Journal of the American Association for Laboratory Animal Science
Volume 46, Number 2, March 2007

ORIGINAL RESEARCH
Biology

Kastenmayer and Perdue.  Variation in Organ Volumes of Matched BALB/c Mice by Microcomputed Tomography Analysis, pp. 7-12

Introduction: The use of microCT results in the creation of a three-dimensional image constructed from a series of two-dimensional X-ray images.  It is used most commonly to scan tissues that are surrounded by tissue of significantly different density (e.g. lung and bone).  One of the main advantages of microCT is that longitudinal studies can be conducted on one animal that is serving as its own control, examples of both refinement and reduction.  The purpose of the study was to determine the amount of variation within a mouse, between mice, within an evaluator (i.e. the person analyzing the scan results), and between evaluators.  The two organs studied were lung and stomach into which contrast had been administered.

Materials and Methods: 10 Female 21-day-old littermate BALB/c mice were used for this study.  The mice were gavaged with 0.3 ml of iodinated contrast at 5 minutes prior to anesthesia with ketamine and xylazine.  After the mice were anesthetized, three scans were performed on each mouse, after which time the animal was euthanized by cervical dislocation and the tissues examined grossly and microscopically for any abnormalities that could potentially affect the study.  Each image generated was analyzed independently in triplicate by an experienced evaluator.  Additionally, the images from three mice chosen at random were analyzed in triplicate by a novice evaluator.

Results
1. 	There was less variability in three scans of the same mouse than in the triplicate analysis of a single scan by one evaluator.
2. 	Triplicate reads of all of the stomach scans showed comparable variability with multiple scans of a single mouse and scans of all mice.
3. 	Standard deviation for organ volume was larger for lung than stomach.
4. 	In the lungs, scan-to-scan variance (intramouse) was the same as mouse-to-mouse (intermouse) variance.
5. 	In the stomach, most of the variability was read-to-read variance.
6. 	When multiple scans were performed, the coefficient of variation decreased by 20% for the lung and by 1% for the stomach.
7. 	When analyzed by two different evaluators, there was more variability for the lung but not the stomach.
8. 	Mean lung and stomach volumes are listed.

Discussion: The authors hypothesized that the most variability would be amongst the organ volume of different mice; however, the results show that most of the variation occurred amongst scans of the same mouse.  Additionally, there was a significant bias between the evaluators, particularly when determining lung volume size.  The authors propose multiple reasons for these unexpected findings and conclude that microCT can be used as a method for both refinement and reduction in animal usage.

Questions: 
1.  	What does microCT stand for?
2.  	How is a microCT image generated?
3.  	True or False: The most variation occurred amongst multiple scans of the same mouse, rather than amongst multiple scans of different mice.

Answers:
1.  	Microcomputed tomography?
2.  	By a series of 2-dimensional X-ray images that are reconstructed with computer software to yield a 3-dimensional image.
3.  	True

Stutler et al.  Effect of Method of Euthanasia on Sperm Motility of Mature Sprague-Dawley Rats, pp. 13-20

Safety testing of pharmaceuticals and chemicals often includes potential impact on human fertility and reproduction, namely analysis of sperm viability. Histopathology of male reproductive organs alone can result in failure to detect deficits in sperm motility, velocity and concentration. Today an automated system (CASA), is used for evaluation of sperm concentration, percentage motility and movement. Commonly recorded parameters include curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), amplitude of lateral sperm head displacement (ALH), beat cross frequency (BCF) and straightness (STR). VCL, VSL and VAP are used to detect adverse kinematic effects. Swimming patterns are determined by ALH and BCF.   
 
In this study, seven approved methods for rat euthanasia (isoflurane, halothane, sevoflurane, enflurane, CO2 asphyxiation, Beuthanasia-D and Sleepaway) were evaluated for impact on sperm motility, concentration and overall viability. Rats euthanasia by rapid decapitation served as negative controls, while positive controls were euthanized by a combination of alpha chlorohydrin and rapid decapitation.  
 
For 5 of the methods, time to ataxia, recumbency, respiratory and cardiac arrest were measured. For rats euthanized by CO2 and Sleepaway, phases of euthanasia were not measured as rigorously in the other methods. Post euthanasia, distal caudal epididymides were harvested, and sperm motility and concentration analyzed.       
 
Of the 4 volatile anesthetics, only isoflurane and halothane resulted in a reasonable time to death (5-8 min). Although time to ataxia and recumbency using CO2 was prolonged as compared to the volatile anesthetics, clinical death was more rapid. Beuthanasia (pentobarbital sodium, 390 mg/ml) at 150 mg/kg IP resulted in the longest times for all phases of euthanasia, and is incompatible for sperm motion studies due to prolonged time to death (24 to 133 min). Of the volatile anesthetics, isoflurane had the least impact on sperm motility and sevoflurane the greatest adverse effect. 
 
The ideal method for euthanasia of rats destined for sperm motility studies is one that is capable of causing very rapid clinical death while minimizing chemical contact with epididymal tissues. In addition to meeting these criteria, CO2 is also easy to use, safe to handle and cost effective. The other agents may be used for sperm motion studies if instead of a euthanasia dose, and anesthetic dose followed by physical euthanasia was used, thus minimizing the duration of agent exposure and adverse effects on sperm motility.      
 
Questions: 
1. 	Increasing pH increases sperm motility. True or false? 
2. 	Circulating glucocorticoid levels affect sperm motility. True or false? 
3. 	There is a positive correlation between sperm motility and time to clinical death. True or false?    
 
Answers: 
1. 	True.
2. 	False.
3. 	False. The % motility and % progressively motile sperm were least affected by agents causing the most rapid euthanasia. 

