Contemporary Topics in Laboratory Animal Science

Volume 42 (5)

Frozen sperm as an alternative to shipping live mice 8-11

OVERVIEW: Authors tested the hypothesis that sperm cryopreserved at one location and then transported transcontinentally via a common package delivery service using both air and ground transport to a second location could be recovered for in vitro fertilization (IVF) to successfully derive live born offspring.

MATERIALS, METHODS, etc.: Split aliquots of fresh and cryopreserved sperm from individual male B6D2F1 mice were tested at both senders’ and recipients’ locations by using similar cryopreservation and IVF procedures to control for differences in handling procedures.  Using oocytes freshly collected from female B6D2F1 mice, IVF was performed with freshly harvested sperm, sperm collected, frozen and thawed on-site for IVF (non-shipped cryopreserved sperm) and sperm collected and frozen at one site, then shipped to a second site for thawing and IVF(shipped cryopreserved sperm).  Shipped cryopreserved sperm was transported transcontinentally in a LN2 dry-vapor shipper with an environmental temperature maintained at ~ -150°C via FedEx overnight delivery.

RESULTS: At both senders’ locations, fertilization rates using cryopreserved sperm were lower than those using fresh sperm, but transfer rates of embryos successfully fertilized using cryopreserved sperm were not significantly different from those using fresh sperm. Fertilization and transfer rates were not significantly different for shipped vs. non-shipped cryopreserved sperm and the weaning rate of pups born were identical.

CONCLUSIONS: Cryopreserved sperm can be transported between different facilities and used for IVF to successfully derive live born mice, and may be a more economical, efficient, and safer alternative to shipping live animals. In this study, achieved results were similar despite slight variations in cryopreservation and IVF protocols methods employed at the two sites. Cryopreserved sperm samples are routinely stored at -196°C, and -150°C worked well for transport and subsequent IVF in this study, thus ultra-low temperatures (-150°C to -196°C) are recommended for transport and storage of cryopreserved sperm.

QUESTIONS: 

1. The authors list multiple economic, efficiency and safety-related shortcomings inherent to shipping live animals; all are intuitive... can you list at least five?

a.

b.

c.

d.

e.

2. T/F Cryopreservation and IVF protocols must be identical at two separate locations in order to achieve similar results

3. What is the ultra-low temperature range recommended for transport and storage of cryopreserved sperm?

ANSWERS:

1. Economic, efficiency and safety-related shortcomings inherent to shipping live animals:

a. Slow method for sharing new animal models to ‘rapidly advance’ research, e.g., only a subset of mice of certain age/gender may be available for dispersal at any one time, and due to space limitations, senders may not maintain all strains of live animals at all times

b. Live mice have vital needs (food, water, environmental temperature, etc.) that must be addressed during shipping and may impact how/when shipping can occur

c. Live mice may –or may not- be subject to handling in transit which might allow exposure from feral or other co-shipped mice with potential disease-causing pathogens

d. Live mice can carry transmissible diseases from the sender’s to the recipient’s colony

e. Shipped live mice typically require quarantine and/or acclimation upon arrival, resulting in lost research time, typically while per diem charges accrue… further, shipments can fail quarantine, resulting in unusable live mice after receipt

2. F

3. -150°C to -196°C

Variable susceptibility of the owl monkey (Aotus nancymae) to four serotypes of Dengue virus 12-20

Summary:
There were two goals in this study:  to determine whether Owl Monkeys could serve as a model of Dengue virus infection for vaccine development studies and to determine the effects of chronic blood sampling.
Goal 1:  20 monkeys were used and divided into five groups; 4 were inculation groups and one was the control.  Each of the four inoculation groups received a subcutaneous dose of either Dengue virus type 1, type 2, type 3, or type 4.  Dose was determined by that dose already established to cause viremia in owl monkeys and rhesus macaques for type 1.  Blood samples were collected on days 0-12, 21, 28, and day 60 and were tested for viremia and antibody presence.  The results revealed that only dengue type 1 resulted in an appreciable viremia, the other 3 types resulted in a short and inconsistent viremia, however,  all 4 types resulted in appropiate antibody responses.  Furthermore, the only consistent clinical sign was lymphadenopathy in half of the animals.

