Download Number 2 - Laboratory Animal Boards Study Group

Journal of the American Association for Laboratory Animal Science
Volume 48, Number 2, March 2009
OVERVIEW
Patino. Intellectual Property Rights and Research Disclosure in the University
Environment: Preserving the Commercialization Option and Optimizing Market
Interest, pp. 138-143
Domain: 3, Research
Task 1: Facilitate or provide research support
SUMMARY: Clinical and basic science research being conducted at research institutions
has potentially huge benefits to help human kind and receive monetary return. It is,
however, not useful when this information isn’t shared broadly due to concern regarding
property rights. It is therefore important to learn the basics about intellectual property
protection to enable the investigators to share their invaluable knowledge without the
concern of losing property protection over their novel, research findings.
There are several concerns regarding the implementation of intellectual property
protection within the scientific community. First, there is the thought that it may slow
down the advancement or use of new technologies as property rights may mandate
money in return for knowledge. Second, there is concern regarding industry support of
research and bias that may be involved based on certain product performance results.
Net, there are some scientists that believe pure science should have no financial
agenda.
Among university settings, scientists and faculty members are required to publish their
work. The number of publications directly impacts their tenure and salary. This is
another reason why it is important to be familiar with what intellectual property rights are
available. The 1980 Bayh-Dole Act allows universities and other nonprofit groups to
take ownership interest of patents that are developed using federal grant money.
There are several forms of intellectual property protection. The most common form for
drugs and devices is the patent. The invention must be useful, novel, and not obvious to
be eligible for patent protection. The scope of claim protection is an important element
in determining if the invention is eligible for patent protection. Scope refers to evaluating
the prior art including published journal articles, issued patents, published patent
applications, abstracts and public grant information. In addition, territory of protection
must be considered in evaluating the value of a patent. If it is deemed necessary to
seek international patent protection, several approaches can be used in the interim for
protection. The most cost effective is to file a Patent Cooperation Treaty (PCT) which
allows for a 30 month delay for international patent prosecutions.
While the prior art and international patent protection are being considered, a simple
strategy is to file a provisional patent application. These can be submitted quickly if the
investigator meets the requirements. Other advantages include they are inexpensive to
file and they are not made publicly available for inspection.
In summary, there are many benefits and economic advantages of having intellectual
property protection for a technology or discovery. A key benefit is it gives institutions a
defined instrument that allows them to promote their idea to a third party for technology
advancement or product development. It is important to keep in mind, however, that
securing intellectual property does not guarantee that the idea or product will succeed in
the marketplace.
QUESTIONS:
1. How and what does intellectual property protection provide?
2. Who grants patents within the US?
3. What act enables universities to take ownership interest in patents that are
developed using federal grant money?
4. What type of application may be filed to provide a 30 month patent protection
internationally?
5. What university office typically manages any negotiations that need to be made with
the patent office?
6. According to a 2003 publication, how much money and time do US drug companies
spend in research and development before a drug goes to market?
7. What should a researcher do when they initially have property that is potentially
eligible for protection?
ANSWERS:
1. Intellectual property protection guards ownership and use rights for concepts and
ideas by using patents, copyrights, trademarks, trade secrets, and know-how
agreements.
2. The US Government grants the patent, known as the property right, to the inventor to
exclude others from making, using, offering for sale, or selling the invention
throughout the US.
3. The 1980 Bayh-Dole Act
4. Patent Cooperation Treaty (PCT)
5. The Technology Transfer Office at a university or institute typically manages the
transfer of assets and represents the best interests of the institution.
6. Estimates are USD $800 million in research and development and more than 90
months from the start of clinical testing are typical for getting a drug to market.
7. They should contact a competent technology transfer professional or intellectual
property attorney to map out the best strategies for moving forward and obtaining
protection for their idea. In most academic and research settings, this may begin
with a visit to the university’s Technology Transfer Office.
