Journal of the American Association for Laboratory Animal Science
Volume 48, Number 5, September 2009
ORIGINAL RESEARCH
Biology
Garcia et al. Growth-Associated Changes in the Periodontal Bone and Molar
Teeth of Male Rats, pp. 475-481
Task 3 – Provide Research Support, Information and Services; K2 Normative biology
Primary Species - Rat (Rattus norvegicus)
SUMMARY: This article presents an examination of changes in the composition and size
of mandibular molars, and the growth, apposition and resorption characteristics of the
associated mandibular bone in young growing rats. The first and second molars erupted
sooner, and initially grew faster than the third molar. As would be expected tooth
mineral density increased with age, nearing maximum density at approximately 3 weeks
for the 1st and 2nd molars, and at approximately 6 weeks for the 3rd molar. Similarly,
tooth root height also increased with age. The rate of mandible mineralization, distance
between the cementoenamel junction to alveolar bone, and number of resorptive lesions
in the mandible all followed a sigmoidal curve allowing for the calculation of the point of
inflection (POI) (i.e. the point at which growth is maximal). The POI for bone resorption
lesions occurred at 4.5 weeks while the POI for mandible mineralization occurred at 4.7
weeks. The POI for cementoenamel junction occurred latest with times of 5.7 weeks for
the 1st and 2nd molars, and 6.5 weeks for the 3rd molar. In general the data reported here
shows that tooth and mandibular growth is harmonious with whole body growth of young
rats wherein the POI is approximately 7 weeks. This research provides important
information on the normative biology of the mandible and teeth in young rats.
QUESTIONS:
1. In terms of body or tissue growth what is the point of inflection (POI) as described in
this paper?
2. Name three methods used to evaluate tooth and bone growth in young rats?
ANSWERS:
1. The POI is the point of maximum body growth. As described as a growth spurt.
2. a. Bone densitometry from radiographic density
b. Distance between cementoenamel junction and alveolar bone crest
a. Height of molar crowns
b. Length of molar root
c. Histological analysis and quantification of osteoblasts
Goldschmidt et al. Variation in Clitoral Length in Rhesus Macaques (Macaca
mulatta), pp. 482-485
Domain 1: Task 3, K1
Species: Primary Rhesus Macaques (Macaca mulatta)
SUMMARY: This article sought to determine clitoral size in normal rhesus monkeys and
possible effects of age and inheritance. They also wanted to see if any correlation could
be found between clitoral length and fertility, and to determine if there was an
association in the distribution of daughters with and without clitoris between mothers with
and without clitoris. A colony of 285 female rhesus monkeys at the Center for Laboratory
Animal Breeding in Brazil had physical examinations performed, along with clitoral length
measurements. The clitoral lengths varied widely and were heterogeneous across age
groupings. Based on observations of behavior and reproductive failure, clitoral size was
not associated with either fertility or reproductive behavior in this colony. No association
was detected when comparing the distribution of daughters with and without clitoris with
mothers with and without clitoris.
QUESTIONS:
1. T/F - Differentiation of the external genitalia in rhesus monkeys occurs in the
beginning of gestation period, at about day 55 to 60, and is complete by day 80.
2. T/F - the earlier the hyperandrogenism occurs during pregnancy, the higher the risk
of fetal virilization.
3. T/F - no correlation was noted in this study with clitoral lengths between mothers and
daughters.
ANSWERS:
1. T
2. T
3. T
Zamora et al. Decreased Blastocyst Production in Mice Exposed to Increased
Rack Noise, pp. 486-491
Domain 3: Research; Task 2: Manage or provide indirect management/oversight of
animal husbandry programs; Knowledge 11: environmental causes of physiological
alterations in animals and their effects on research
Species: Primary, Mouse
SUMMARY: This study investigates the effect of rack type (ventilated vs. static) on the
blastocyst yield of C57BL/6 mouse embryo donors in response to a previous observation
that embryo donor mice appeared to produce fewer blastocysts when housed in
ventilated cages.
After superovulation and breeding, all mice were housed for three days in their
designated rack type. In the first phase, one group (A) was housed in a ventilated rack
and a second group (B) was housed in a static rack in the same room. In the second
phase, a third group (C) was housed in a static rack in the absence of the ventilated
rack. Noise within each rack, blastocyst yield and plasma corticosterone levels were
measured.
Noise levels from the racks housing groups A-C were progressively lower.