Goal 2:  Total blood volume was estimated to be 55-60 ml/kg, similar to other NHP species.  The total amount of blood drawn was 8 ml in the first 12 days and 9.5 ml in the first 30 days of the study.  An inhouse normal reference range was established and ranged from 36.9% to 59.0%.  All animals started with a hematocrit of at least 40% and all animals except for 1 ended the 12 day sample period with hematocrits of at least 40%.  Furthermore, all but one animal had a maximum hematocrit decrease (the difference between the day 0 hematocrit and the lowest hematocrit recording) of 12.9% or less over this same time period.

In summary, viremia can be established in Aotus only with Dengue type 1 virus at the dose used in this study.  Antibody responses however, occurs with all 4 Dengue virus types. Aotus tolerate repeated blood-letting quite well with up to 17% of the total blood volume taken per week and up to 20% total blood volume taken per month.

Questions:

1.  How is Dengue virus transmitted?

2.  Once a mosquito becomes infective, how long does it retain this capacity to infect humans or other mammals?

3.  What are the two clinical diseases of Dengue in humans?

4.  What are the traditional NHP models of Dengue virus infection?

Answers:
1.  mosquito:  Aedes, subgenus Stegomyia
2.  For the life of the mosquito
3. Dengue Fever (DF) and Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS)
4.  rhesus and cynomologus macaques

Treatment of persistent self-injurious behavior in rhesus monkeys through socialization: a preliminary report 21-23

Long term single-housing of captive primates may lead to display of abnormal activity including self-injurious behavior (SIB). Few laboratory interventions have been successful in complete elimination of this complex behavioral pathology. Treatment regimens (e.g., drug therapy, enrichment manipulations) have had only limited success in eliminating SIB. The etiology of these destructive behaviors is not completely understood but has been associated with stressful social experience in early life. Novak suggests that SIB may serve as coping strategy, reducing the animal's arousal by lowering its heart rate. Although an effective means for a monkey to control escalating hear rate, SIB may lead to wounding, requiring Veterinary intervention and extensive clinical care. Social companionship has been recommended for captive laboratory primates because it is well understood and accepted that primates are social animals. Social companionship may also provide a means for decreasing or eliminating self-biting behavior. In addition, social companionship is highly recommended as an effective means of satisfying enrichment requirements for captive nonhuman primates and should be implemented whenever possible.
Materials and methods.
Subjects: Six rhesus monkeys (Macaca mulata) , all adult males exhibiting self injurious behavior were used for social pairing with female partners.
Housing: Prior to surgeries and socialization, five of the adult male rhesus monkeys were housed individually . The sixth had already undergone successful pairing with an apparently infertile female . Unwanted pregnancy of the female in this pairing placed this male in the group selected for vasectomy surgery. After the surgeries, all six male monkeys were paired with female partners.
Socialization: Subjects remained singly housed for up to four weeks after surgery. Individual housing was implemented prior to socialization to allow for close post-surgical observation and sufficient time for healing. Males were placed in cages adjacent to females and separated by either a Plexiglass or stainless-steel mesh panel prior to introduction to a partner. Animal care personnel or behavior techs record initial interactions between newly established pairs. Compatible pairs that were formed after the surgery remained intact except when studies terminated and/or animals were transferred out of the facility.
 
Results: Although not all pairs were able to remain together permanently, due to relocation, protocol change, study termination, all of the SIB male primates were successfully paired with female monkeys after vasectomy and observed for 1, 1.5, 2, 8, 12, and 32 months. Animal record showed that behavioral referral or clinical intervention for SIB incidents were markedly reduced for all primates after social pairing.
 
Questions: True or false
1. SIB is a coping strategy, reducing the animal's arousal by lowering its heart rate.
2.Mixed sex pairing promotes the expression of species typical behavior.
3. Section 3.81 of the Animal Welfare Act requires that all research facilities, dealers and exhibitors develop, document and follow an appropriate environmental enhancement program adequate to promote the psychological well-being of NHP.
 
