoid the amputation of part of the restrainers finger.  The use of plastic restraint cones to restrain animals appears to reduce distress from handling, minimizes the chance of injury to personnel, and improves positioning of the animal in the guillotine.  Rats may become aggressive if the guillotine is not cleaned well between animals and the smell of blood is present.  Animals should not be guillotined in the presence of others to avoid unnecessary distress.  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 with inhaled halothane or isoflurane or injectable pentobarbital prior to decapitation or these drugs may be used as the sole agent for euthanasia.  As with adult rats, 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 if the thoracic cavity has not been 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.