ORIGINAL RESEARCH
Biology
Reisinger et al. Dietary Ammonium Chloride for the Acidification of Mouse Urine,
pp. 144-146
Domain: Task 1 K9, K10; Task 3, K1
Species- Mouse- Crl: CD-1(ICR)BR
SUMMARY: During a routine carcinogenicity study in mice and rats, a compound was
found to induce transitional cell carcinomas in the urinary bladders of male rats. It was
hypothesized that the tumorigenesis was caused by epithelial irritation secondary to
urine crystal formation rather than a primary effect of the drug compound itself.
Ammonium chloride has been used to acidify urine in a variety of species, and the
authors felt that it would be more convenient to use if it could be administered via the
diet. Therefore, the purpose of this study was to test the efficacy of adding 1% NH 4Cl to
a standard rodent diet for urinary acidification, in hopes of creating an alternative route of
daily administration rather than its labor intensive addition to water bottles.
One group of 10 mice were fed a standard rodent diet, and the other group of ten mice
were fed the same diet manufactured with 1% NH4Cl added. Urine samples were
collected and the pH was measured after 1 week post diet administration, 1 hr after the
start of the light cycle, and 2 wks post diet administration both 1 and 8hrs after the start
of the light cycle.
The mean urinary pH of mice fed the acidifying diet was significantly (P < 0.01) lower
than that of concurrent control mice at both intervals and collection time points. At week
2, for moth groups, samples measured at the 8 hr time point were significantly more
acidic than samples measured at the 1 hour time point. This finding reflects the normal
diurnal variation of urine pH in mice due to their primarily nocturnal consumption of food.
Investigators should be mindful of this.
The authors concluded that the use of 1% NH4Cl containing feed provides a simple, cost
effective mechanism for acidifying the urine of large groups of mice in long-term studies.
QUESTIONS:
1. The formation of endogenous calcium and magnesium salts in rat urine is facilitated
at a urinary pH greater that what?
a. 7
b. 6.5
c. 6
2. T or F. Rodents are more susceptible to both the formation and the carcinogenic
effects of calculi than are humans?
3. T or F. The formation of crystals and sedimentation of solids in the urine increase
urinary bladder tumor in rodents
4. List three factors, other than pH, that also affect the formation of solids or calculi in
the urine:
ANSWERS:
1. b
2. T
3. T
4. Changes in water consumption, the administration of renally excreted test articles,
and dietary mineral intake
McBrier et al. Magnetic Resonance Imaging of Acute Injury in Rats and the Effects
of Buprenorphine on Limb Volume, pp. 147-151
Domain 1: Task 3: K1c.
Species: Primary - rats
SUMMARY: The purpose of this study was to compare the ability of MRI-based T2
mapping and measuring limb volume to differentiate injured and uninjured tissues
following blunt trauma on rat hind limbs, and to see if buprenorphine administration
influenced these assessments. A reproducible drop mass technique was utilized to
cause blunt trauma to the gastrocnemius muscle of male Wistar rats, and MRI was used
at different time points after injury to compare T2 mapping and limb volume. Although T2
mapping is not explained in great detail in the article, it states that changes in the T2
signal are generally accepted as a measure of tissue damage, specifically inflammation,
edema and hemorrhage. The addition of buprenorphine as an analgesic was also
studied, to see if there was any effect on the ability to differentiate injured and uninjured
tissues. The contralateral limb was used as the control in each of the study animals.
Results: Buprenorphine showed an increase in the limb volume of the control limb, and
thus blunted the relative increase in the injured limb. Buprenorphine administered rats
showed an average relative increase of 20%, versus 31% of rats that did not receive
buprenorphine. Administration of the drug had no effect on the T2 signal, with an
average of 23% higher signal in the injured tissue than the uninjured tissue. The
discussion section provides potential causes for this “proinflammatory” effect of
buprenorphine in acute local injury to be either the analgesic effect causing more activity
by the rats and thus inhibiting the normal healing process or an inhibition of
corticosteroid release by the opioid preventing the normal healing response. Extensive
sensitivities and specificities were performed to back up the conclusion that the use of
T2 maps will likely provide a more accurate assessment to track injury initially and over
time than will the use of limb volume.
QUESTIONS:
1. True or False: In this study it was determined that the administration of
buprenorphine in rats increased the overall volume of both healthy limbs and limbs
with contusion injury.
