Contemporary Topics 43(2)

Use of CO2/O2 Anesthesia in the Collection of Samples for Serum Corticosterone Analysis from Fischer 344 Rats.  Pages 8-12
Summary:  
Serum corticosterone is used as a biomarker of stress in laboratory rats for evaluation of the response to experimental stressors. The purpose of the study was to evaluate the use of CO2/O2 anesthesia, with two different handling techniques, in serum sample collection procedures in male and female Fischer 344 rats.  The study mimicked the initiation of a toxicity study after the fourth dose of sham administration of 0.5% aqueous methylcellulose. Rats were divided into acclimated and non-acclimated groups of male and female Fischer 344 rats.  Non-acclimated animals were moved to a separate anesthesia chamber while acclimated animals remained in their home cages. Blood was collected via retro-orbital plexus at least two hours after methylcellulose dosing. During this time room noise was kept to a minimum since Fischer F344 rats are sensitive to stress. 
Male and female acclimated rats had lower serum corticosterone levels, approximately 25% and 50% respective to the non-acclimated animals. These results were compared to methods often cited in the literature including decapitation, tail cutting, exsanguination after pentobarbital anesthesia, cervical dislocation and unspecified techniques. This method will eliminate all the disadvantages of the cited methods. Decapitation necessitates increased costs, extra animals and loss of observations in individual animal clinical pathology between bleeds. Tail cutting requires extra animals and is not likely to produce sufficient volume of blood.
 	This reported method of blood collection allows rapid recovery from anesthesia and will eliminate the induction of potential chemical interaction which can occur if inhalation or injectable anesthesia agents are used. The authors hypothesized that this technique is transferable to other rat strains. This study indicates a reliable baseline level of corticosterone in male and female Fischer 344 rats. This refinement of this technique will decrease animal stress and animal numbers and allow reuse of animals for future bleeds. These improvements in animal use can lead to better corticosterone data and lower variability on a given toxicology study.
Questions:
1.    How much lower was the corticosterone serum levels in acclimated male and female Fischer 344 rats versus non-acclimated male and female Fischer 344 rats?
2.    What two advantages does this method of serum collection provide in toxicological studies?
3.    What are three methods of serum collection previously stated in the literature that has greater variability?
Answers:
1.    Acclimated Fischer 344 rats corticosterone levels were 25% and 50%, respectively, to the non-acclimated rats.
2.    Two major advantages noted in this study were consistent corticosterone serum baseline data and decreased stress levels in study animals.
3.    Decapitation, tail cut and cervical dislocation are the three methods of serum collection stated previously in the literature. 
 
Effect of Activation Regimens for Rat Oocytes on Full-term Development after Round Spermatid Injection. Pages 13-15
 
Summary:  This study compares two methods for fusion of mouse round spermatids with oocytes for production of full-term mice.  Round spermatids are the youngest haploid spermatogenic cells.  Elongated spermatids and spermatozoa contain sperm-borne oocyte-activating factor (SOAF), but not round spermatids.  Metaphase promoting factor (MPF) in oocytes causes premature chromosome condensation in injected spermatid nuclei.  During the physiological process of mammalian fertilization, there are oscillations in Ca++ that cause a subsequent decrease in MPF activity.  Chemicals, such as ionophore A23187, puromycin, 6-dimethylaminopurine (6-DMAP) and SrCl2 have been used for parthenogenetic acativation and subsequent development of rodent oocytes.  A direct current (DC) pulse causes a single increase in the intracellular Ca++ concentration of an oocyte, while SrCl2 causes multiple Ca++ increases or oscillations.  In this study, use of SrCl2 for activation of oocytes was compared  with use of 6-DMAP plus two direct current pulses (DC+6-DMAP).  While the proportions of oocytes surviving injection and exhibiting pronuclear formation were comparable between the SrCl2 group and the DC+6-DMAP, significantly more two-cell embryos developed to morulae/blastocysts and live offspring in the DC+6-DMAP group (36.1 and 14.6%) compared with the SrCl2 group (12.3 and 6.1%).
No questions or answers 
Nylon ear tags for individual identification of guinea pigs.  Page 16-20

