In vivo studies with highly pathogenic viruses prompt concerns regarding the persistence of infectious virus in pathology specimens. Although formalin fixation of tissues may inactivate infectious virus, fixation may also degrade viral nucleic acid and antigens, thereby limiting detection of virus in tissues by polymerase chain reaction (PCR) amplification or immunohistochemistry (IHC). We sought to: 1) assess the rate of inactivation of infectious virus in tissue specimens during formalin fixation, 2) assess IHC recognition of viral antigens and PCR detection of viral DNA after long-term (14 d) formalin fixation, and 3) investigate microtome contamination by DNA carry-over to subsequently sectioned tissues. Infectious baboon herpesvirus HVP2 could be recovered from fresh tissues of infected mice but not those fixed in formalin for ≥24 h. The intensity of IHC staining of viral antigen was unaffected by the duration of formalin fixation. PCR detection of viral DNA was negatively impacted by formalin fixation and/or heat inherent to paraffin processing; however, amplification of very short DNA sequences using real-time PCR was not affected. Lastly, microtome contamination by viral DNA was demonstrated by PCR screening of uninoculated control tissues that were sectioned after sectioning infected tissues. In summary, infectious virus is inactivated after only 24 h of formalin fixation whereas IHC staining remains sensitive in tissues fixed for up to 14 d. Formalin fixation does degrade DNA, but viral DNA can be detected by PCR amplification of very short DNA sequences. In addition, viral DNA can contaminate a microtome knife such that subsequently sectioned uninoculated control tissues exhibit false positive PCR amplification.

We evaluated the effect of ketamine–xylazine–acepromazine anesthesia (31.25, 6.25, and 1.25 mg/kg subcutaneously, respectively) on postsurgical recovery in male Sprague–Dawley (Crl:SD) rats undergoing laparotomy with and without the postoperative analgesic ketorolac. Recovery was determined by changes in body weight (BW) and water intake. The time of ketorolac administration (5 mg/kg intramuscularly), 60 min after anesthetic injection, was based on return of the pedal withdrawal reflex in Long–Evans (HsdBlu:LE) rats undergoing stereotaxic surgery in a separate experiment. Results were compared with those of housing and anesthesia controls as well as of laparotomized rats receiving a single sugared treat for nonpharmacologic management of postoperative pain. Surgery took place on day 0; the first 24 h postsurgery was considered the 'acute phase,' and days 1 through 4 comprised the 'recovery phase.' Results suggest that 1) the anesthetic mixture is fast- and long-acting and provides sufficient immobility, loss of consciousness, and analgesia; 2) during the acute phase, rats subjected to laparotomy did not lose more BW than rats exposed to anesthesia alone; 3) water intake during both phases did not significantly differ between treatment groups; 4) postsurgical ketorolac administration did not minimize BW loss during the acute phase nor cause any adverse effects under this anesthetic regimen; and 5) provision of single sugared treats had salutary effects on BW recovery. This finding suggests that postsurgical BW loss after use of this anesthetic mixture is due to distress unrelated to pain; this nonpain distress may have masked potential beneficial effects of ketorolac.

We compared CO₂, Ar, and N₂ for inducing unconsciousness and euthanasia of Sprague-Dawley rats. We determined time to unconsciousness and monitored heart rate (HR) and mean arterial blood pressure (MAP) by radiotelemetry to assess stress, recovery after exposure, and time of death. Unconsciousness (mean ± standard error) occurred 24 ± 3, 87 ± 8, and 93 ± 8 s after short-term exposure to CO₂, Ar, and N₂, respectively. During exposure, CO₂ depressed HR, whereas Ar and N₂ increased HR. Upon removal from the chamber, rats' HR rapidly normalized after CO₂ or N₂ but remained elevated for 60 min after Ar. During exposure, all agents depressed MAP, which returned to resting levels 10 to 50 min after rats' removal from the chamber. For euthanasia, CO₂ at approximately 100% induced unconsciousness in 37 ± 3 s, increased and then depressed MAP and HR, and caused death at 188 ± 15 s. CO₂ at approximately 30% induced unconsciousness in 150 ± 15 s, decreased HR and MAP, and induced death at 440 ± 9 s. Ar at approximately 100% increased MAP but decreased HR, induced unconsciousness with hyperreflexia at 54 ± 4 s, and caused death at 197 ± 20 s. N₂ at approximately 100% decreased MAP but not HR and produced unconsciousness with hyperreflexia at 164 ± 17 s and death at 426 ± 28 s. We conclude that CO₂ effectively produced unconsciousness and euthanasia, but we were unable to ascertain distress. Ar also appears effective but produced hyperreflexia and tachycardia. N₂ was ineffective.