2. True or False: Swelling is listed as a side effect of morphine derivatives in general.
3. Sensitivity or Specificity: _____________ is the test’s ability to detect a condition or
disease when the condition or disease is present.
ANSWERS:
1. True
2. False
3. Sensitivity
Skrajnar et al. A Flow Cytometric Method of Determination of the Blood
Neutrophil Fraction in Rats, pp. 152-156
Task 1: Prevent, Diagnose, Control, and Treat Disease 18 ± 4%
K10 Diagnostic tests – Performance and Evaluation
a) Clinical pathology (e.g., hematology [CBC], clinical chemistries and
urinalysis) – especially species uniqueness.
Task 10: Design and Conduct Research 4 + 1%
K2 Data collection techniques
K3 Data analysis techniques
Species - Rat, Primary (70+5%)
Abstract: Determination of the proportion of neutrophils in the peripheral blood is
important for diagnostic purposes in medicine and for evaluating new drugs in the
pharmaceutical industry. To measure the neutrophil concentration in rat blood, a fast and
accurate flow cytometric method was developed. Rat neutrophils were quantified by
using primary antibodies that recognize the RP1 antigen and secondary antibodies
conjugated with fluorescein isothiocyanate. The flow cytometric method was calibrated
by comparing cytometric results with data from a manual differential count. The results
obtained by these 2 methods correlated with a Pearson correlation coefficient of 0.91
and were in agreement according to subsequent statistical analysis. To confirm the
usefulness of the method in preclinical applications, the production of neutrophils in rats
was stimulated by pegfilgrastim. Blood samples were taken at predetermined time
points, and the pharmacodynamic profile was determined. These results confirmed that
the flow cytometric method for neutrophil quantification is accurate and much faster than
the manual microscopic method. Moreover, the flow cytometric method is easy to use,
suggesting that it could become the method of choice for preclinical applications.
Results: Please see evaluate Figures 1 and 2. To best understand the basic concept of
Flow Cytometry, please see Technology Update by Kendall et al. Flow Cytometry.
Contemporary Topics, 41(5), September 2002. Of interest, the determination of blood
neutrophil fraction done by flow cytometry was in close agreement with microscopic
enumeration (Figure 3). However, the authors state that the flow cytometry method
could evaluate more samples in a shorter period of time, making it a more efficient
technique.
Discussion: Rats are the most widely used rodents in toxicology and pharmacokinetic
studies, and the rat peripheral blood test have great potential for providing investigators
with useful information. One of the key benefits of the described flow cytometric method
over the microscopic method is speed. The authors also state that though many medical
laboratories use automated blood cell counters, the appropriate software to evaluate
multiple animal species has only recently been evaluated (Read manuscript introduction,
Page 152).
Conclusion: Flow cytometry can accurately quantify neutrophils in a short period of time.
The authors’ state that the flow cytometry method used in this study is a valuable
screening tool to evaluate neutrophil granulocyte counts in rats during preclinical drug
testing studies.
QUESTIONS:
1. What is flow cytometry?
2. What is the most widely used rodent in toxicology and pharmacokinetic studies?
a. Rabbit
b. Guinea pig
c. Rat
d. Hamster
e. Deer mouse
ANSWERS:
1. Flow cytometry is a laser-based technology that enables the user to examine the
phenotype and morphologic characteristics of individual cells.
2. C; Rat.
Kemppinen et al. The Effect of Dividing Walls, a Tunnel, and Restricted Feeding
on Cardiovascular Responses to Cage Change and Gavage in Rats (Rattus
norvegicus), pp. 157-165
Domain: Animal Care, T1
Primary species: Rat
SUMMARY: The authors hypothesized that cage objects would alter the effect of cage
change and gavage on telemetrically recorded cardiovascular parameters and locomotor
activity. The study was designed to evaluate the effect of an aspen wall divider with or
without restricted feeding and the presence of an aspen tunnel in the cage on these
measures after routine cage change and gavage of laboratory rats in both open-top
cages and IVCs.