Answer:
1. T
2. T
3. T

Social conflict reduction program for male mice 24-26

Summary:
 In natural environments, a dominant mouse will establish dominance and will have variable numbers of subdominant mice and subordinates. Whenever possible, the non-dominant mice avoid the dominant mouse and it's urine-marked areas. However, in lab animal settings, placing mice in shoeboxes leaves them no place to hide. Dominant mice will constantly monitor the cage space and will often attack others.  This defeat of the other non-dominant mice has been proven to induce a profound opioid analgesia. Also, some dominants will attack and kill the subordinates. Because animals should be housed with the goal of maximizing species-specific behavior and minimizing stress-induced behaviors, these researchers wanted to eliminate dominant mice from social groups to reduce or eliminate the number of aggressive acts seen in cages of mice. Reducing the numbers of aggressive attacks in cages, they felt, would result in less-stressed animals, less fighting injuries, less animal loss and less rejection for experimental usage.
 Male mice were used (Crl:CD-1) weighing 25-30 grams. Mice were housed in accordance with CCAC guidelines. Animals were observed soon after initial housing, as the mice were determining social organization was being determined. Periods of increased mice activity were defined as between 8-9am & 2-3 pm. They ended up being able to do these observations within 5 minutes during this period, after proper training and getting comfortable with the signs to look for. Dominant mice behavior was: attacking, tail rattling, chasing, threatening, biting, fighting, unilaterally attacking, show sideways offensive posture to all cagemates.
 Subordinate mice were usually found in a huddle with other nondominant mice. They never retaliate, but show distress, flight, submissive & defensive postures, hiding & squealing. Wounds from fighting are often on the tail base, back, feet, prepuce, anus, tail, and shoulders.
 Subdominant mice are in good condition, few injuries, and usually found alone.
 When they housed the mice, they used CCAC guidelines to avoid overcrowding, and did not put only two mice in a cage because the non-dominant mouse will get a lot of attacks. They would observe all newly arriving animals and separated out aggressive mice to a new clean cage. Dominant mice were either left in room or removed completely. If they are left in the room, the urine smell from a dominant mouse may encourage other mice to act dominantly. After the researchers started moving dominant mice to a completely different room, the percent dominants found in new cages dropped by almost «.
 Over the course of the study, the number of mice removed that were lost on study from non-study related characteristics decreased from 0.3% to 0.13%. Also, number of clinical incidences and deaths from non-study related procedures also was decreased. The number of mice removed from social conflict reduction program, however, was higher than this figure, however, the authors were first thinking about the welfare of the animals, and not the fact that they would have to remove a lot of dominant mice. They also thought the lack of attacks and injuries was definitely an advantage to their program from a humane perspective.
 
Question 1: FYI the amount of cage space required for group housed mice according to CCAC guidelines in 160 cm2 per mouse, with a height of 15 cm. By the end of the study, how long did animal techs have to observe the new groups of mice in order to assess aggressive dominant behavior?
Answer 1: 5 minutes
 
Question 2: Name behaviors of dominant mice:
Answer 2: Attacking, tail rattling, chasing, threatening, biting, fighting
 
Question 3: Name behaviors of subdominant mice:
Question 3: Usually found alone, have few bite marks or scars
 
Question 4: Name behaviors of subordinate mice:
Answer 4: Found in a huddle with other subordinates, flight, submissive postures, defensive postures, hiding, squealing.
 
Question 5: What was one of the complications involving personal protective equipment associated with seeing a dominant mouse and not being able to immediately separate it?
Answer 5: If animal techs did not don gloves upon entering the room, if they saw a dominant mouse, then went to grab gloves to separate it, they often could not distinguish which mouse it was until he displayed another dominant behavior. The lesson learned was to always have proper PPE for mice handling if going to observe mouse groups for dominance.

The effect of an environmental enrichment device on individually caged rabbits in a safety assessment facility 27-30

An eight-week study was performed to evaluate the effects of stainless steel rattles on spring clips as environmental enrichment for individually houses rabbits.  No significant differences were found between study (rabbits with the enrichment device) and control rabbits (no enrichment device) when body weight, food consumption, and hematologic parameters were analyzed.  A one-hour peak device manipulation time was determined to be 7:30 to 8:30 a.m.  The observations occurred weekdays for eight weeks at this time.  Interaction with the device decreased over the eight-week period in both sexes.  The authors concluded that because no adverse effects were noted in the analyzed parameters, this enrichment device is suitable for safety assessment studies and other studies in which the introduction of new variables is unacceptable.