Lamacchia et al.  Discrimination of C57BL/6J Rj and 129S2/SvPasCrl Inbred Mouse Strains by Use of Simple Sequence Length Polymorphisms, pp. 21-24

Primary Species: Mouse
Task 3.1: Provide Research Support, Information, and Services- biomethodology

The majority of mutant mouse strains are derived from embryonic stem cell lines of 129 strain mice the chimeras of which are bred with C57Bl/6 mice to propagate the mutation. 
Unfortunately genetic strain can modulate the phenotypic expression of the mutation hence it becomes necessary to back cross into a pure genetic background resulting in congenic mutant mice which will have the desired phenotypical expression. Traditional backcrossing takes 2-3 years because it takes 8-12 sequential generations of matings to move the genes of interest to the desired strain. 

Speed congenics which uses a Marker Selection Protocol (MASP) can reduce the time by identifying offspring with the lowest amount of donor alleles. Those with the lowest are used for breeding and the process continues until the genes are ‘fully’ transferred.

Simple sequence length polymorphism (SSLP) markers are very short nucleotide tandem repeats (CA)n    which are nonfunctional. Strains differ genetically and this difference is reflected in the distribution of these SSLP markers. PCR can amplify these markers as a means of genetic mapping to distinguish strains. Information on the SSLP map of the mouse genome is available in the Mouse Genome Database.

In this study SSLP markers were used to evaluate the PCR products obtained on NuSieve agarose or polyacrylamide gels.  The use of these markers helped successfully create the C57Bl/6j Rj congenic strain from the 129S2/SvPasCrl x C57Bl/6j Rj.

Questions
1. 	Speed congenics 
a) 	Incorporates SSLP and PCR
b) 	Refers to a harem breed scheme
c) 	Utilizes functional nucleotide sequences
d) 	None
2. 	A marker selection protocol (MASP)
a) 	Is used to select animals with the greatest donor alleles
b)	Is the principle behind speed congenics
c) 	Both
d) 	None
3. 	Which statement is not true?
a) 	Genetic strain can modulate phenotypical expression
b) 	SSLP markers are nucleotide repeats that code for enzymes
d) 	Congenic strains are produced with 8-12 generations of sequential matings
d) 	SSLP are amplified via PCR during screening

Answers:
1.	a
2.	b
3.	b

Callicott et al.  Genomic Comparison of Lewis and Wistar-Furth Rat Substrains by Use of Microsatellite Markers, pp. 25-29

Summary:  Inbred strains of rats are commonly used and many users are aware of strain differences between vendors.  This paper examines that genetic variation through the use of microsatellite markers.  3 Lewis substrains and  2 Wistar-Furth substrains were obtained from 3 different suppliers.  (See  historic chart on page 29 of lineage of Lewis and Wistar-Furth substrains.)  Lewis and Wistar-Furth strains were both created from outbred Wistar stock.  The Lewis substrains have been separated since the late 1950s.  Overall the  Wistar-Furth substrains have been transferred to fewer locations than the  various Lewis substrains.  Lewis rat substrains differed as much as 45%.  Wistar-Furth substrains differed by 8%.  Investigators should consider the  background genetics of the particular strains for their research projects  and should use strains from a  single source when feasible.

Questions:
1.  	Name several factors that can contribute to substrain divergence.
2.  	What are SSLPs?

Answers:
1. 	Breeding errors, incomplete inbreeding with residual allogenicity, mutation and genetic drift
2.	Simple sequence-length polymorphisms - selected markers used to compare substrains 

Kaiser et al.  Anesthesia for Cardiovascular Interventions and Magnetic Resonance Imaging in Pigs, pp. 30-33

Summary: The aim of this study was to develop an effective anesthetic regimen for pigs undergoing vascular interventional procedures under magnetic resonance imaging (MRI) guidance. Inhalation anesthesia was not a viable option due to the potential interaction of MRI and ferromagnetic equipment. The anesthetic regimen needed to be adequate for transportation, surgical placement of catheters, sedation during imaging, and investigator-induced intermittent breath-hold episodes to reduce motion artifact. 32 domestic pigs (German Landrace Universal, 40-125 kg) were given ketamine (30 mg/kg), azaperone (2 mg/kg), and atropine (0.025 mg/kg). When sedate, the animals were induced with propofol (2 mg/kg) and fentanyl (0.005 mg/kg), and anesthesia was maintained by continuous intravenous infusion of propofol (2.29 mg/kg/h), midazolam (1.14 mg/kg/h) and fentanyl (0.009 mg/kg/h) for a duration of 8-10 hours. An endotracheal tube was placed and pigs were ventilated with 50% oxygen at 10-12 breaths/min. For MR procedures, all infusion pumps and ventilation equipment were placed beyond the 5-m Gauss line using 6-m lines and ventilation tube. This anesthetic regimen was adequate for all procedures and no complications were reported. For MR breath-hold procedures, apnea was achieved for a maximum of 2 min without signs of hypoxia. 

Questions:
1. 	Which of these anesthetic regimens has been shown to be well-suited for MR-guided vascular interventional procedures in pigs?
a. 	Isoflurane
b. 	IV infusion of propofol, midazolam, and fentanyl
c. 	IV infusion of ketamine, midazolam, and fentanyl
d. 	IV infusion of midazolam
2. 	When administering anesthesia for MR procedures, how far away from the magnet must ferromagnetic equipment be placed?
a. 	More than 5 meters
b. 	More than 10 meters
c. 	Beyond the 5-m Gauss line
d. beyond the 10-m Gauss line
3. 	True or false: Apnea for MR breath-hold episodes cannot be achieved with IV infusion anesthesia.