Two strains of 12 male rats were used; BN (BN/RijHsd) and Fischer 344 (F344/NHsd)
and they were housed 3 to a cage with one animal being surgically implanted with a
telemetry device that measured blood pressure (MAP), heart rate (HR) and locomotor
activity. A crossover design was utilized with 2-week periods and rotational order, the
first item was allocated randomly. 2 kinds of dividers made of 2 intersecting aspen
boards and a rectangular aspen tunnel - one divider included round holes for food pellets
from which rats had to gnaw wood to obtain food (diet board). The other divider did not
contain food holes. On day 8 of each 2-week period, the cages were changed; on day
11, rats were gavaged. Baseline values for each rat were subtracted from the
corresponding response values.
The presence of objects did not affect the responses of F344 rats to cage changing or
gavage. In BN rats with IVCs, the presence of the plain divider modified the response to
both procedures. Responses to procedures appeared to be dependent on both the strain
and the cage object which complicates establishing valid general guidelines for rats. The
new European regulations on laboratory rodents mandates the provision of sufficient
nest material to build a complete or covered nest or, if doing so is not possible, providing
a nest-box. Species and strain differences raise the question as to whether general
guidelines are warranted due to potential for negative effects on some strains and/or
species.
QUESTIONS:
1. Why are generalized guidelines for laboratory rodent enrichment questionable?
a. Species-specific behavior is extremely variable; what may have a positive effect
on one species, may negatively affect another
b. Establishing generalized guidelines may create greater stresses on individual
strains of rats
c. The cage object and cage type may positively impact one strain and negatively
stress another
d. The age of the rat may affect its response to husbandry procedures
e. All of the above
2. What two strains of rats were used in this study and are they inbred or outbred
strains?
a. BN (Brown Norway) F344 (Fischer 344) - inbred strains.
b. BN (Brown Nigerian) F344 (Flatlander 344) - outbred strains
c. BN (Brown Norway) F344 (Fischer 344) - outbred strains
d. BN (Brown Nigerian) F344 (Flatlander 344) - inbred strains
3. The time of day cage changes were made can affect the stress reaction of rats. T/F
ANSWERS:
1. e
2. a
3. True
Stephans DeValle. Successful Management of Rabbit Anesthesia Through the
Use of Nasotracheal Intubation, pp. 166-170
Domain 1, Task 2: Management of experimentally induced condition (control)
Domain 6, Task 2: Education (maintain current knowledge and continued competence in
laboratory animal medicine)
Species: Primary, rabbit
SUMMARY: The objective of this report was to present a method of nasotracheal
intubation in rabbits, compare the technique to orotracheal intubation and describe key
points for its successful use.
Rabbit intubation is considered technically difficult. Nasotracheal intubation takes
advantage of the fact that rabbits are obligate nasal breathers; a tube placed nasally will
traverse the pathway of air from the nasopharynx into the trachea. However, previous
studies have reported the introduction of pathogens into the lungs and the need for high
oxygen flow rates with this technique.
38 male, New Zealand White rabbits were each intubated twice for two surgical
procedures 2 months apart. After 7 rabbits underwent orotracheal intubation with various
difficulties for the first procedure, all other intubations were performed using the
described nasotracheal technique. SpO_2 levels for the two techniques were compared,
and rabbits were monitored for clinical signs of respiratory disease between intubations.
The nasotracheal intubation technique was described (Figure 1). The rabbit was place in
dorsal recumbency with the head held up and in dorsoflexion by the handler’s
subordinate hand. The nasal fold was lifted, and the tube was advanced medially and
ventrally until placement was confirmed by condensation. After application of tape to its
free end, the tube was secured by suturing both sides of the tape to the dorsum of the
nose (Figure 2). Anesthesia was maintained with 3% isoflurane and 1.5 L/min oxygen.
Mean oxygen saturation between rabbits intubated by both techniques did not differ
among all rabbits or among procedures in rabbits intubated both ways. No rabbits
exhibited clinical signs of respiratory disease during the 2 months of observation
between surgical procedures, but neither respiratory tract cultures nor histopathology
were performed.
Several criteria were considered necessary to effective nasotracheal intubation:
1. Total relaxation of the rabbits, achieved with ketamine/xylazine and isoflurane via
mask
2. Modification of the classical blind method by placing rabbits in dorsal recumbency
similar to human pediatric patients undergoing intubation
3. A medial-ventral approach with the tube
4. A unique method of securing the tube with tape and suture to the dorsum of the
nose.