We monitored the development of hepatocellular carcinoma due to chronic infection with woodchuck hepatitis virus by using monthly serum samples, physical examination, and magnetic resonance imaging. The same woodchucks can be imaged repeatedly over a 1-y period by allowing the animals to recover after each experiment, thus reducing the number of animals required without compromising the quality of the data obtained. Age- and sex-matched uninfected control (n = 5) and chronically infected (n = 5) woodchucks were group-housed according to sex and infection status. Woodchucks were anaesthetized using an inhalation anesthetic (isoflurane) without premedication. During imaging, we regularly monitored heart rate, body temperature, and respiration. Tumor growth was observed using MRI, whereas the extent of hepatocyte injury was followed using serum liver enzymes. Elevated serum gamma glutamyltransferase and aspartate aminotransferase levels indicated hepatocyte injury due to tumor growth. On magnetic resonance images, the liver should appear as a well-defined, homogenous organ with defined regions of hyperintensities from larger blood vessels. Within tumor nodules, the liver appeared irregularly shaped, having heterogeneous intensity from unregulated cellular proliferation. Changes in tumor size can be monitored by imaging infected woodchucks on a regular basis. Using the imaging techniques we describe, the development of hepatocellular carcinoma can be visualized using magnetic resonance imaging, correlated to serum tests, and compared with the results from uninfected control woodchucks, thereby improving the understanding of the disease progress.

The cynomolgus monkey (Macaca fascicularis) may be the species of choice for nonclinical reproductive toxicology studies because of the comparability of its reproductive processes to man, similarity of pharmacologic response to various test substances, and decreased probability of immunogenic response to biological therapeutic products. For success in conducting these studies, the male and female monkeys used need to be characterized thoroughly. This study focused on the evaluation of 42 male cynomolgus monkeys as potential breeders for reproductive toxicology studies. Parameters evaluated included age, body weight, testicular volume, serum testosterone levels, ejaculate volume, and sperm parameters (count, motility, and morphology). The results showed that physical parameters (age, body weight, testicular volume) were a good starting point for selection of potential breeder males. However, additional endpoints (testosterone; ejaculate volume; sperm count, motility and morphology; mating behavior) were also helpful as part of an overall 'weight of evidence' approach to optimize selection of breeder males. In light of the data obtained in this study, 29 of 42 of the males evaluated were used with excellent success as breeder males for reproductive toxicology studies, resulting in an overall pregnancy rate of 36% when mated with sexually mature females. The successful breeder males were at least 6 y old, weighed at least 5 kg, had testicular volumes of at least 25 ml and serum testosterone levels of 1 to 10 ng/ml, and produced ejaculates with large numbers of sperm (median: 502 × 10⁶ per ejaculate) of high quality (few morphologic defects and most sperm highly motile).

An adult sooty mangabey (Cercocebus atys) with a solid mass arising from the skin of the dorsolateral cervical area was presented to the veterinary clinical staff. Grossly, the mass was firm, elongated, ulcerated at the tip, and measured 2.7 × 2.0 × 2.3 cm. It was surgically excised and then submitted for histopathologic evaluation. On histopathology, this tumor was composed of irregular masses and cords of neoplastic squamous epithelial cells that invaded the dermis and subcutis, often undergoing keratinization and forming numerous keratin pearls. On the basis of these histologic findings, the mass was diagnosed as a squamous cell carcinoma. Additional tests, including hematologic evaluations and radiographic views of the abdominal, thoracic, and cervical areas, were normal. Sections of the tumor were analyzed by electron microscopy and showed no evidence of viral particles. To the authors' knowledge, this is the first reported case of a spontaneous cutaneous squamous cell carcinoma in a sooty mangabey.

A 4.3-y-old, colony-bred female baboon (Papio hamadryas anubis) of low social rank and exhibiting no clinically significant signs of illness or distress was found dead at the Oklahoma University Health Sciences Center baboon breeding facility at El Reno, OK. Prior to death she exhibited excessive grooming behavior both toward herself and other baboons. In addition, she was consistently shy, timid, reclusive, and prone to minimal sustained movement (that is, generally lethargic behavior). Animals of low social rank typically exhibit some degree of these behaviors in order to avoid surplus interactions with other animals within their groups, which can lead to conflict and injury. Accordingly, her death was surprising in view of the apparent lack of clinical signs. Necropsy established the cause for death as systemic shock with resultant cardiovascular collapse resulting from a massive jejunal intussusception. This intussusception and resulting entrapment of the jejunal mesenteric vasculature caused total occlusion of the small bowel blood supply, with resulting hemorrhage and ischemic necrosis (small bowel infarction). Jejunal intussusceptions generally are considered to be uncommon and therefore are rarely reported in either the veterinary or human literature. Of special interest was the cause for this intussusception, determined to have been a large hairball located at the most proximal portion of the jejunum. Extending from this hairball and traversing essentially the entire length of the jejunum was a braided strand of hair acting as a string foreign body about which the intussusception formed. In light of our findings we suggest that animals of low social rank exhibiting excessive grooming behavior and lethargy might merit clinical evaluation to rule out possible abdominal disorders.