Answers:
1. 	b
2. 	c
3. 	False

FACILITY MANAGEMENT

Burke et al.  Use of Environmentally Enriched Housing for Rats with Spinal Cord Injury: The Need for Standardization, pp. 34-41

Task: 2 Prevent, alleviate, and minimize pain and distress
Task:  8 Educate Scientific, Animal Care and Ancillary Staff
Primary Species: Rats

Summary: Rehabilitation methods that increase the social interaction and locomotor activity have been shown to improve the recovery of animals and humans with spinal cord injuries (SCI). This study evaluated the effects of environmental enrichment and group housing of 4 rats in thoracic SCI model in S-D rats. The effects of the rehabilitation methods were assessed using electrophysiology, behavior [Basso-Beattie-Bresnahan (BBB) open field locomotor test] and morphologic methods. Further, trans-cranial magnetic motor evoked potential (tcMMEP) and magnetic-evoked interlimb reflex (MILR) were used to assess the neurotransmission in the thoraco-lumbar spinal cord. Results of this study indicated that the environmental enrichment has no effect for either electrophysiological or morphologic (amount of spared white matter at the epicenter) measures. Also, in contrast to previous study, the environmental enrichment did not improve the locomotion of rats with contusion thoracic SCI. This may due to the fact that present study differed in several ways from previous investigations, such as differences in the rat strain, gender, injury level, study duration and animal density, cage size and available cage space per animal. Thus, to determine the effectiveness of environmental enrichment to improve locomotor recovery in SCI rats requires standardization of housing procedures, outcome measures and analyses. 

Questions: 
1.	Name the two electrophysiological measurements used in this study to assess the neurotransmission through the thoracic and lumbar spinal cord. 
2.	Define BBB 
3.	T or F. Low housing density is reported to influence the statistical power and effect size as well as outcome measures of the experiment. 

Answers:
1.	Trans-cranial magnetic motor evoked potential (tcMMEP) and Magnetic-evoked interlimb reflex (MILR) 
2.	Basso-Beattie-Bresnahan (BBB) open-field locomotor scale. It is a measure that assigns points to some components of walking characteristics assessed during the open field test, including the toe clearance, paw position, trunk instability and tail position. 
3.	True 

Reichard et al.  Efficacy of Fenbendazole and Milbemycin Oxime for Treating Baboons (Papio cynocephalus anubis) Infected with Trichuris trichuria, pp. 42-45

Task 1 - Prevent, Diagnose, Control and Treat Disease
Primary Species - Olive Baboon (Papio cynocephalus anubis)

Summary: This article focused on the efficacy of fenbendazole and milbemycin oxime in the treatment of baboons with naturally acquired Trichuris trichuria infection.  All baboons that received fenbendazole stopped shedding T. trichuria eggs within 6 days of treatment.  The milbemycin oxime regimen never totally eliminated the eggs of T. trichuria.

Questions:
1. 	T or F. Trichuris trichuria as zoonotic.
2.  	For this study, the eggs shed per gram of feces was determined up to 
a. 	20 days prior to treatment
b. 	Every other day for 30 days
c. 	65 days after treatment
3. 	The standard treatment of whipworm infection in animals includes all of the following except:
a. 	Fenbendazole
b. 	Mebendazole
c. 	Milbemycin oxime
4. 	Antihelmintic efficacy was determined by _____________________________ and ______________________________________.
5. 	Fenbendazole exerts ________________ by binding to nematode __________ and disrupting mitosis and mitochondria function.
6. 	Milbemycin oxime is similar to __________________ in structure and activity by binding to _________________________ causing          paralysis and death of nematode.

Answers:
1. 	True
2. 	C
3. 	B
4. 	Descriptive statistics, percentage fecal egg count reduction (FECR)
5. 	Nematocidal activity, tubulin
6. 	Ivermectin, glutamate-gated chloride channels

EXPERIMENTAL TECHNIQUE

Johnston et al.  Strategies for Refinement of Abdominal Device Implantation in Mice: Strain, Carboxymethylcellulose, Thermal Support, and Atipamezole, pp.46-53

Summary: Abdominal implantation of transmitters for telemetric monitoring that are relatively large in comparison to the size of the mouse abdomen can lead to anorexia and inactivity, abdominal adhesions, intestinal obstruction, and death. Authors therefore evaluated refinement strategies associated with the abdominal implantation of these transmitters during a primary study involving cranial and abdominal surgery for a Candida albicans infection model. Mouse strain and body weight were found to influence the proportion of mice that survived, causing this group of researchers to now require a minimum weight of 25-26 g for a mouse undergoing dual cranial and abdominal surgery. Atipamezole administration was associated with increased mortality. Carboxymethylcelluose (CMC), an abdominal lubricant that may decrease adhesion formation, was detrimental to treated mice, causing decreased activity and lower core body temperatures, though none of the mice treated with CMC had adhesions or intestinal surface changes at necropsy. Post-surgical thermal support and ibuprofen treatment had no appreciable effect on recovery. 

Questions:
1. 	A) A/J mice or B) C57BL/6 mice are more sensitive to carrageenan-induced hypersensitivity.
2. 	T/F   Carboxymethylcellulose treatment of the abdominal cavities during surgery greatly improved the survival of mice implanted with a telemetry device.
3. 	Bowl obstructions typically occur within ___ days after abdominal surgery to implant telemetry transponders.
	A) 	1
B) 	2
C) 	5
D) 	10
4. T/F   Atipamezole competitively inhibits alpha-2 adrenergic receptors, making it useful as a reversal agent for medetomidine and potentially xylazine.

Answers:
1. 	A) A/J mice
2. 	F – CMC appeared to interfere with, rather than promote, recuperation.
3. 	C) 5 days
4. 	T

Hayashimoto et al.  A Specific Polymerase Chain Reaction Based on the gyrB Gene Sequence and Subsequent Restriction Fragment Length Polymorphism Analysis of Pasteurella pneumotropica Isolates from Laboratory Mice, pp. 54-58

Task 1 - Prevent, Diagnose, Control, and Treat Disease 
Primary species – mice

Summary: A polymerase chain reaction (PCR) was developed base on gyrB gene sequence, and the amplified product from the PCR was used on a restriction fragment length polymorphism (RFLP) analysis to facilitate the differentiation of biotypes of Pasteurella pneumotropica.

The polymorphism in the gyrB gene has a higher ability to discriminate among closely related bacterial species even when the 16S rRNA sequence is almost identical. This molecular identification and typing tools was validated using 105 isolates of Pasteurella pneumotropica.