Questions:

1.      True or False:  The introduction of stainless steel rabbit rattles as environmental enrichment had no effect on body weight, food consumption, or hematologic parameters in a recent study.

2.      In a recent study evaluating the use of stainless steel rabbit rattles for enrichment it was found that:

a.      Manipulation of the rattle remained constant throughout the study
b.      Males manipulated the rattle more frequently than females
c.      Manipulation of the rattle declined over time
d.      Ingestion of the rattle was a frequent complication
e.      Manipulation of the rattle induced significant eosinophilia

3.      What is the dental formula of the rabbit?

Answers:

1.      True
2.      C
3.      (i2/1, c0/0, pm3/2, m2-3/3) X 2 = 26 to 28 teeth

Reproductive output in the hatchery-reared California sea hare at different stocking densities 31-35

This study characterized the reproductive period of hatchery-born and -raised Aplysia californica at different stocking densities. Although it had little influence on time to maturity and on average number of days of reproductive life, stocking density (and thus animal size) strongly influenced their lifetime fecundity.

Housing environment:

Stocking densities: 2, 5, 10, 15, and 20 animals per 16-liter cage

Temperature: controlled at 13^oC – 15^oC

Photoperiod: 14:10 hours light:dark cycle

Seawater flow through cages to optimize seawater O₂ concentration, pH, and salinity

Findings:

· Feeding algal diet ad libitum (compared to scheduled feedings) produced early sexual maturity

· Despite different growth rates, age at first sexual maturity (first egg mass) averaged 210 ± 15 days of age among the different stocking densities

· As stocking density increased:

o Number of spawning episodes per animal decreased

o Average weight of spawn per animal over its reproductive lifetime decreased

o Spawn weight per animal decreased

o Animal size decreased

· Larger animals produced more eggs during the 10 spawns at the peak of the spawning period

Question:

1. What is the scientific name for the California Sea Hare? What types of studies are they used for?

2. What is “first egg mass”? When is it in the California Sea Hare?

3. T/F Stocking density strongly influences the lifetime fecundity of California Sea Hare, as well as their time to maturity and average reproductive period.

Answer:

1. Aplysia californica, used in neurobiological studies due to its simple and well-characterized nervous system.

2. Age at first sexual maturity, which averaged 210 ± 15 days of age.

3. False

Subcutaneous continuous glucose monitoring and dose adjustment decreases glycosylated hemoglobin in spontaneously diabetic cynomolgus monkeys 36-40

The CGMS (continuous glucose monitoring system) can provide profiles of blood glucose trends and identify level of glucose in the blood that were not identified by periodic blood sampling in cynomolgus monkeys with spontaneous diabetes mellitus, which are similar to type II diabetes in humans. By this system it is possible to monitor the level of glucose level continuously and helps in the accurate level of insulin that should be administered to the animal.
 The instrument consists of glucose sensors were inserted in the subcutaneous tissues at the waistline of each animal by using introducer needles. The sensors were held against the skin with sterile transparent tape (IV3000, MiniMed, Inc.). The animals wore primate jackets to prevent the animals from removing the sensors. The glucose monitor was located in the pocket at the back of the jacket.
 By using CGMS different glucose profiles among diabetic monkeys can be identified and dosage of insulin can be adjusted. Excessive hyperglycemia and hypoglycemia could be prevented each day. Furthermore, by applying CGMS to healthy and glucose-tolerant monkeys as well as diabetic monkeys, the pharmacological effect and toxicity of various antidiabetic agents can be evaluated.
Questions:
1. Spontaneous diabetes in cynomolgus monkeys (Macaca fascicularis) exhibit a condition similar to what type of diabetes in humans.
2. Name a system through which the glucose level in the body of animal can be continuously monitored
3. What do you mean by glycosylated heamoglobin and how does it reflect the blood glucose level in our body.
Answers
1.Type II
2. continuous glucose monitoring system
3.HgA1c is a hemoglobin bound with D-glucose and associated mucopolysaccharides and is used as an indicator for controlling blood glucose levels because its concentration reflects the status of blood glucose during the past one to two months.