Blastocyst yield for groups A-C was progressively higher. A negative linear relationship
with good but not significant correlation was seen.
Representative plasma corticosterone levels were not significantly different between
groups A and B but were generally high, which the authors attributed to transport and
handling.
The authors conclude that results support the previous observation that decreased
blastocyst yield occurs following housing of bred C57BL/6 donor mice in ventilated
cages. Noise generated by the ventilated rack used was implicated as a potential source
of stress.
QUESTIONS:
1. Using standard methods, mice are super-ovulated by injection of which two
hormones approximately 2 days apart?
2. In this study, which of the following was true for bred embryo donor mice housed in
ventilated racks versus those housed in static racks?
a. They produced more blastocysts and had lower serum corticosterone levels
b. They produced fewer blastocysts and had higher serum corticosterone levels
c. They produced more blastocysts and had higher serum corticosterone levels
d. They produced fewer blastocysts and serum corticosterone levels did not differ
3. According to the Guide, what is the sound pressure level exposure above which
rodents are subject to auditory and non-auditory effects?
a. 100 dB
b. 80 dB
c. 85 dB
e. None of the above
ANSWERS:
1. Pregnant mare serum gonadotropin; human chorionic gonadotropin
2. d
3. c
Pritchett-Corning et al. Breeding and Housing Laboratory Rats and Mice in the
Same Room Does Not Affect the Growth or Reproduction of Either Species, pp.
492-498
Species: primary, mouse
Domain 4: Animal Care
SUMMARY: The authors collected data to support housing rats and mice with the same
health status in the same barrier. They measured reproductive parameters such as litter
size at birth, litter size at weaning, and pups missing at weaning, and weight gain from
weaning to adulthood. Data was collected on 33 colonies of mice and 28 colonies of rats
with several breeding females in both groups. The presence or absence of other species
was not associated with statistically significant changes in any parameter measured.
There were no significant negative effects on parameters measured. The authors were
careful to house the different species based on a similar health status.
QUESTIONS:
1. The Guide for the Care and Use of Laboratory Animals suggests that species should
be physically separated based on which of the following parameters:
a. To prevent transmission of disease between species
b. To prevent interspecies conflict as a source of anxiety or behavioral change
c. To prevent interbreeding of species
d. All of the above
e. a and b
f. b and c
2. T/F Mice can be fostered by rats except that weanling rats are likely to cannibalize
the mice weanlings
3. Did the study support that rats are not negatively affected by the presence of mice in
the same room?
a. No, there were some variations on growth rate depending on the number of mice
in the room
b. Yes, only on the parameters measured: growth and reproductive parameters
c. Yes, rats showed increased resistance to stressors based on having mice
housed in the same room
ANSWERS:
1. e, a and b are correct
2. False, rat-fostered mice have their behavior altered and show preference to rats,
were not reported to be cannibalized.
3. b
Experimental Technique
Varisli et al. Effect of Chilling on Motility and Acrosomal Integrity of Rat Sperm in
the Presence of Various Extenders, pp. 499-505
Primary Species: Rat
Task 3, K1
SUMMARY: Studies were conducted to determine the effect of chilling on rat sperm and
optimal components (extenders) to avoid chilling-induced injury.
The following
experiments were conducted:
Experiment 1: The effects of chilling (at 4, 10 or 22⁰C) on the motility and acrosomal
integrity of sperm from 2 strains of rats were compared.
Experiment 2: The motility of Sprague-Dawley rat sperm after exposure to extenders
(HEPES-buffered Tyrode lactate, skim milk, lactose monohydrate, Tris-citrate, and
TEST) and cooling and warming was determined. The effects of supplementing these
solutions with 20% lactose-egg yolk alone or in combination with a commercial SDS
based paste in preventing chilling injury was determined.
Experiment 3:
The motility of Sprague-Dawley sperm were compared after
supplementing the base extenders with either 0.4M permeating cryoprotective
agent(glycerol, propylene glycol, or DMSO) or a 0.1M nonpermeating CPA (sucrose or
raffinose)
Results: The results show that chilling significantly reduced the motility but not the
integrity of Sprague-Dawley and F344 rat sperm strains. The addition of LEY into each
extender significantly prevented motility loss after chilling. The current data suggests
that the addition of an SDS based product to egg yolk during cold storage has no
additional benefit on the motility of rat sperm.
QUESTIONS
1. Name some of the most commonly used semen extenders.
2. T/F Extenders are used to provide temperature protection for sperm while reducing
heir metabolic
rate in cool storage.