Questions:
1. 	PCR and RFLP analysis has been successfully used to differentiate biotypes of Pasteurella pneumotropica. 
a.	True 
b.	False
2. 	The polymorphism in the gyrB gene has a higher ability to discriminate among closely related bacterial species even when the 16S rRNA sequence is almost identical.
a.	True 
b.	False

Answers:
1.	a 
2.	a

Wiedmeyer et al.  Complete Blood Count, Clinical Chemistry, and Serology Profile by Using a Single Tube of Whole Blood from Mice, pp. 59-64

Task 1; Diagnose and control disease
Species = Mouse

Summary: Traditional recommendations for handling blood for analysis is to put part of the sample into an EDTA tube for CBC analysis and a serum clot tube to obtain serum for chemistry and serological analysis. Because of the small size of mice, obtaining enough blood for an adequate sample in both tubes can at times be problematic. This study compared doing all three analyses from blood in a single heparin tube verses the EDTA and serum clot tubes. The study found that a much higher percentage of all tests could be performed when one tube was used instead of 2 tubes. A minimum of 500 μl of blood was needed for the single tube to perform all of the tests. 

The greatest difference in test results was in platelet counts when the blood was stored for 24 hours before being analyzed. It appeared that a number of platelets clumped with the heparin tubes, thereby reducing the number that was counted, whereas this was not a problem with the EDTA blood. Other blood parameters were not significantly different between the two methods of anticoagulant. Final recommendations were to use a heparin tube when you need to run both a CBC and chemistry and/or serology.

The study also compared amount of blood that could be obtained from mice that received drinking water from a gel pack vs. a water bottle. An average of 672 μl of blood was obtained from mice on a water bottle and 660 μl from the mice on gel packs, which indicates that mice on gel packs do not become dehydrated.

Questions:
1. 	Name a use for gel packs
2. 	The main difference on CBCs from the heparin tube and EDTA tubes was? 

Answers:
1.  	Shipping 
2.  	Decreased platelet counts after storage for 24 hrs. with the heparin tubes due to platelet clumping

McIntyre et al.  Automated Mouse Euthanasia in an Individually Ventilated Caging System: System Development and Assessment, pp. 65-73
Task 2: Prevent, Alleviate, and Minimize Pain And Distress 
Summary: The authors developed a CO2 euthanasia system that functions as an individually ventilated caging system and accommodates the simultaneous euthanasia of as many as 70 cages of mice.

The AVMA panel considers CO2 euthanasia to be acceptable for rodent euthanasia under specific conditions. CO2 delivery should be predictable, controllable, and displace air by mixing and dilution. 2005 ACLAM Task Force on Rodent Euthanasia report reiterates several of AVMA Panel on Euthanasia points. Chambers should be filled at a flow rate that balances time to unconsciousness with such associated aversive stimuli as noise or high-velocity air movement. Air should be replaced with CO2 at a rate equal to 20% of the chamber volume per minute.  Animals should be closely observed to be able to adjust optimum flow rate.
System developed consisted of a 2-min fill, 15 -min soak and a 5-min exhaust phase. Extensive testing resulted in 100% efficacy of euthanasia for nongravid adult and juvenile (older than 6 days) mice. It failed to euthanize neonates 6 d and younger and late-term gravid females with 100% efficacy. Resistance of neonates to the effect of CO2 is consistent with reports stating that extended exposure to CO2 is necessary to cause death in neonates. Fetal and neonatal mice are adapted to the low-oxygen uterine environment and possess a reflex whereby metabolic, respiratory and heart rates decrease during hypoxic conditions. Mechanism of resistance from gravid females not elucidated - may have O2 from fetal blood, dam may adapt to lower O2 during pregnancy - they used 100% Co2 - further work needed to understand mechanism of resistance.
Questions: 
1. 	CO2 delivery for euthanasia should be: 
a. 	Controllable 
b.  	Predictable 
c.  	Cooled before delivery to the system 
d.  	Displace air by mixing and dilution 
e.  	a, b and d 
f.  	All of the above 
2.	The ACLAM Task Force on Rodent Euthanasia recommends flow rates of CO2 should displace ______ of the chamber volume per minute:
a. 	30 % 
b.  	25% 
c.  	20% 
d.  	50% 
3. 	T or F.  Mice less than 6 d of age cannot be euthanized in this system due to a reflex adaptation to low-oxygen environment developed in the uterus where their metabolic, heart and respiratory rate decrease.

Answers: 
1. 	e 
2. 	c 
3. 	T 

Saha et al.  Comparison of Cardiovascular Effects of Tiletamine-Zolazepam, Pentobarbital, and Ketamine-Xylazine in Male Rats, pp. 74-80

Task 3: Provide Research Support, Information, and Services 
Primary Species

Anesthetics can cause profound physiologic alterations, especially in respiratory and cardiovascular systems.  The ideal anesthetic protocol balances the effects of the anesthetic regimen on study results and the comfort and welfare of the animals.  

Traditionally, barbiturates such as pentobarbital have been the anesthetic of choice for rats because of ease of administration and minimal cardiovascular depression.   However, barbiturates have poor analgesic characteristics.  Barbiturates cause generalized central nervous system depression, prolonged respiratory depression, hypothermia, hypotension, tachycardia, and prolonged recovery.  Surgical anesthesia with pentobarbital can be maintained only with doses close to causing respiratory failure.  Ketamine is a dissociative anesthetic has been thought to have minimal cardiac and respiratory effects.  It is short- acting, produces variable analgesia, and causes muscle rigidity, which can be minimized by using it with xylazine or diazepam.  Telazol consists of tiletamine (a dissociative anesthetic) and zolazepam (benzodiazepine tranquilizer).  

Although Telazol has been reported to be an effective anesthetic in rats, the cardiovascular effects of this agent had not been explored previously in rats.  The purpose of the present study was to assess the cardiovascular effects of Telazol in comparison with ketamine and pentobarbital in rats.

The size and sex of an animal can contribute to different responses to anesthetics (e.g. female rats are more sensitive to barbiturates than male rats.  Fat can be associated with prolonged recovery time because fat can serve as a storage site.   Thus, in this study only male rats of similar weight were used.  