Survival anesthetic and injection procedures for neonatal opossums 41-43

OBJECTIVES: To develop a protocol for short-term inhalation anesthesia protocol for Monodelphis mothers without simultaneously anesthetizing the neonates.
 
BACKGROUND: 
Taxonomy of the opossum 
 family: Didelphidae
 Genus and species: Monodelphis domestica 
Origin: South America (Brazil, Bolivia, and Paraguay)
Size: adult females weigh 60 - 100g, adult males weight 90 - 150g
Mean litter size: 8
Parity: Can have up to 3 litters per year
Gestation time:  13.5 days. Because this is so brief they are essentially extrauterine fetuses, equivalent to 12.5 day-old mouse or a 6-week old human embryo.
Lungs are precociously developed, but most others are not
Neonates are completely exposed, as the female has no pouch, making manipulation easy
 
Uses in research: Mammalian developmental processes early in life (those that occur after birth in marsupials and before birth in eutherian mammals). Examples include
 1. Research on function of embryonic nervous system in long-term cell culture
 2. Healing of neonatal spinal cord after complete crushing or transection
 3. Effects of estrogen on testicular development
 4. Ontogeny of skin healing and development of scarring
 5. Development of neuropeptides, steroid receptors, and visual system
 6. UV-induced melanoma
 7. Allogeneic and xenogenic models for melanoma research
 
Challenges:
Neonatal opossums (<5 days of age) tend to release the nipples during anesthesia or when the mother recovers and behaves in an agitated manner
 
HYPOTHESIS:
Release of the nipples by neonatal opossums was due to the neonate's becoming anesthetized by inhalation or via respiration through the skin, rather than by consumption of the milk. 
 
METHODS:
Inhalation Anesthesia - 
 2 ml of halothane was added to cotton balls in a 2-L beaker, and the beaker covered for 3-5 minutes. The mother with the neonatal litter was gently suspended upside down by holding its tail, so that its body was 1/3- to ½-way into the beaker while keeping the babies outside of the beaker. After the mother was lightly anesthetized, she was removed from the beaker, placed on the table, and a 50ml conical tube with a cotton ball in the end (pre-equilabrated with 1ml halothane) was used as a nose cone to completely anesthetize the mother.
Injection of the neonates 
 - Neonates are exposed by laying the mother on her back. 
 - Using surgical scissors to hold down the limbs allows good exposure 
so that one person can do the injections.
 - Use a 29-guage needle for injections
 - Neonates were manipulated using a pair of fine forceps, so as not to disturb the mother
Experimental Groups:
 - 5 litters of pups (1 litter on the day they were born, 1 litter 1 day old, 3 litters that were 2 days old)
 - Control - 1 litter. The mother was anesthesized by placing her in a beaker of halothane and removed when muscle relaxation was observed.
 
RESULTS:
Conventional anesthesia 
 - 8 of 11 pups detached from the mother during induction. During the injection procedure, the remaining 3 detached. None of the neonates reattached to the mother, and all died.
Experimental Method
 - None of the neonates using the new method detached from the mother.
 - Day 0 litter - the skin was extremely friable. Safely injected 5-10ul of PBS with no deaths.
 - Day 1 litter - the skin was more developed and 15-20ul was safely injected.
 - Day 2 litters - up to 15-20ul was injected safely with no deaths. 
 - 7 neonates died when 25 - 30ul were injected. Cause unknown
 
CONCLUSIONS:
 - Lowering the mother upside down into the halothane-containing beaker for about 30 seconds to lightly anesthesize the animal before applying the nose cone is quick and effective for brief procedures
 - Injection volumes should be minimal (due to small size and underdeveloped state of skin)
 - Some injection volume may be lost by post-injection leakage at the injection site
 