3. Why are non-permeating sugars (such as Sucrose and Raffinose) added to
extenders?
4. Name some cryoprotective agents used with extenders.
5. T/F. Sperm cryopreservation protocols developed for 1 species will work for sperm of
all species.
ANSWERS:
1. Tris-citrate, skim milk, egg yolk, lactose and N-tris
2. True
3. Sugars are added to withdraw intracellular water by increasing osmotic pressure and
providing cryoprotection.
4. Glycerol, ethylene glycol, propylene glycol, or DMSO
5. False
Schoell et al. Euthanasia Method for Mice in Rapid time-Course Pulmonary
Pharmacokinetic Studies, pp. 506-511
Domain 2, Task 3
Primary Species: Mouse
SUMMARY: This was a two-part study to 1) compare retro-orbital and tail vein injection
of ketamine-xylazine with regard to preparation time, utility, tissue distribution, and time
to onset of euthanasia; 2) compare retro-orbital ketamine-xylazine, CO2 gas, and
intraperitoneal pentobarbital as euthanasia agents in a rapid time-point pharmacokinetic
study. For the first part, two groups of 7 naïve mice were euthanized with 100 µL of a
10:1 mixture of Ketamine-xylazine (ketamine 100mg/ml and xylazine 100 mg/ml)
delivered either via tail-vein or retroorbitally. Both methods resulted in rapid death (3
seconds for tail-vein injection vs. 5 seconds for retroorbital injection), however the total
time between removal of the mouse from its cage to death was 50s longer in tail vein
injected mice due to the time needed for tail warming and mouse restraint. There were
no significant differences in average concentrations of ketamine and xylazine in the
brain, lung, heart, and plasma after tail vein and retroorbital injections. Because
concentrations did not differ between the two methods, the authors concluded that
retroorbital ketamine-xylazine is superior to tail vein ketamine-xylazine due to the
reduced time needed to complete the procedure.
For the second part, 72 naïve BALB/c mice received 1 mg/kg fluticasone intratracheally.
The animals were then divided into 3 arms with 6 mice from each group euthanized at 1
of 4 time points (5, 15, 30, and 60 min) by either CO2 inhalation, 100mg/kg
intraperitoneal pentobarbital, or retroorbital injection of ketamine-xylazine. At death,
blood, BAL fluid, and lung tissue were harvested for pharmacokinetics analysis. In this
part of the study, the clinical time to death was 262s for CO2 inhalation, 253s for IP
pentobarbital, and 5.3s for retroorbital ketamine-xylazine. At 5 minutes after dosing, the
lung fluticasone levels were highest and the plasma fluticasone lowest in the ketamine-
xylazine group. By 30 minutes, the plasma and lung levels were nearly the same for all
3 groups. Because the ketamine-xylazine method was so quick, more of the fluticasone
was captured in the lungs before clearance into the plasma. This demonstrated how the
choice of euthanasia may influence the data collected at early closely spaced time points
and change the perceived pharmacokinetics profile.
The retroorbital ketamine-xylazine euthanasia method may be an option where rapid,
nontraumatic, technically simple, and consistent means of euthanasia is required. This
technique satisfies the principles set forth in the AVMA Guidelines on Euthanasia and
the ACLAM Task Force Guidelines on Euthanasia such as 1) rapid loss of
consciousness; 2) reliability; 3) safety of personnel; 4) irreversibility; 5) compatibility with
study requirements; 6) minimal negative emotional effect on observers and personnel; 7)
compatibility with subsequent evaluation, examination, or use of tissue
QUESTIONS:
1. The average time to clinical death was shortest using which method of euthanasia?
a. Retroorbital ketamine-xylazine
b. Intraperitoneal pentobarbital
c. CO2 inhalation
2. The average concentrations of Ketamine and xylazine in the brain, lung, heart, and
plasma were significantly different between intravenous and retroorbital methods
a. True
b. False
3. Which is NOT a characteristic of an acceptable euthanasia method?
a. Rapid loss of consciousness
b. Reversibility
c. Reliability
d. Safety of Personnel
ANSWERS:
1. a
2. b
3. b
Torreilles et al. Evaluation and Refinement of Euthanasia Methods for Xenopus
laevis, pp. 512-516
Domains: D4, K7
Species: Xenopus laevis
SUMMARY: This article evaluated the current methods of euthanasia for Xenopus
laevis and reported newly found acceptable doses for those methods. The methods
evaluated were, immersion in MS222, intracoelomic injection of MS222, intracoelomic
injection of sodium pentobarbital with phenytoin, and ventral cutaneous application of
benzocaine gel. They found, that intracoelomic injection of sodium pentobarbital (1100
mg/kg) with phenytoin was the most effective and rapid method for euthanizing small
numbers of frogs; and immersion fro 1 hour in MS222 solution at 5 g/l was the most
effective and rapid method for euthanizing larger numbers of frogs. Ventral cutaneous
application of benzocaine gel (182 mg/kg) proved to be a reliable method, although it
was slower than pentobarbital and immersion in MS222. However; they did feel as if it
would be suitable and convenient, particularly for field experiments, or when only a few
frogs are euthanized at a time. All of the doses found to be effective were considerably
higher than those previously published.
QUESTIONS:
1. What is the only anesthetic approved by the FDA for anesthesia in fish?
2. What is a disadvantage of using MS222? (think occur health)
3. How should MS222 be stored?
4. Pentobarbital is a Schedule ____ controlled drug.
ANSWERS:
1. MS222
2. Its potential toxicity to humans. If it is inhaled or absorbed chronically through the
skin, it can induce reversible retinal toxicity.
3. In a light proof container, preferably in a freezer.
4. Schedule III
Domain 2: Management of Pain and Distress; Task 3. Euthanatize
Secondary Species: Xenopus spp.
SUMMARY: Xenopus are fully aquatic lung breathers and euthanasia methods common
to mammals are unsuitable. AVMA guidelines for physical euthanasia of amphibians
include decapitation and pithing of the brain following pithing of the spinal cord.
Anesthetic injection or immersion techniques are less stressful for both the operator and
the animal. The three common anesthetic agents used for euthanasia are benzocaine,
tricaine methane sulfonate (MS222) and sodium pentobarbital with the most common
being MS222. Current dosing of MS222 has a very wide range for both immersion and
for intracoelomic injection. The authors set out to compare a range of doses of MS222
for both immersion and intracoelomic injection along with injection of sodium
pentobarbital/sodium phenytoin and lastly a novel method of ventral application of 20%
benzocaine gel.
Results: Deep anesthesia was achieved within 4 minutes of immersion in 1, 2, 3, and 5
g/L of MS222. Frogs were removed after 1 hr of immersion with variability in response
over time. Some of the frogs in 1 g/L were not euthanized and recovered. Immersion in
3 g/L resulted in euthanasia of all frogs by 5hr and by 3hr in frogs immersed in 5 g/L.
Results were similar between frogs immersed individually or frogs immersed in groups.
Intracoelomic injection of MS222 at a high dose of 2590 mg/kg did not result in
euthanasia within 5 hr. Combined sodium pentobarbital (1100 mg/kg) and sodium
phenytoin (141 mg/kg) euthanized 17 of 20 frogs within 1 hr and all frogs within 3 hr.
Ventral application of 20% benzocaine gel euthanized 18 of 20 frogs within 3 hr and all
frogs within 5 hr.
Discussion: The authors conclude that intracoelomic injection of sodium
pentobarbital/sodium phenytoin and immersion for 1hr in 5 g/L MS222 were the most
effective and consistent methods of euthanizing Xenopus laevis. It was noted that the
effective doses reported in this paper are much higher then those previously reported in
the literature. MS222 shows a variable response at lower doses. Further, MS222 is a
potential toxin to humans with the risk of renal toxicity following inhalation or skin
exposure. MS222 powder can degrade over time and should be stored in a lightproof
container and preferably in a freezer. Unbuffered MS222 is acidic and as a result is
irritating to the skin and coelomic cavity. The benzocaine gel was determined to be
suitable for euthanasia and may have application in the field or when very small
numbers of frogs are being euthanized. When doses of MS222 lower then 5 g/L or
immersion times less then 1 hr are used secondary methods of euthanasia including
quick freezing, double pithing, heart removal, or decapitation are recommended.
QUESTIONS:
1. T/F. Results of immersion techniques vary dramatically between frogs immersed
individually and frogs immersed in groups.
2. What methods of euthanasia did the authors believe was most effective?
3. T/F. Intracoelomic injection of MS222 at a high dose was an effective method of
euthanasia
ANSWERS:
1. F
2. The authors conclude that intracoelomic injection of sodium pentobarbital/sodium
phenytoin and immersion for 1hr in 5 g/L MS222 were the most effective and
consistent methods of euthanizing Xenopus laevis.