The following anesthetic groups were compared:  ketamine 50 mg/kg and xylazine 5 mg/kg; ketamine 75 mg/kg and xylazine 5 mg/kg; pentobarbital 45 mg/kg; Telazol 30 mg/kg; Telazol 40 mg/kg, Telazol 50 mg/kg, and Telazol 60 mg/kg.

Animals were re-dosed as needed.  Respiratory rate (RR), heart rate (HR), mean arterial pressure (MAP), cardiac index (CI), and stroke index (SI) were measured every 30 min for 4 h.  Once anesthetized, the femoral artery and jugular vein were cannulated.   HR determined from the arterial pulse.  Core body temperature (CT) from the intravascular thermistor probe.  Cardiac output (CO) was measured by using the thermodilution technique.  All measurements were taken q 30 minutes beginning 90 minutes after initial anesthetic dose.  The CO and stroke volume were normalized to body weight and reported as cardiac index and stroke index, respectively. 
  
The Telazol groups showed a dose-dependent increase in duration of anesthesia.  Duration of anesthesia was significantly shorter for the ketamine and pentobarbital groups than for any of the Telazol doses.  Heart rate showed a dose-dependent decrease among the Telazol groups , but overall heart rate in these groups was higher than in the ketamine and pentobarbital groups.  MAP in the Telazol 40 and 50 groups were significantly higher than the pentobarbital and higher ketamine groups yet lower than the Telazol 60 group.  Overall, animals anesthetized with Telazol showed the highest cardiac index, ketamine intermediate, and pentobarbital the lowest.  The pentobarbital group exhibited the lowest stroke index, whereas ketamine-treated animals had an intermediate stroke index.  Telazol groups showed stable RRs.  RRs for Telazol groups were highest followed by ketamine groups and then pentobarbital group.   

These differing effects of anesthetics on cardiovascular parameters must be considered when choosing an anesthesia regimen or comparing data from different studies.  In the authors model, the Telazol 40 and 50 groups appeared to exhibit the fewest adverse cardiovascular effects. 

Questions:
1.	What are the constituent pharmaceutical agents that comprise Telazol? 
2.	Describe these agents.
3.	Which dosing regimen evaluated caused the greatest depression in the RR?
4.	Which dosing regimens appeared to exhibit the fewest adverse cardiovascular effects?

Answers:
1.	Tiletamine and zolazepam
2.	Tiletamine (a dissociative anesthetic) and zolazepam (benzodiazepine tranquilizer)	
3.	Pentobarbital
4.	Telazol 40 and 50 groups  

Tashiro et al.  The Evaluation of Whole-body Plethysmography as a Semiautomated Method for Analysis of Emesis in the House Musk Shrew (Suncus murinus), pp. 81-85

Task 3 - Provide Research Support, Information, And Services 
Task 9 - Collaborate on the Selection and Development of Animal Models 

Tertiary Species – Other (house musk shrew, Suncus murinus)

Summary: Emesis associated with chemotherapy and radiotherapy is a serious problem for cancer patients.  Research with animal emesis models allows for screening of new anti-emetic agents.  However, current preclinical animal models require subjective and time- and labor-intensive methods to evaluate emetic behaviors.  In this study, whole-body plethysmography analysis of cisplatin-induced emetic responses in the house musk shrew (Suncus murinus) was compared with behavioral observation and pharmacologic validation.  Changes in peak expiratory flow (PEF) were easily and semiautomatically detected by whole-body plethysmography and were coincident with emesis.  Plethysmography can be used as a novel, objective, sensitive, and efficient semi-automated method for analyzing emesis.  The technique also provides scientific and cost advantages as compared to existing methodology for emesis research.  

Questions
1.	All of the following agents are considered emetogens EXCEPT?
a.	Cisplatin
b.	Veratrine hydrochloride
c.	Ondansetron
d.	Nicotine bitartrate
e.	Copper sulfate
2.	All of the following animals have been studied as emesis models EXCEPT?
a.	Pigeons
b.	Dogs
c.	Ferrets
d.	House musk shrews
e.	Rats
3.	All of the following techniques are ways of detecting emetic behaviors automatically in preclinical animal models EXCEPT?
a.	Arterial pressure
b.	Electroencephalogram recordings
c.	Phrenic nerve activity
d.	Electrocardiogram recordings
e.	Intratracheal pressure
4.	Which of the following statements best describes Suncus murinus?
a.	Carnivore
b.	Herbivore
c.	Insectivore
d.	Omnivore
5.	Name some advantages and disadvantages in using whole-body plethysmography in emesis studies.
6.	To which order and family does Suncus murinus belong?

Answers: 
1. 	c
2. 	e
3. 	b
4. 	c
5.	Advantages: 1) provides more objective measurement than counting emetic episodes; 2) offers a high-throughput approach for analysis of emetic behaviors; 3) allows PIs to test more animals and compounds daily; and 4) eliminates operator fatigue that results during analysis of video recordings.  
	Disadvantages: 1) plethysmography cannot discriminate between retching and vomiting; 2) unknown whether defecation, micturition, or jumping affects PEF values; and 3) unknown whether drugs or agents that cause sedation or affect respiratory function alter plethysmographic analysis (possible source of false-positives or false-negatives)
6.	Order Insectivora, Family Soricidae

CASE REPORTS

Pritchett and Saunders.  Epistylididae Ectoparasites in a Colony of African Clawed Frogs (Xenopus laevis), pp. 86-91

Secondary Species - African clawed frog
Tertiary Species - Invertebrates

Task 1 – Prevent, Diagnose, Control, and Treat Disease
Task 8 – Educate Scientific, Animal Care and Ancillary Staff

The African clawed frog, Xenopus laevis, is used in research for oocyte production for cell and molecular biology research, teratogen screening, and toxicant susceptibility.  The tiger salamander, Amblystoma tigrinum, is used for developmental research, limb regeneration studies, and comparative anatomy studies. Both animals are captured from the wild routinely for research use however, only Xenopus are bred in captivity.