The authors concluded that the described technique for nasotracheal intubation can be
used as a humane, clinically preferred method of rabbit intubation and that additional
research is needed to determine any pathology resulting from this technique.
QUESTIONS:
1. Imagining figures 1 and 2 (p. 167) on the practical exam, what technique is being
shown and in what species?
2. T/F: In this study, high oxygen flow rates were necessary to maintain oxygen
saturation in rabbits undergoing nasotracheal versus orotracheal intubation.
3. What descriptive phrase explains why rabbits normally have their epiglottis
entrapped on the dorsal surface of the soft palate?
4. Complications of orotracheal intubation in rabbits include:
a. Difficult placement
b. Trauma to the oropharynx
c. Difficulty with positive-pressure ventilation
d. a and b
e. None of the above
ANSWERS:
1. Nasotracheal intubation in a rabbit
2. F
3. Obligate nasal breather
4. d
Koeller. Comparison of Buprenorphine and Butorphanol Analgesia in the Eastern
Red-Spotted Newt (Notophthalmus viridescens), pp. 171-175
Domain 2: Management of Pain and Distress
Tertiary Species: Amphibians
SUMMARY: Authors conducted this study to learn about analgesia in newts.
Authors observed signs to detect whether newts indicated changes in behavior to
assess pain or distress. Intracoelomic injected buprenorphine and butorphanol added to
the tank water were compared for analgesic effect in newts after bilateral forelimb
amputations.
The following 4 groups were studied:
1. Surgery with buprenorphine analgesia
2. Surgery with butorphanol analgesia
3. Anesthesia, with no surgery
4. Surgery without analgesia.
Newts were anesthetized, surgery (or lack of surgery), recovery with or without
analgesia, and testing for behavioral changes were performed. Newts were tested for
food consumption, spontaneous movement, response to tapping on the tank, response
to being touched, and body posture.
Authors concluded that both forms of analgesic administration (buprenorphine given
intracoelomically and butorphanol provided in tank water) significantly promoted the
resumption of normal behavior after bilateral surgical amputation of the forelimbs in
newts.
QUESTIONS:
1. Which are uses of amphibians in biomedical research?
a. Developmental biology
b. Pain research
c. Toxicology
d. All of the above
2. Which covers amphibian use in research?
a. AWA, PHS
b. AWA
c. PHS
d. None of the above
3. Which term best describes the perception of noxious stimuli?
a. Kinesthesia
b. Nociception
c. Pain
d. Hyperalgesia
ANSWERS:
1. d
2. c
3. b
Fontenot et al. Dose-Finding Study of Fluoxetine and Venlafaxine for the
Treatment of Self-Injurious and Stereotypic Behavior in Rhesus Macaques
(Macaca mulatta), pp. 176-184
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and
Conditions
Primary – Macaques (Macaca spp.)
SUMMARY: In a continuing effort to address treatment for NHPs with self-injurious
behavior (SIB), the authors report the success of different doses of two different drugs.
Both fluoxetine (FLX) and venlafaxine (VEN) were used in a trial of four different doses
to determine efficacy in NHPs diagnosed with SIB. A macaque-specific ethogram was
used in assessing drug response as well as measurement of platelet serotonin and
cerebrospinal fluid 5-hydroxyindoleacetic acid (CSF 5HIAA) concentrations. Self-biting
behavior that did not result in injury was differentiated from self-wounding with the use of
the ethogram. Only FLX at a dose of 2 mg/kg was effective in reducing SIB and
correlated with reductions in platelet serotonin and CSF 5HIAA levels.