We investigated the effect of ketamine hydrochloride anesthesia on hematologic and serum biochemical values in 10 aged female bonnet macaques (Macaca radiata) before and 120 min after intramuscular administration of ketamine hydrochloride (15 mg/kg body weight). Ketamine anesthesia caused significant reduction in the total leukocyte count, lymphocyte count, red blood cell count, hemoglobin concentration, packed cell volume, and serum concentrations of glucose, total protein, alkaline phosphatase, calcium, sodium, and potassium. Although the effects of ketamine hydrochloride on hematologic and serum biochemical values have been reported for most of the nonhuman primates, no literature on bonnet macaques is available. These findings will be useful in designing experiments assessing pathologic and toxicologic changes in blood and serum parameters and interpreting data obtained from aged bonnet monkeys.

Reference values of blood volume (BV) and plasma volume (PV) of animal species are given as functions of body weight and gender specification generally is not given. Considering the common observation of a decreased hematocrit (Hct) in the females of many species, the BV, the PV, or both must differ between genders. The present study was performed to determine the magnitude of those differences. We measured Hct and PV in 24 female and 23 male Sprague-Dawley rats in their 12th week of life. The rats were surgically prepared with indwelling femoral arterial catheters 4 d prior to the determination of BV. Evan's Blue dye dilution was used to determine PV in conscious, quietly resting animals. BV was calculated as PV/(1 – Hct). Mean Hct was 2% lower in female rats than males, and PV (mean ± 1 standard deviation) was 4.86 ± 0.54 ml/100 g in females compared with 4.12 ± 0.32 ml/100 g in males. Calculated BV in female rats was 7.84 ± 0.70 ml/100 g compared with 6.86 ± 0.53 ml/100 g in males. When precise estimates of BV or PV are needed for research or dosing purposes, gender differences of 18% for PV and 14% for BV must be considered. In addition, species other than the rat may have similar discrepancies between sexes, and the prudent investigator must determine individual volume assessments of both sexes before assumptions of BV and PV for a species can be made.

Bilateral hindlimb paresis occurred in 3 guinea pigs after immunization with an adjuvant–antigen mixture containing complete Freund's adjuvant. Doses were injected into unanaesthetized animals, divided among 3 or 4 sites, and given slightly off midline in the subcutaneous tissues of the back. Neurologic examination of affected animals revealed intact flexor and panniculus responses and limited voluntary movement of the hindlimbs. Histopathologic interpretation of 2 affected animals showed fibrogranulomatous material effacing the skeletal muscle and vertebral bone, with marked bone lysis and infiltration into the marrow space and spinal canal. In addition, multiple granulomas in the pulmonary parenchyma were noted. A postmortem radiograph of the excised thoracolumbar spine of 1 animal revealed a soft tissue swelling and 'moth-eaten' and geographic osteolysis of 2 spinous processes. Hindlimb paresis and osteolysis likely resulted from accidental injection of the adjuvant–antigen mixture into the epaxial musculature and subsequent extension of injection site granulomas into the spinal canal.

Mice at our institution were hypophysectomized to evaluate the effects of growth hormone on the expression of a transfected human factor IX gene. The hypophysectomy was performed in-house by using a parapharyngeal approach modified from previously published surgical techniques. Modifications included: 1) choice of ketamine–xylazine and isoflurane for anesthesia, with butorphanol for postoperative analgesia; 2) use of a V-trough for positioning mice correctly and consistently; 3) selection of increasing sizes of dental burrs to create a foramen in the cranial base through which the pituitary gland was removed; and 4) disuse of a tracheotomy for airway patency. In addition, verification of successful gland removal was assessed by measuring major urinary protein (MUP) in the urine; presence of MUP indicated incomplete hypophysectomy. This assessment enabled antemortem determination of surgical success by using a single urine collection. Each of these modifications contributed to the success of the surgical procedure. We had a safe and reliable anesthetic regimen, consistent positioning of the surgical patient, and smooth and rapid penetration of the cranium. In our experience, the tracheotomy described in previous techniques was unnecessary, as the mice tolerated brief periods of apnea (approximately 5 sec maximum) while the trachea was retracted. Here we seek to provide details that will assist those interested in learning this technique and that will reduce the number of mice needed for practice. Other applications include a method of evaluating the production of growth hormone without euthanizing the animal.