QUESTIONS:
 1. What is the genus and species of the opossum?
 2. How is the opossum used in medical research? Give some examples. 
 3. What is the mean gestation period of the opossum? Neonatal opossums are equivalent to what age neonates in mice and in humans?
 4. Which organs are precociously developed in the opossum neonate?
 5. What is the problem most often encountered during inhalation anesthesia of mother and neonatal opossums?
 6. Describe a method by which mother opossums can be safely 
anesthesized so that neonatal pups do not become detached.
ANSWERS:
 1. Monodelphis domestica
 2. Uses in research: Mammalian developmental processes early in life (those that occur after birth in marsupials and before birth in eutherian mammals). Examples include
 - Research on function of embryonic nervous system in long-term cell culture
 - Healing of neonatal spinal cord after complete crushing or transection
 - Effects of estrogen on testicular development
 - Ontogeny of skin healing and development of scarring
 - Development of neuropeptides, steroid receptors, and visual system
 - UV-induced melanoma
 - Allogeneic and xenogenic models for melanoma research
 3. 13.5 days. Because this is so brief they are essentially extrauterine fetuses, equivalent to 12.5 day-old mouse or a 6-week old human embryo.
 4. Lungs. Most other organs are not precociously developed.
 5. The neonates become detached from the nipples either during anesthesia or during recovery if the mother becomes agitated. Once detached, it is difficult to reattach them and they will die.
 6. The mother can be lowered upside down into a beaker containing halothane for about 30 seconds (neonates remain outside of the beaker). Once the animal is lightly anesthesized, a nose cone containing a cotton ball soaked in halothane can be used. 

24-hour intravenous infusion via the marginal ear vein in the New Zealand white rabbit 44-46

The authors developed a method of 24 hour intravenous infusion into the marginal ear vein to permit unlimited access for oncology drug safety assessment while minimizing the need for animal restraint. 32 NZW rabbits of both sexes were preconditioned to Lycra jackets, Elizabethan collars, and nylon restraint bags daily for 7 days.

Technicians inserted a human infant 15 cm 24 gauge catheter into the marginal ear vein to permit placement of the tip into the cervical jugular vein, using sterile technique and no pharmacologic intervention. The catheters were attached to a small, lightweight, disposable ambulatory pumps (Infu Disk) set to administer either 1 or 2 ml/hr. Pumps were changed every 5-10 hours, depending upon infusion rate. The catheter was flushed to check patency every 3 hours with 0.05 M isotonic citrate buffer (test article vehicle). If patency was in question, a heparin flush was used, and if that failed to restore patency, the catheters were replaced (6 of 32 rabbits). The rabbits wore the pumps in Lycra jackets on their backs to permit normal activity.

Blood samples were collected from the contralateral marginal ear vein using a 22 gauge angiocath at predetermined timepoints over an eight hour period. After eight hours, the catheter was replaced in the central ear artery for sampling at 24 h, 25 h, 26 h, and 32 h. After 32 h the catheter was removed, but replaced again at 48 h for additional sampling.

ECGs were recorded at various timepoints from animals restrained in nylon restraint bags.

Animals were euthanized following completion of sampling. Necropsies performed confirmed catheter tip placement into the cervical jugular vein, and indicated no abnormalities at gross and histomorphologic evaluation due to catheter placement.

Calculations of pump accuracy revealed diminished (>15%) accuracy after at 20-24 h post infusion, but a mean accuracy rate of within 5% up to 20 hours.

Question:

1. Why acclimatize animals to jackets and restraint systems prior to experimentation?
2. Why was such a long catheter inserted?
3. What is the benefit of wearing a pump in a jacket?
4. How long did pumps accurately dose drug?

Answers:

1. To decrease stress on animals, and avoid alteration in physiologic parameters due to stress that might adversely affect study data.
2. To permit drug infusion into the cervical jugular vein.
3. The jacket permitted normal activity of the animal to occur, and eliminated the need to use a more bulky and restrictive tether system that would require more restraint and more monitoring of the animal.
4. 20 hours.

Vascular microdissection, perfusion, and excision of the murine arterial tree for use in atherogenic disease investigations 47-52

The article is a descriptive document on the dissection, perfusion and removal of the intact aortic vasculature tree in C57BL/6J(^{apo -/-}) mice. Please see the diagrams in the article further describing the dissection process as well as the anatomy of the vascular tree.