Captive housing systems, water quality, handling, and manipulations associated with research or general husbandry can cause stress to amphibians.  This stress can lead to perturbation in immune system function making them susceptible to opportunistic infections.  The authors point out the amphibians has a natural barrier to infection in the mucus layer produced by the skin that provides a physical barrier to pathogens and also contains biologically active peptides.  Of the family of antimicrobial peptides evaluated in Xenopus laevis, the magainins found in the mucous layer are antibacterial, antifungal, and antiprotozoal in function.  Therefore, a disturbance of the protective mucous layer due to research or husbandry practices may leave the animal susceptible to multiple opportunistic infections.

Case Report: Two different species of amphibians were housed in the same room at a research facility. 1) A colony of female African clawed frogs (Xenopus laevis) obtained from a vender (bred by the vender) for oocyte production.  These frogs were housed in a static housing system.  2) A colony of tiger salamanders, Amblystoma tigrinum, was acquired from a vendor.  These animals were previously captured in the wild and were sorted by size.  The salamanders housing units had Reef Builder (compound increases alkalinity and total hardness of the water).  The salamanders were also kept refrigerated at 4 degrees centigrade to prevent metamorphosis and minimize trauma from cage mates’ aggression.  These animals were also kept in static housing units.  See the Animals and Husbandry section of this paper for a detailed description of the housing and husbandry practices for these amphibians (page 86-87).

On routine examination, animal care staff reported several salamanders were reported for having a white, flocculent material adherent to their gills and tails.  Wet-mount microscopic examination of this material was shown to be Saprolegnia sp.  Treatment for this condition was initiated using malachite green-formalin added to the water every other day for 5 days (total of 3 treatments) at a concentration of 0.15 mg/l cubed.  A complete water exchange was performed after each bah treatment.  Clinical signs of Saprolegnia resolved after treatment.

Eleven days post treating the salamanders for Saprolegnia, half of the resident frog population was reported to veterinary staff for tuffs of flocculent material on the toes and claws of these animals (Figure 2).  Water samples were taken from both the tap and the frog tanks (carboys) for microbiological culture and water quality testing.  The white flocculent material was evaluated by wet-mount microscopy revealed Saprolegnia sp. and a ciliated, sessile protozoan which is classified as Epistylis sp., (most likely E. plicatilis).  Histopathological examination of an affected frog showed hepatocellular degeneration, moderate tubular necrosis, and protozoans consistent with Epistylis sp in hyperkeratotic skin.  The Epistylis infection was treated with a once a week 30 minute bath in noniodized sodium chloride (25 g/l).  This protocol was modified from a treatment previously used in fish.  This treatment was administered once weekly for 3 week, an additional 3 week course of sodium chloride baths was continued for frog tanks with persistent infections.  Frogs were also treated with malachite green-formalin (0.15 mg/l) every other day for 2 weeks.  

One week after the frogs were treated, a decrease in oocyte quality was noted.  Investigators removed these affected frogs from study and used new un-affected animals to complete research projects.  No frog mortality was noted during the first 3 treatments however, frogs were found dead after the 5th sodium chloride treatment.  Clinical signs of Saprolegnia evidence did not exist so malachite green-formalin treatment was discontinued.  The frog colony was monitored for a month after the last sodium chloride treatment; they were shown to have persistent Epistylis infection due to sodium chloride resistance.  Because the Epistylis infection could not be eradicated in these frogs, all frogs in the affected room were euthanized.

Summary:  Please read the well written discussion section for exam review.  Note Table 1 and Figure 3.  Saprolegnia are aquatic fungi found in soil and fresh water.  The organisms infect animals through direct contact.  These species of fungi can infect amphibians and fish.  It is a primarily opportunistic pathogens and only able to infect animals with areas of injured skin or damaged mucous layers.  Infections with Saprolegnia sp probably are acquired from poor water quality or water with high organic load.  Epistylis sp, subclass Peritrichia, suborder Sessilina, and family Epistylididae is classified as a freshwater sessile protozoan.  The Authors conclude that inconsistent husbandry practices (inadequate disinfection of the scoop between tank transfers), poor water quality in the static tank systems for both frogs and salamanders, and the housing of two different species of amphibian in the same room lead to the Saprolegnia and Epistylis infections.  Additionally, the housing requirements for the salamanders (4 degrees centigrade, high water alkalinity and hardness), prevented the proliferation of Epistylis in this species.  Housing these two species separately would have prevent the cross infection with Saprolegnia.  The authors state that Epistylis infection in healthy Xenopus laevis is nonpathogenic, eradication is likely not essential with sound husbandry practices.  Frogs treated with sodium chloride should receive no more than three treatments; remain isolated from non-infected frogs until used for terminal procedures.

Questions:
1.	What is the common name of Xenopus laevis?
2.	Name the genus and species of the tiger salamander?
3.	What type of kidney does Xenopus sp. have?  How does this type of kidney function?
4.	Why were tiger salamanders housed at 4 degree centigrade temperatures?
5.	What was the disadvantage of extensive use of the sodium chloride treatments to treat Epistylis infections?