3. F
CASE REPORTS
Brossia et al. Interstrain Differences in the Development of Pyometra after
Estrogen Treatment of Rats, pp. 517-520
SUMMARY: This is a case report describing development of pyometra in ovariectomized
Brown Norway rats after long-term estrogen administration. At the time of ovariectomy
15 Brown Norway and 15 Sprague Dawley rats were implanted with a capsule
containing estradiol 17-B providing a slow release (2ug/d). A control group of 15
ovariectomized Brown Norway without estrogen supplementation was also used as part
of the original experiment involving irradiation of the right eye to induce cataract
development in the presence or absence of estrogens. One hundred percent of the
estrogen treated Brown Norway Rats developed severe infectious (E. coli) pyometra
between 51 and 255 post-implantation vs. none of the control animals or the Sprague
Dawley group. The authors acknowledge that this finding is consistent with other studies
with Brown Norway rats developing pyometra with estrogen supplementation. They go
on to suggest that the strain susceptibility is a result of a complex interplay between
genes and hormones and that estrogen alters the expression of pyometra-susceptibility
genes in Brown Norway strain. According to the conclusion of this study, caution should
be taken when designing estrogen-related experiments involving Brown Norway rats and
other potentially sensitive strains.
QUESITONS
1. T/F - This and other studies suggest that the Brown-Norway rat is at an increased
susceptibility to estrogen-induced pyometra.
2. As in dogs, estrogens increase the susceptibility of pyometra in the rat by:
a. Increasing the risk of allowing bacteria to cross the cervix into the uterine lumen
b. Altering leukocyte permeability of the uterine mucosa
c. Altering the expression of pyometra-susceptibility genes
d. All the of the above
3. T or F - E. coli is not a common bacterial isolate from estrogen-induced pyometra in
conventionally housed rats when appropriate environmental sanitation measures are
adhered to.
ANSWERS
1. T
2. d.
3. F
Wathen et al. Enoxaparin Treatment of Spontaneous Deep Vein Thrombosis in a
Chronically Catheterized Rhesus Macaque (Macaca mulatta), pp. 521-526
Primary Species - Macaques
SUMMARY: Nonhuman primates are commonly catheterized for prolonged periods of
time for a variety of reasons including blood collection, drug administration and
parenteral nutrition. A number of complications are consistent with chronic
catheterization including leakage of the catheter, catheter migration, infection, catheterassociated thrombus formation and vascular injury. In the current study deep vein
thrombosis (DVT) was diagnosed via ultrasonography in a nonhuman primate that had
been chronically catheterized. This animal had presented with scrotal swelling that
moderately resolved with warm hydrotherapy and massage. The condition progressed
to the point of severe scrotal swelling, prepucial swelling and thickening of the
associated skin. As the problem persisted a normal battery of tests did not reveal any
abnormalities. As a follow up to a possible vascular event the animal was referred for
ultrasonography. The left femoral vein and bilateral iliac vessels did not "flatten" as
expected when compressed. Adherent venous thrombi were observed in the same
vessels. This information provided a diagnosis of DVT. It was hypothesized that the
presence of a DVT was impeding venous drainage and responsible for the swelling
observed clinically. As follow up to the diagnosis of DVT blood was collected for a
coagulation panel. The panel revealed an increase in the D-dimer level which measures
secondary fibrinolysis. Based on therapeutic approaches used in humans the animal
was started on low-molecular-weight heparin (LMWH) at a daily dose of 20mg for 10
days. There was a clinical response but not a complete resolution of clinical signs. As a
result the Enoxaprin was repeated for an additional 10 days, 2 months after the initial
therapy. Two weeks after the second round of therapy clinical signs has resolved. Six
months later ultrasonography revealed resolution of the DVT and the D-dimer test was
normal. LMWH inactivates factor Xa which prevents or lessens thrombus formation.
Contraindications for the use of enoxaprin include any bleeding conditions or conditions
that predispose to bleeding. Enoxaprin has the advantage over heparin of being longer
acting and having a more predictable effect after dosing.
QUESTIONS:
1. List 3 complications of chronic catheterization
2. T/F. Venous thrombi can affect the ability of a vessel to "flatten"
3. T/F. Heparin has a duration of effect longer then LMWH
ANSWERS:
1. Leakage of the catheter, catheter migration, infection, catheter-associated thrombus
formation and vascular injury
2. T
3. F