Summary:  This article describes the use of nylon ear tags as an alternative to the traditional metal ear tags for individual identification of guinea pigs (Cavia porcellus).  Using an application gun similar to that used for attaching price tags to clothing merchandise, nylon tags were placed as close to the center of the pinna as possible.  Metal tags were placed either at the center of the ear (resulting in folded pinna), or near the edge of the pinna.   
Animals with metal tags: saw cartilaginous nodules, scratch and pull at tags, proliferative changes, neoplastic lesions, chronic inflammation and infection, redness and swelling during entire observation period.  On histopath, epidermal ulceration, granulomatous inflammation, hypertrophied keratinocytes with no evidence of epidermal regeneration or healing.
Pros of using Nylong tags:
1.	Loss of nylon tag over the 8-week study period was significantly less.
2.	Replacement nylon tag may be placed in the same ear/hole.
3.	No local bacterial infection or persistent local irritation.
4.	Multicolor.
5.	Light.
6.	Economical.
Cons: 
1.	May be unsuitable for group-housed animals (avoid obliteration of serial numbers and subsequent loss of nylon tags due to aggressive behaviors, such as fighting or chewing of the ear.
Conclusion: Nylon ear tags are more suitable method for guinea pig identification than metal tags due to low incidence of loss and minimal tissue damage around the tagged site.
Questions
1.	Name at least three identification options for guinea pigs.
2.	What are some side effects associated with metal ear tags in guinea pigs? 
Answers
1.	Electronic microchips, metal ear tags, nylon ear tags, tattoo, marking/coding.
2.	Lose tag, cartilaginous nodules, scratch and pull at tags, proliferative changes, neoplastic lesions, chronic inflammation and infection, redness and swelling during observation period.

Activated Clotting Time and Heparin Administration in Sprague-Dawley Rats and Syrian Golden Hamsters.  Page 21-24

Summary:  Activated clotting times (ACT) were determined in rats and hamsters in response to different heparin doses in both species using a Medtronic HemoTec ACT measurement system.  The mean ACT for non-heparinized rats was 48.0 sec, for hamsters it was 42.5 sec.  (for humans it ranges between 84 and 107 sec.) Heparin was given IV at doses from 500-1200 IU for rats and 300-1000 IU for hamsters.the upper dose for each animal respectively allowed for an ACT >480 sec.  When blood was collected at 30 minutes post heparin administration.
	Important points:  ACT is used when assessing patients treated with anti-coagulants and reversal agents for cardiopulmonary bypass (CPB) surgery.
	In humans, the goal of anticoagulation therapy for CPB is an ACT >480 sec. for at least 30 minutes.
	Heparin is the most widely used anti-coagulant. it is obtained from bovine lung or porcine gut.
	Heparin creates a complex with antithrombin III, inactivating thrombin and deactivating factors X, XII, XI, and IX.
	Methods of measurement of ACT generally rely on a clotting activator such as Kaolin or celite.
	They reference a paper indicating that circadian variation has an effect on clotting times and coagulation factors.
Conclusion:  Rats and Hamsters have a short ACT and low sensitivity to Heparin when compared to humans.  Rats and hamsters like humans, have a wide variation in ACT values.
Questions:
1)  Heparin is an anionic heterogeneous sulfated glycosaminoglycan found naturally in:
a.  red blood cells
b.  platelets
c.  mast cells
d.  cartilage
2)  Calcium Heparin is extracted from:
a.  porcine intestinal mucosa
b.  bovine lung
c.  snake venom
d.  kelp
3)  Heparin inactivates:
a.  intrinsic coagulation pathway
b.  extrinsic coagulation pathway
c.  all of the above
d.  none of the above
Answers:
1) c.
2) a.  (porcine intestine contains both sodium and calcium heparin, bovine lung contains only sodium heparin)
3) c.  at low doses heparin binds antithrombin III, inactivating factor Xa, and preventing conversion of prothrombin to thrombin.  At higher doses, heparin inactivates thrombin and blocks the conversion of fibrinogen to fibrin.  it also inactivates factors IX, X, XI, XII when bound to antithrombin III.

Improving the Results of Small-Bore Vascular Graft Testing in
Rabbits:  A Technical Primer. Pages 25-27.
 