Answers:
1.	African clawed frog
2.	Amblystoma tigrinum
3.	Mesonephric kidneys, have no loops of Henle, cannot concentrate urine because frogs are hypertonic to their environment
4.	To prevent metamorphosis and minimize trauma from cage mate aggression
5.	Persistent Epistylis infection due to sodium chloride resistance, increased frog mortality after three treatments, decreased oocyte quality

Chu et al.  Uterine Epithelioid Trophoblastic Tumor in an African Green Monkey (Chlorocebus aethiops sabaeus), pp. 92-96

Summary:  This article is the first documented case of spontaneous uterine epithelioid trophoblastic tumor (ETT) in an African green monkey. ETT is a rare malignant neoplasm of the chorionic-type intermediate trophoblast origin. Trophoblasts originate as a thin layer of cells within a blastocyst.  The blastocyst consists of an inner cell mass and the trophoblast. The inner cell mass eventually develops into the fetal embryo whereas trophoblastic cells contribute to chorionic placenta formation. Subpopulations of trophoblasts include cytototrophoblasts the undifferentiated stem cell population; synctiotrophoblasts the terminally differentiated trophoblasts which produce placental hormones; and intermediate trophoblasts which are functionally and morphologically in between the first two cell types. Intermediate trophoblasts are classified into various subpopulations of villous intermediate trophoblasts called trophoblasts at the placental anchoring villi. Intermediate trophoblasts differentiate into either implantation-site intermediate trophoblastic cells of the chorion frondosum or degenerate into chorionic-type intermediate trophoblasts of the chorionic laeve. ETT arises from chorionic-type intermediate trophoblasts. Only 90 human cases have been documented since characterization of ETT in the late 1990’s. Only two nonhuman primate cases of ETT have been published prior to this report. They are an ovarian ETT in a cynomologus monkey and a uterine ETT in a red-tailed guenon. In the current case the tumor mass was round, tan, firm, minimally compressible, well-circumscribed, solid, measured 4.5 x 5 cm, occupied the fundus plus the lower uterine segment, and was tightly adhered to the endometrium. It was surgically removed along with the entire uterus. The animal suffered no postoperative symptoms to date after 27 months. Histologically, the uterine mass was composed of sheets, nests, and cords of infiltrating medium to large sized prominently mononuclear cells in myometrial tissue, with a background of abundant “geographic” necrosis. The tumor cells were of two morphologic types 1) some cells were relatively uniform with mild nuclear pleomorphism and nuclear enlargement, clear to lightly eosinophilic cytoplasm and 2) the other cells had more abundant eosinophilic cytoplasm, with moderate to focally severe nuclear pleomorphism, scattered enlarge bizarre nuclei, and numerous atypical and typical mitotic figures (4 mitotic figures/high-power field). The tumor cells’ cytoplasm stained intensely immunohistochemically with pancytokeratin marker AE1/3, weakly and in patches with human placental lactogen. Tumor cell nuclei were diffusely labeled with transcription factor p63 antibody. Moderate membrane staining was detected using the placental alkaline phosphatase antibody. ETT is a rare tumor that may be clinically asymptomatic. In humans ETT has a 25% metastatic rate and a 10% mortality rate. 

Questions:
1) Which species of nonhuman primates has not had a reported diagnosis of an epithelioid trophoblastic tumor (ETT)?
a. 	Cynomolgus monkey (Macaca fasicularis)
b. 	African green monkey (Chlorocebus aethiops sabaeus)
c. 	Rhesus monkey (Macaca mulatta)
d. 	Red-tailed guenon (Cercopithecus ascanius)
2) 	Which is the cell type of origin of epithelioid trophoblastic tumors (ETT)?
a. 	Cytototrophoblasts, the undifferentiated stem cell population
b. Synctiotrophoblasts the terminally differentiated trophoblasts which produce placental hormones
c. 	Chorionic-type intermediate trophoblasts of the chorionic leave
d. 	Intermediate trophoblastic cells of the chorion frondosum
3) 	Which of the following immunohistochemical staining patterns can be used to assist in the diagnosis of epithelioid trophoblastic tumors?
a. 	Nuclear staining of p63 and cytoplasmic staining of smooth muscle actin
b. 	Positive cytoplasmic staining of pancytokeratin A1/3 and negative staining with alpha inhibin 
c. 	Diffuse cytoplasmic staining with placental alkaline phosphatase and p63
d. 	Nuclear staining of p63 and cytoplasmic staining of pancytokeratin A1/3

Answers
1.  c
2.  c
3.  d

ACLAM Task Force Members.  Guidelines for the Assessment and Management of Pain in Rodents and Rabbits, pp. 97-108

Task 2: Prevent, Alleviate and Minimize Distress

Summary: ACLAM public statement regarding pain recognition and management in rodents and rabbits. Mediators of inflammation and pain: At time of tissue injury, a host of substances are released locally and contribute to the process of inflammation and increased sensitivity of nociceptors.  Bradykinins, prostaglandins and serotonin activate and sensitize nociceptors. This causes release of substance P (a tachykinin) and other peptides which causes mast cell degranulation and histamine release which further excites nociceptors. At level of dorsal horn, 2 endogenous opioid peptides, enkephalin and dynorphin are released after nociceptive stimulation from descending fibers originating in brain stem. Opioid containing neurons can have an excitatory or inhibitory effect, but primarily act to inhibit transmission of nociceptive activity. 

Another modulating mechanism, known as the "pain gate" phenomenon, occurs when stimulation of thick A beta fibers of peripheral nerves decreases the sensitivity of postsynaptic cells in the dorsal horn to incoming signals from C and A gamma fibers. This property helps to explain how mechanical stimulation such as stroking or rubbing the skin can provide pain relief. 

Descending control pathways: These inhibit pain perception and are called antinociceptive, and include supraspinal modulating loops. Central modulation of pain perception response involves parts of cortex, the periaqueductal gray region of the midbrain, the lateral pons and ventromedial medulla. Stimulation of these sites can result in inhibition of dorsal horn neuronal activity. 

Pain response can be modified at the peripheral level, spinal cord, or CNS. Anti-inflammatories may dramatically reduce sensitization of nociceptive fibers, administration of epidural and intrathecal agents work at the level of the dorsal horn by reducing excitation of nociceptive neurons and stimulating inhibitory pathways. 

Non-pharmacological considerations for pain management can be enhanced by providing appropriate housing, handling, and restraint and by using appropriate experimental techniques such as proper surgical technique.  Increased environmental temperature is generally required during anesthesia recovery, removal of wet materials, easy access to food and water, nesting materials, soft food, bandaging and expression of bladder with spinal cord dysfunction are examples of these methods.