Perfusion: The right atrium is excised to release the perfusate. A 25 gauge ¾ inch needle with the bevel removed was inserted into the mid left ventricle and left to continue perfusion for 3-5 minutes until all the blood drained from the right atrium and the perfusate (Dulbecco’s PBS) flowed clear. Satisfactory perfusion is indicated when the liver and spleen turn pale during the perfusion process. It was very important for the perfusion to be performed for a sufficient amount of time (5-minutes) otherwise blood can remain in the aorta after dissection.

Lesion assessment in the aortic root: Atherosclerosis in the aortic root is assessed by intimal area whereas lesions present in the descending aorta are assessed in intimal volume. In the aortic arch major lesions usually are present at the branching vessels and along the inner curvature of the aorta. Lesions are measured only along the inner curvature because the vessels normally show little variation between individual animals.

Proposed time assessment required per animal: The entire process is reported to take about 60 minutes per animal if performed by 2-3 persons dividing up the steps indicated in figure 1 and table 1.

Results: There were differences in the rate of lesion development along the aortic tree with more extensive lesions in the aortic arch than those in the descending thoracic aorta. Figure 4 demonstrates the increasing areas of atherosclerosis are associated with the duration of diet provision.

Discussion: The assessment of atherosclerotic lesions involved analyses of cross-sections taken from the aortic sinus (root) aortic arch, and the thoracic and abdominal aorta. ‘En face’ analysis is another method used to assess lesion formation and is suitable for analysis of the entire arterial tree. This method requires the removal of the arterial tree and commonly involves Sudan Black staining and computer-assisted image analysis to quantify the proportion of stained (diseased) and unstained (healthy) surface areas. En face lesion assessment is the method of choice for intervention studies involving large numbers of animals, as computer-assisted evaluation of the entire aorta is relatively quick (~1 hour/aorta) and allows comparison with results obtained from other species. A disadvantage of this method is that lesion volume cannot be determined and three-dimensional reconstruction of the lesions is not possible.

No questions provided.

Other tests of strength & coordination 53-54

Summary:
This article detailed the grip strength test and beam walking as methods to quantitatively measure strength, coordination, and locomotor ability in rodents. These tests are useful in the study of both spontaneous and induced musculoskeletal and nervous system lesions, and have been suggested as a component of behavioral tests used in phenotyping genetically modified mice.
 The grip strength test is used to measure forelimb strength in mice and rats. The basic test involves a triangular strain gauge fastenend to a platform. The rodent is lowered to allow grasping of the gauge with the forepaws, the rodent is then pulled by its tail in a horizontal direction. The gauge records the force at which the rodent detaches from the gauge. A variation of this test measures hindlimb strength additionally. A T-rod guage is placed behind the triangular strain gauge. As the rodent is pulled backwards, the forelimbs detach from the triangular gauge as before. With continued backwards traction, the hindlimbs come into contact with and grasp the T-rod gauge; the force is measured when the hindlimbs detach from the T-rod gauge.
 The beam walking test provides a measure of hind and forelimb coordination. The rodent is stimulated to cross a flat, elevated beam via a bright light and loud noise at the start. A trial is successful if the rodent reaches the dark goal box at the end of the beam within 10-60 sec. If the rodent falls from the beam, the time from start until falling is recorded. Variations of this test include simple balancing (without forward movement) and walking along a horizontal ladder.
 Animal welfare concerns were addressed by describing how to hold the tail during the grip strength test, and proper gripping techniques to prevent tail degloving. Also, documented was a description of the soft surface required underneath the balance beam.
 
Questions:
1) How much force should the strain gauge record for mice?
 A up to 50-100g
  B up to 100-250g
 C up to 250-500g
 D up to 500-1kg
2) How much force should the strain gauge record for rats?
 A up to 250-500kg
 B up to 500-1kg
 C up to 1kg
 D up to 1.5kg
3) What material is recommended for construction of the balance beam?
 A wood
 B plastic
 C metal
 D all the above
4) True or false:
 Monitoring rodent movement on a flat surface will give the same
coordination results as using the balance beam
Answers:
1) B
2) C
3) D
4) False. In may cases, animals with sensorimotor cortex lesions show
no deficits when walking on a flat surface, while these deficits are
readily apparent when a balance beam is used. Movement along the beam
requires the forelimbs and hindlimbs to move together.