Summary:  The rabbit is an ideal model for experimental small-diameter vascular graft studies.  The thrombogenic properties of rabbit blood are similar to those of humans, as are the thromboplastic and fibrinolytic properties of rabbit arteries.  The rabbit also develops intimal hyperplasia as a result of vascular injury, which is a recognized mode of failure for small-bore grafts implanted in humans.  The authors used the rabbit abdominal aorta model to evaluate the behavior of small-diameter bovine-derived grafts in the arterial circulation.  They modified the model by adding hypothermia (32-34 C) and supplemented Lactated Ringers solution after several cases of postoperative paraplegia and death thought to be related to normothermic ischemic injury of the spinal chord.  The mild hypothermia was achieved by external and internal cooling of the animal by the use of ice bags, cool intravenous Lactated Ringers, and saline slush.  Neuroprotective effects of agents contained in the Lactated Ringers solution (sodium bicarbonate, mannitol, and dexamethasone sodium phosphate) were taken into account.  A 3- to 4-mm internal diameter bovine-derived conduit was implanted into the infrarenal abdominal aortic position.  End to end anastomoses were performed at both ends.  The authors achieved a 100 % immediate patency rate and low incidence of postoperative complications in the study group.  They previously determined that a cross-clamp time exceeding 30 min under normothermic or mild hypothermic conditions contributed to a high rate of postoperative paralysis.  They obtained their favorable results because of improvements in surgical technique, use of mild hypothermia, and limiting of ischemic periods to <30 min as well as to the perioperative administration of agents with metabolic, rheologic, and neuroprotective properties.   
Questions:      
1.  What makes the rabbit an attractive model for the in vivo evaluation of small-bore vascular grafts? 
2.  T/F:  Flunixin is COX 1 and 2 inhibitor 
Answers:  
1.  The similarity of the thrombogenic properties of rabbit blood, thromboplastic and fibrinolytic properties of rabbit arteries to those of humans. 
2.  T 

Cytologic Assessment of the vaginal Epithelium in the Common Marmoset (Callithrix jacchus): A Preliminary New Approach to Reproductive Screening. pages 28-31

The common marmoset does not display any overt signs of estrus.  Analysis of plasma progesterone levels is currently the most reliable and readily used indicator of ovulation occurrence.  Vaginal cytology is useful in the measure of cyclic hormonal events and correlates well with endometrial biopsies and urinary estrogen profiles. The authors wanted to determine if vaginal cytology correlates to circulating plasma hormones in the common marmoset.  If a correlation exists, vaginal cytology may be a useful alternative to blood sampling for ovulation detection.

Six female common marmosets were used in this study.  Animals underwent vaginal swabbing and blood collection at least twice weekly for 7 weeks.  Blood was collected for plasma progesterone determinations.  Vaginal swabs were rolled onto clean slides, stained with Wright Giemsa.  100 cells were examined for nuclear morphology.  Maturation of the vaginal epithelium was determined by the karyopyknotic index (KPI) which is the ratio of superficial (mature) squamous epithelial cells to intermediate (immature) squamous epithelial cells.

In three of the consistently cycling females the increased in KPI did correlate to rising  plasma progesterone levels.  The other three females had irregular estrus cycles and the irregularity of their plasma progesterone levels correlated with inconsistent KPIs.

Questions
1.  Genus and species of the common marmoset
2.  What is the KPI?
3.  What agent was used to terminate pregnancies in this study?

Answers
1.  Callithrix jacchus
2.  The ratio of superficial (mature) squamous epithelial cells to intermediate (immature) squamous epithelial cells.
3.  Cloprostenol sodium