Assessment of pain: Detection of pain in rodents and rabbits is based on subjective evaluation of behavioral and attitudinal changes as well as an objective analysis of physiologic parameters. Behavioral signs indicative of pain perception may include reluctance to move, abnormal posturing, teeth grinding, decreased appetite and vocalization.  Attitudinal changes include anxiety, apprehension and hypersensitivity as well as depression or, uncommonly overt aggression. Signs indicative of pain-induced distress characteristic of rodents include polyphagia of bedding that may progress to self-mutilation.  Physiologic signs of pain perception include fluctuations in blood pressure, heart rate, and body temperature. Food, water consumption and body weight are others. 

Pain scoring system: Adopted from human work, the visual analog pain scale is used by the observer to rate each behavior/criteria chosen on a scale from 1-5. Scales of pain scores are created from this and higher numbers are used to justify analgesics, humane endpoints or euthanasia.  This must be used efficiently however and allow the staff doing the scoring time to administer analgesics if needed. 

Analgesics: Local anesthetics can be very useful for rodents and rabbits used solely or as an adjunct to other agents. Lidocaine and bupivicaine are the two referred to by the authors.  NSAIDs such as carprofen and meloxicam can be used for mild to severe pain and can be combined with an opioid such as buprenorphine, morphine and fentanyl patch (rabbit).  Scant information is known about efficacy of analgesics in hamsters, gerbils and guinea pigs. Buprenorphine is the most commonly used analgesic for these species. 

Questions:
1.	Visual analog pain scoring system is used to predict the amount of pain derived from experimental procedure? T F
2.	Administration of a local anesthetic should be carefully done to avoid IV placement? T F
3.	C-section in a rat would be expected to cause what severity of pain
a.	Mild
b.	Moderate
c.	Severe
d.	None
4.	Cool, moist bedding materials are very important for rapid recovery of rodents and rabbits following anesthesia. T F

Answers:
1.	F, evaluation only
2.	T
3.	b
4.	F

PUBLIC STATEMENT

ACLAM task force.  Guidelines for the Assessment and Management of Pain in Rodents and Rabbits, pp. 97-108

Task 2: Prevent, Alleviate and Minimize Distress

Summary: ACLAM public statement regarding pain recognition and management in rodents and rabbits. Mediators of inflammation and pain: At time of tissue injury, a host of substances are released locally and contribute to the process of inflammation and increased sensitivity of nociceptors.  Bradykinins, prostaglandins and serotonin activate and sensitize nociceptors. This causes release of substance P (a tachykinin) and other peptides which causes mast cell degranulation and histamine release which further excites nociceptors. At level of dorsal horn, 2 endogenous opioid peptides, enkephalin and dynorphin are released after nociceptive stimulation from descending fibers originating in brain stem. Opioid containing neurons can have an excitatory or inhibitory effect, but primarily act to inhibit transmission of nociceptive activity.

Another modulating mechanism, known as the "pain gate" phenomenon, occurs when stimulation of thick A beta fibers of peripheral nerves decreases the sensitivity of postsynaptic cells in the dorsal horn to incoming signals from C and A gamma fibers. This property helps to explain how mechanical stimulation such as stroking or rubbing the skin can provide pain relief.

Descending control pathways: These inhibit pain perception and are called antinociceptive, and include supraspinal modulating loops.  Central modulation of pain perception response involves parts of cortex, the periaqueductal gray region of the midbrain, the lateral pons and 
ventromedial medulla. Stimulation of these sites can result in inhibition of dorsal horn neuronal activity.

Pain response can be modified at the peripheral level, spinal cord, or CNS. Anti-inflammatories may dramatically reduce sensitization of nociceptive fibers, administration of epidural and intrathecal agents work at the level of the dorsal horn by reducing excitation of nociceptive neurons and stimulating inhibitory pathways.

Non-pharmacological considerations for pain management can be enhanced by providing appropriate housing, handling, and restraint and by using appropriate experimental techniques such as proper surgical technique.  Increased environmental temperature is generally required during anesthesia recovery, removal of wet materials, easy access to food and water, nesting materials, soft food, bandaging and expression of bladder with spinal cord dysfunction are examples of these methods.  

Assessment of pain: Detection of pain in rodents and rabbits is based on subjective evaluation of behavioral and attitudinal changes as well as an objective analysis of physiologic parameters. Behavioral signs indicative of pain perception may include reluctance to move, abnormal posturing, teeth grinding, decreased appetite and vocalization. 
Attitudinal changes include anxiety, apprehension and hypersensitivity as well as depression or, uncommonly overt aggression. Signs indicative of pain-induced distress characteristic of rodents include polyphagia of bedding that may progress to self-mutilation.  Physiologic signs of pain perception include fluctuations in blood pressure, heart rate, and body temperature. Food, water consumption and body weight are others.

Pain scoring system: Adopted from human work, the visual analog pain scale is used by the observer to rate each behavior/criteria chosen on a scale from 1-5. Scales of pain scores are created from this and higher numbers are used to justify analgesics, humane endpoints or euthanasia.  This must be used efficiently however and allow the staff doing the scoring time to administer analgesics if needed.

Analgesics: Local anesthetics can be very useful for rodents and rabbits used solely or as an adjunct to other agents. Lidocaine and bupivicaine are the two referred to by the authors.  NSAIDs such as carprofen and meloxicam can be used for mild to severe pain and can be combined with an opioid such as buprenorphine, morphine and fentanyl patch (rabbit).  Scant information is known about efficacy of analgesics in hamsters, gerbils and guinea pigs. Buprenorphine is the most commonly used analgesic for these species.=20

Questions:
1.	Visual analog pain scoring system is used to predict the amount of pain derived from experimental procedure? T F
2.	Administration of a local anesthetic should be carefully done to avoid IV placement? T F
3.	C-section in a rat would be expected to cause what severity of pain
a.	Mild
b.	Moderate
c.	Severe
d.	None
4.	Cool, moist bedding materials are very important for rapid recovery of rodents and rabbits following anesthesia. T F

Answers:
1.	F, evaluation only
2.	T
3.	b
4.	F