QUESTIONS:
1. What type of drug is fluoxetine and what condition has it been effectively treated?
2. What type of drug is venlafaxine and what is it used for in humans?
3. What are the risk factors for macaques in developing SIB?
4. What other treatments have found to be effective for SIB in rhesus?
ANSWERS:
1. Selective serotonin reuptake inhibitor (SSRI); efficacious in treating acral lick
dermatitis in dogs
2. Serotonin-norepinephrine reuptake inhibitor (SNRI); efficacy in the treatment of
depression, panic attacks, and obsessive-compulsive behavior in humans
3. Stressful social experiences in the first 2 years of life, such as early weaning
4. Tryptophan, buspirone, guanfacine, and valium are a few examples
Watson et al. L-Tryptophan and Correlates of Self-Injurious Behavior in SmallEared Bushbabies (Otolemur garnettii), pp. 185-191
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and
Conditions
T2. Control spontaneous or unintended disease or condition
Tertiary Species – Other non-human primates
SUMMARY: In this study, dietary supplementation with L-tryptophan (100mg/kg daily)
was evaluated for treatment of Self-Injurious Behavior (SIB) in small-eared bushbabies
(a prosimian primate). The treatment was chosen based on prior studies in rhesus
macaques in which a 21 day treatment period (100mg/kg twice daily) reduced the
duration of self-biting behavior in monkeys with recent histories of SIB and results from
other laboratories that promote the view that serotonergic agents may have potential for
treatment of SIB. Increasing the availability of tryptophan, the precursor to serotonin is
one way to facilitate serotonin synthesis in the CNS. Plasma cortisol levels were also
monitored in this study since frequency of SIB in rhesus monkeys has been negatively
correlated with cortisol levels in prior studies. Supplementation of the bushbabies’ diet
with L-tryptophan significantly reduced stereotypy, wound area, and severity. High
plasma cortisol levels in bushbabies with SIB was reduced after treatment whereas
serotonin concentrations were increased. Therefore, L-tryptophan as a dietary
supplement may be a viable option for addressing animals exhibiting stereotypy and
SIB.
Experimental Design: Twenty-two adult small-eared bushbabies exhibiting SIB were
supplemented with L-tryptophan 100 mg/kg daily for 4 weeks. Eleven untreated adult
bushbabies with no evidence of SIB were used as controls. Blood sampling for
determination of baseline levels of plasma cortisol and serum serotonin were collected in
week 1; baseline behavioral data was recorded during weeks 1 and 2. Behavioral
parameters included frequency of stereotypy (pacing and swaying for 3 or more
repetitions) and grooming (normal grooming and wound grooming). Self-inflicted
wounds were measured and their severity scored by 2 independent investigators (score
of 1 = mild-moderate, score of 2 = moderate to severe). Behavior data was collected
throughout the experimental period; blood sampling and wound assessment was
repeated at the end of the experimental period (6 weeks total – 2 weeks of baseline data
collection followed by 4 weeks of treatment).
QUESTIONS:
1. How does SIB typically manifest itself in NHPs? What is the typical sign of SIB in
bushbabies as reported by the authors?
2. Give 2 examples of other serotonergic agents that have been used to treat SIB in
non-human primates and their mechanism of action.
ANSWERS:
1. In NHPs SIB typically is manifested as self-biting, overgrooming, or hair plucking. In
bushbabies, SIB takes the form of obsessive licking at an existing wound site.
2. Fluoxetine (serotonin reuptake inhibitor) and buspirone (serotonin 1-alpha agonist)
Veeder et al.
Positive Reinforcement Training to Enhance the Voluntary
Movement of Group-Housed Sooty Mangabeys (Cercocebus atys atys), pp. 192195
Domain 4: Animal Care
Task 2: Manage or provide indirect management/oversight of animal husbandry
programs
Tertiary Species: Other non-human primates
SUMMARY: Positive reinforcement training (PRT) is a technique in which the animal
receives a positive reward, often a food treat, for exhibiting a particular behavior, thereby
increasing the likelihood that the animal will perform the desired behavior again in the
future. PRT has been used successfully in previous reports to train animals to
cooperate with multiple veterinary and research procedures such as injections,
presentation for voluntary blood, semen, and urine collection, and to improve social
dynamics in primate groups by reducing competition and aggression over food and
increasing affiliative behaviors between conspecifics. Since little information is available
about training sooty mangabeys specifically, this study was conducted to analyze the
use of PRT to train these monkeys to move from one part of their enclosure to another.