Gill Trematodes (flukes) in Wild Caught Killifish ( Fundulus heteroclitus).  Page 32-34 
Sixty-two wild caught killifish were group-housed in aquaria upon arrival at the research institution. One day after arrival two fish were found dead, the next day another fish was found dead, as well as two more that appeared inactive. The water in the two aquaria was tested for pH, salinity, nitrate, nitrite, and ammonia levels and all values were within acceptable levels. The two fish that were inactive were euthanized with an anesthetic overdose of NaOH buffered MS222 for diagnostic evaluation and necropsy. 
Gross external exam revealed multiple, scattered, ulcerated dorsolateral skin lesion. Skin lesions were cultured on 6 different types of agar plates. No gross lesions were present on the gills, although the gills did appear red and inflamed. No other lesions were found. Saline wet mounts were made of the skin lesions and of the gills. No abnormalities were noted in the wet mounts of the skin. In the wet mounts of the gill multiple, encysted, motile organisms located in the lamellae of the gills were observed. 
A diagnosis of trematodiasis was made. Due to the location and morphologic characteristics the encapsulated parasites were diagnosed as metacercariae of a digenetic trematode. No pathogenic bacteria were cultured from  the skin. 
Trematodes are divided into two subclasses, monogenetic and digenetic. 
Monogenetic complete their entire development in one host. Digenetic trematodes require at least one intermediate host in order to mature. 
On page 33 there is a nice color chart showing how in this case the killifish could be either an intermediate host or a final host. 
Life cycle of Digenetic trematodes 
1. eggs, large and operculated, pass in the feces of the definitive host(fish, bird, mammal) 
2. a ciliated, free-swimming miracidium hatches in 21 days after the eggs enter the water. 
3. The miracidium infects the intermediate host, usually a snail. 
4. Asexual reproduction takes place and sporocysts are formed 
5. After 6 weeks free-swimming cercariae are released into the water from the first intermediate host 
6. The released cercariae penetrate or are ingested by either the definitive host or a second intermediate host. 
7. If the cercariae infect a second intermediate host then they migrate to the host's target tissue and the cercariae differentiate into metacercariae. 
8. The metacercariae encyst in various tissues, depending on the type of digenean. 
Usually larval digenetic trematodes do more tissue damage to the host than the adult digenetic trematodes. Two that are common are Diplostomomum and Neascus.  The adult stages of the digeneans must be seen histologically to determine a species, and in this case only the larval forms were seen so it was not possible to speciate the trematode. 
The combination of  recent capture, stress of transport may have made the fish more susceptible to the effects of the parasites. It is possible to treat fish with Praziquantal by prolonged immersion ( 24 hours) 2 to 10 mg  praziquantal/ liter 
to reduce metacercariae numbers. 
Questions:
 How many subclasses of trematodes are there? 
1.two 
2.four 
3. six 
4. 8 
What did they use to euthanize the fish? 
1. M 50/50 
2. MS 222 
3 T-61 
4. guillotine 
Wet mounts of of gills stained with methylene blue showed? 
1. multiple encysted motile organisms in the gills 
2. pathogenic bacteria in the skin 
3. an operculated egg in the gill lamellae 
4.sporocysts surrounded by giant cells   
 Answers 1 Two, monogenetic, and digenetic 
               2. MS-222 
               1 Multiple encysted motile organisms in the gills 

Long-term Results of Dietary Fenbendazole to Eradicate Syphacia muris from Rat Colonies.  Pages 35-36
	This paper summarizes the use of Fenbendazole treated rodent feed as a treatment for the pinworm, Syphacia muris, in rats and demonstrates the importance of compliance with the treatment SOP in achieving the treatment goal.
	Fenbendazole medicated feed (150 ppm) was fed on alternate weeks for five rounds of administrations without environmental decontamination.  Follow-up tape tests of the perianal skin were collected at least quarterly for 47 months after the treatment to evaluate efficacy of treatment.  Two populations were treated - one managed by the central animal facility personnel and one managed by the researchers in the unit.  Both populations were supposed to be treated identically.  The treatment was very effective in the first population over a total time of 54 months, however in the satellite facility with research management a new outbreak was found 17 months after the initial treatment while animal facility animals on the same floor remained negative.
	Several reasons for the latter ineffective results are discussed.  Only one follow-up test at six months was conducted before the 17 month test which came up positive.  Sentinel animals were not collected as scheduled due to a short staffing problem.  The feed might not have been uniformly constituted (unlikely since the larger colony treatment was successful).  More likely a few cages were not treated during each cycle of treatment, allowing a very low number of parasites to survive and gradually reinfect the colony.   A second round of treatment with animal care personnel working with the research personnel was successful.
	The point is made that intensive follow-up testing is essential following treatment, in order to detect treatment failures in a timely manner.  
Questions:
1.  The parasite Syphacia muris found in these rats is commonly known as a ________________.  In mice the analogous parasite most often found is ____________________.
2.  Fenbendazole is a _________________________.
	A.  benzodiazepine
	B.  benzimadazole
	C.  pyrethroid
3.  The eggs of Syphacia spp pinworms are shaped like a _______________, while those of Aspiculuris are shaped like ____________________.
4.  The major factor believed to account for the failure of treatment in the satellite facility in this report was ________________.  Why might this have seemed justified to the team doing the treatments?
Answers:
1.  Pinworm, Syphacia obvelata
2.  B
3.  banana,  oval - (similar to the hook-worm egg of dogs)
4.  incomplete treatment of the full colony or inconsistent compliance.  The research team may have been concerned that the drug could confound research results and thus may have skipped treatment on some animals during one or more treatment cycles.

