2.0 RESPONSIBILITY: It is responsibility of Principal Investigator (PI), Animal House In-Charge and Animal House Personals.
3.0 PROCEDURE:
3.1 Acceptable Blood Collection Procedures- Pre-Anesthetic Fasting
Rat and mice do not vomit, and as they also possess a high metabolic rate pre-anesthetic fasting is not recommended. Therefore, water is not withheld.
Volume less than 100µl:
Method 1: Tail tip – Rat and mice
a) Blood sample was performed on un-anaesthetized animal if mouse was less than three (3) weeks of age and rat less than two (2) weeks of age. Anesthesia was required if mouse was found older than 2 weeks.
b) Animal was placed in an approved animal restrainer, in order to vasodilate the blood vessels of the tail. The tail of animal was carefully warmed with a warm air gun for 2-3 minutes or the tail was immersed in warm water. The animal was placed on a clean work surface.
c) The tail of animal was held tightly at base of the tail.
d) Sing heparinized 30 G needle and 1 ml syringe blood were collected for lateral tail vein.
e) After collection of recommended quantity of blood, gentle digital pressure was applied with a clean gauze pad for 30-45 second to site to stop blood flow and prevent hematoma formulation.
B) Terminal Methods:
Method 1: Retro-orbital-Rat and Mice
a) The animal was anaesthetized with an appropriate quantity of anesthesia prior to commencement. Signs that indicate a satisfactory plane of anesthesia include a lack of response to toe pinch and respiration that should be relaxed and regular.
b) The anaesthetized rat was laid on its back with the head facing the surgeon.
c) The head was fixed with the thumb and the forefinger, tightening the skin over the side of the face. This retracts the eyelids and protracts the globe.
d) A microhematocrit (capillary) tube was inserted into the canthus and was twist to break through the bulbar conjunctiva. The tube was directed towards the medical aspect of the bony orbit.
e) The blood was collected into tube and the sealed. This was continued till the blood does not ooze. If there was difficulty then the eye was changed.
f) The animal was euthanized when blood collection was completed.
Method 2: Cardiac puncture-Rats and Mice
a) The animal was anaesthetized at an appropriate quantity of anesthesia prior to commencement. Signs that indicate a satisfactory plane of anaesthesia included a lack of response to toe pinch and respiration that were relaxed and regular.
b) A 21 gauge needle was fitted to 10 ml Syringe (rat, guinea pigs) or a 23 gauge needle to a 3ml syringe (mice).
c) There are 2 approaches:
(i) Sternal
(i.i) The animal was placed in dorsal recumbence.
(i.ii) The chest was cleaned of dirt and debris.
(i.iii) The heart beat was palpitated and xiphoid process.
(i.iv) A three ml syringe was fitted to a 21 gauge needle (rat) at an angle where the xiphoid process meets the last ribs.
(i.v) A light negative pressure was applied and blood was withdrawn.
(i.vi) The animal was euthanized when the blood collection was completed.
(ii) Lateral
(ii.i) An anomal was placed with its right side down.
(ii.ii) The chest was cleaned of dirt and debris.
(ii.iii) The forelimb was fully flexed and a note should be made of the place where the elbow meets the chest.
(ii.iv) A three ml syringe with a 21gauge needle was inserted (rat, guinea pigs) at the place where the xiphoid process meets the last ribs.
(ii.v) A light negative pressure was applied and the blood was withdrawn.
(ii.vi) The animal was euthanized when blood collection was completed.
3.2 Acceptable Collection Volume For All Species:
3.2.1 Single Collection:
3.2.1.1 Up to 10% of circulating blood volume with fluid replacement in normal, healthy animals.
3.2.1.2 Blood withdrawal and fluid replacement must be performed slowly (over 1-2 minutes) and at a steady rate.
3.2.1.3 Fluid replacement: Sterile, warm physiologic saline equal to the volume of blood collected administered intravenously or intra-peritoneally.
3.2.1.4 Can be repeated only in 3-4 weeks (time required for erythrocyte and blood volume regeneration).
Maximal acceptable volume in a single Bleed (ml) = 10% * circulating blood volume (ml/kg) X weight(kg)
3.2.2 Circulating blood volume:
3.2.2.1 Rat-60 ml/ Kg (e.g. 15 ml for 250 gram rat)
3.2.2.2 Mouse-80 ml/kg (e.g. 2ml for 25 gram mouse)
3.2.3 Repetitive blood collection at short intervals:
3.2.4.1 Up to total of 1% of circulating blood volume should be removed every 24 hours in normal, healthy animals.
3.2.4.2 Blood withdrawal and fluid replacement must be performed slowly (over 1-2 minutes) and at a steady rate.
3.2.5 Fluid replacement: Sterile, warm physiologic saline equal to the volume of blood collected should be administered intraperitoneally.
4.0 INFORMATION REQUIRED:
1. Species: _____________________________
Age: _____________________________
Approximate Weight: __________________
2. Route: Put an X next to the one to be employed:
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Survival procedures: |
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1.
Lateral saphenous vein- preferred |
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2.
Tail artery |
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3.
Jugular Vein (percutaneous) |
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4.
Tail tip |
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Terminal procedures: |
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10. Other, specify:___ |
a.
Volume per collection: ____________
b.
Frequency
of collection: ___________
If
repeated how frequent (interval)? ______
Replacement volume: NO
YES , Specify:
____
c. 
Are there change to this SOP indicate
in the AUP form? YES NO
If yes, specify changes: _____
d. Pl
Signature: _______________________ Date:______________
5.0 PRECAUTIONS:
4.1 The animals need to be handles carefully.
4.2 The readings should be noted down carefully
4.3 The amount of anesthesia administered should be thoroughly checked.
5.0 REFERENCES:
5.1 Dardai E, Heavner JE. Respiratory and cardiovascular effects of halothane, isoflurane and enflurane delivered via a Jackson-Rees breathing system in temperature controlled and uncontrolled rats. Meth Find Exptl Clin Pharmacol. 1987;9:717–720. [PubMed]
5.2 Devitt CM, Cox RE, Hailey JJ. Duration, complications, stress, and pain of open ovariohysterectomy versus a simple method of laparoscopic-assisted ovariohysterectomy in dogs. JAVMA. 2005;227:921–927. [PubMed]
5.3 DOI [Department of the Interior] Revision of Regulations Implementing the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (50 CFR Parts 10, 13, 17, and 23) 2007. [accessed April 8, 2010]. Available at www.fws.gov/policy/library/2007/07-3960.pdf.
5.4 Dubner R. Research on pain mechanisms in animals. JAVMA. 1987;191:1273–1276. [PubMed]
5.5 FASS [Federation of Animal Science Societies] Transport, Guide for the Care and Use of Agricultural Animals in Research and Teaching. 3rd ed. Champlain, IL: FASS; 2010. p. 54.
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