The desired behavior was to move 30 adult males from one part of their enclosure (6
runs connected by guillotine doors), through a tunnel across a corridor, into another set
of connected runs. Monkeys were given visual and verbal cues to move through the
runs. In the early training sessions, the guillotine doors behind the monkeys were closed
as they moved through the runs. When they moved through the final tunnel, verbal
praise and a preferred food was given as a reward for completing the shift. Training
sessions were between 15 and 40 minutes for the group and subjects were trained 3-5
minutes each week during a 4 month period. The goal for the group to be considered
reliably trained was when 90% of the group shifted on cue 5 times in a row.
In the first 4 months, 57 training sessions were completed. In the first 5 sessions, 76%
of the group was compliant to some extent; during the last 5 of the 57 sessions, 86% of
the monkeys cooperated. Inspection of the data revealed that 5 of the subjects complied
less than 75% of the time. A dominance matrix (using behavioral observation data from
the group) and statistical analysis of this data was performed which revealed that
dominant animals had significantly higher compliance than did low-ranking individuals.
The researchers then used problem-solving techniques to help the consistently noncompliant monkeys become more proficient. These techniques included emptying the
other monkeys from the run they were asking the non-compliant monkey to move into,
using preferred food rewards (yogurt, bananas, grapes), and "jackpot" rewards (entire
ear of corn, whole banana) when rapid progress was made.
Data collection was halted after 57 sessions and then resumed 34 weeks later for 34
sessions. These 34 follow-up sessions had greater than 90% compliance, meeting the
established goal. Training the animals to move within the enclosure also markedly
decreased the time for completion of husbandry procedures by facilitating cleaning of
runs and allowed the staff to interact with the animals in a way that was personally
rewarding. The authors’ final conclusion was that PRT can be successfully used to train
nonhuman primates though other factors such as social structure within the group need
to be evaluated carefully to ensure success.
QUESTIONS:
1. Positive reinforcement training is an example of what type of conditioning?
a. Temporal conditioning
b. Operant conditioning
c. Classical conditioning
d. Noncontingent reinforcement
2. When performing positive reinforcement training, the reward (a.k.a. the primary
reinforcer) is typically paired with a cue (e.g., clicking sound) which serves to pinpoint
the moment when the desired behavior occurs. What is the term for this cue?
ANSWERS:
1. b. Operant conditioning
2. Conditioned reinforcer or "bridge"
Experimental Technique
Frynta et al. Apparatus for Collection of Fecal Samples from Undisturbed Spiny
Mice (Acomys cahirinus) Living in a Complex Social Group, pp. 196-201
Domain 3 - Research; Task 3: Design and Conduct Research
Species: Tertiary -Other Rodents
SUMMARY: Stress responses in rodents are assessed by monitoring serum
glucocorticoid levels. The challenge is to obtain samples without stress associated
affects. Measuring glucocorticoid metabolites in the feces can be used as a direct
correlate to blood glucocorticoid levels. Fecal collection eliminates the stress effects
from blood collection but a dilemma exists for obtaining individual samples under
conditions of social grouping. To overcome this problem, an apparatus was constructed
which allowed an animal from a group to enter a small containment voluntarily, remained
detained for 4 hours to pass stool through a grid floor, and then rejoin the group. This
housing posed no stress to the animal and proved not to affect glucocorticoid levels.
Most rodents save the golden hamster exist in pairs or groups. The Egyptian spiny
mouse (Acomys cahirinus) was chosen for this study because its social organization is
more complex than mice. They were also chosen because they do not build nests hence
a grid floor would be better tolerated and less stressful. Cortisol is the main circulating
glucocorticoid in these animals.
Other facts about the spiny mouse: nocturnal desert dwelling rodent closely related to
the gerbil, gestation period approximately 38 days, and the offspring are precocial. They
are used for behavioral and physiologic studies.