Proactive Compliance- the Team Approach to Revitalizing Primate Enrichment.  Page 37-38.
The Division of Laboratory Animal Resources (DLAR) of the University of Pittsburgh instituted the 1999 draft policy to the Animal Welfare Act entitled “ The Final report on Environmental Enrichment to Promote the Psychological Well being of Non Human Primates.” This policy has yet to be adopted and implemented. 
Implementation of a non human primate enrichment program reflecting the guidelines described in the draft policy was accomplished by involving all individuals working with non human primates, namely the research staff, animal care and veterinary personnel, the local IACUC. A position for an environmental specialist was also created.  
The draft policy clearly states that there are five critical elements, known collectively as “The Critical Element Concept” necessary to promote the psychological well being of non human primates. These are 1) social grouping 2) social needs of infants 3) structure and substrate 4) foraging opportunities 5) manipulanda. 
In addition to reviewing their existing enrichment program and incorporating whenever possible all five critical elements, the DLAR also established documentation for enrichment at several levels: room level, individual animals displaying problematic behaviours, individual animals with other medical conditions, group housing attempts, novel enrichment strategies and enrichment provided by the research staff. Behavioural records are now a standard component of the medical record. 
Finally, the IACUC revised the review process for protocols involving non human primates. For instance, investigators using singly housed animals must not only provide scientific justification for the lack of pair/social housing, but also submit to the IACUC an enrichment plan in order to compensate for the missing critical element. All protocols using non human primates must now be in compliance with their new Enrichment Plan. 

Title: The Elevated Plus-Maze. Pages 39-40.
Summary: The elevated plus-maze (EPM) is a popular behavioral test used in the study of fear and anxiety. In the case of mice and rats, freezing behavior and avoidance of brightly lit areas are used as measures to assess anxiety and fear in behavioral tests such as the open field test. The EPM is based on the conflict between the natural tendency of mice and rats to explore a novel environment balanced against the aversive and anxiety producing properties of entering a brightly-lit open area. Animals are provided the opportunity to explore a novel environment consisting of an elevated maze with two intersecting arms: a closed arm with tall walls creating an alley that intersects with an open arm without walls that presents an unprotected walking surface. As one would expect, normal exploratory behavior is generally concentrated in the closed arms. Administration of anxiety-provoking compounds leads to a decrease of time spent in the open areas while anxiolytic compounds tend to reduce the aversion to the open arms and increase the exploratory time spent in the open arms. The EPM is commonly used by the pharmaceutical industry as a screening tool for compounds with anxiolytic potential. In addition, the EPM has been proposed as one of a number of behavioral tests for phenotyping genetically modified mice. 
The basic test is performed by placing the mouse or rat in the center of the maze, typically facing a closed arm. Upon release, the animal is free to explore the apparatus for a set period of time, usually five minutes. Two measures or indices of anxiety are then recorded: 1) the number of entries into each arm, and 2) the time spent in each arm. With the use of computer software, video recordings from test sessions can also provide data on total distance traveled. 
The elevated zero-maze (EZM) is a modification of the EPM. Rather than an intersection of light and dark arms of a maze, the EZM provides alternating light and dark areas in a continuous circle. It is theorized that a continuous circuit of light and dark sections, which avoids an intersection between the open and closed arms, may reduce the ambiguity in scoring. For example, in the EPM an animal may stay in the start area or repeatedly return to the start area rather than fully enter an arm. As the EZM has no start location, animal activity is simpler to score. 
Questions:
1.                   True/False - Fear and anxiety produce freezing behavior in many species.
2.                   What is the definition of an anxiolytic compound?
3.                   How are observations typically performed when conducting the EPM?  
4.                   What is an animal welfare consideration noted in the article related to the use of this equipment?
Answers:
1.                   True
2.                   A compound that relieves anxiety.
3.                   Observations are typically performed by video recording the test sessions, with the video camera mounted     directly above the maze. 
4.         Since behavioral testing apparatuses are often difficult or impossible to disassemble and thoroughly disinfect due to their complexity or construction materials, the use of shared equipment involving animals of different health statuses may expose clean animals to unwanted pathogens.