QUESTIONS:
1. T or F The spiny mouse and the G. pig have precocial offspring.
2. What is not true regarding precocial offspring?
A. Eyes and ears open
B. Nursing not needed
C. Have teeth
D. Ambulatory
3. Of the animals listed, which animal has the longest gestation?
A. Spiny mouse
B. Golden hamster
C. Gerbil
4. T or F. Glucocorticoid levels can be measured in the stool.
5. T or F. Corticosterone is the major circulating glucocorticoid in the spiny mouse
6. T or F. The spiny mouse is nocturnal just like the mouse
ANSWERS:
1. T
2. B see BB pg 211
3. A spiny mouse 38 days; gerbil 24-27 days depending on lactation; golden hamster
15-18 day
4. F Glucocorticoid metabolites
5. F Cortisol
6. T
Cray et al. Effects of Storage Temperature and Time on Clinical Biochemical
Parameters from Rat Serum, pp. 202-204
Domain 3: Research (biomethodology techniques)
Species: Rat (primary)
SUMMARY: Experimental design frequently necessitates the use of frozen serum
samples for retrospective studies. The planning for multiple experimental time points
often results in samples that will be analyzed together at a later date and thus subjected
to different periods of storage before analysis. In addition, samples collected after hours
or on weekends are often stored in a refrigerator until analysis can be performed.
Although studies of human blood samples, including stability analyses for refrigeration
and freezing have been conducted, data from such reports in veterinary medicine are
relatively scarce. In the veterinary literature, reports on canine and avian samples
revealed interspecies differences in storage stability. The authors assessed the stability
of 17 analytes in rat serum during refrigeration at 4 0 C, extended storage at - 200 C
(frost-free and nonfrost-free freezers) and - 700 C. For this study, Sprague-Dawley rats
were euthanized by CO2 overdose, and blood was collected by cardiac puncture. For
each sample, serum was pooled from two individual animals. Aliquots of each sample
were stored at 4 0 C (refrigerator), - 200 C in frost-free and nonfrost-free freezers, and at 700 C. These aliquots, stored under various conditions were analyzed using a dry-slide
chemistry analyzer on days 7, 30, 90, and 360. The following biochemical analytes were
measured: glucose, BUN, sodium, potassium, chloride, total CO2 , amylase, lipase,
calcium, phosphorus, total protein, albumin, aspartate aminotransferase, alanine
aminotransferase, lactate dehydrogenase, creatine kinase, and alkaline phosphatase.
Results: The authors reported that a difference of 10% was statistically significant in this
study. Refrigeration for 7 days: CO2 decreased by 9.4%, creatinine kinase activity by
11.1%, and lactate dehydrogenase by 8.0%. No significant changes were detected at 7
days for samples frozen at - 200 C and at - 700 C. However, samples stored for 360 days
in a frost-free freezer ( - 200 C) showed a 59% drop in creatine kinase activity and
alanine aminotransferase activity decreased by 54%. In the samples stored at - 700 C,
none of the chemistry markers had changed by more than 10% by day 360. There were
no reported differences in samples stored in frost-free and nonfrost-free freezers at - 200
C. Samples in both - 200 C freezers showed decreases of more than 10% in CO2 on day
30 and levels were even lower on day 90. Alanine aminotransferase levels were
significantly decreased after 90 days at - 200 C. Most of the marked changes in analyte
levels for samples held at - 200 C occurred between 90 and 360 days.
Discussion: This study shows that, with the exception of creatine kinase activity,
common analytes in rat serum are stable under refrigeration for 7 days. Whenever
possible, prolonged sample storage should occur at - 700 C. Alternatively, storage at 200 C is acceptable for as long as 90 days for common analytes except CO 2 and alanine
aminotransferase. It should be noted that these samples were obtained from healthy
animals. The authors presume that samples from clinically ill rats would behave similarly,
but differences in enzyme isoforms and perhaps lower or higher starting values may be
associated with particular sample instabilities. Further studies are needed to address this
issue.
QUESTIONS:
1. True or False: According to this study, there's a difference in stability if serum
samples are stored in a frost-free freezer vs. a nonfrost-free freezer (- 200 C).
2. True or False: Short-term storage (30 days) in a - 200 C may be acceptable for the
majority of common analytes measured.
3. Name the analyte in this study that showed significant instability after 7 days of
refrigeration.
4. Name the analyte that was significantly affected by storage in a - 200 C freezer for 30
days (and even lower at 90 days).
ANSWERS:
1. F
2. T
3. Creatine kinase
4. CO2