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XENOPUS
OOCYTE HARVESTING
Scope
Amphibian
oocytes are used for studies in molecular biology, embryology, and
biochemistry. Stage I-VI oocytes are obtained by surgical laparotomy.
Federal regulations require that multiple surgeries on a single
animal must be scientifically justified. The reduction in the total
numbers of animals used over the long term is considered appropriate
justification. However, the total number of animals used must be
considered relative to the pain or distress experienced by an individual
animal. This policy describes considerations that should be used
in deciding laparotomy limits and procedures to alleviate pain and
distress.
Procedure
The
total number of laparotomies should be limited and will depend on
the condition of the animal and quality of the oocytes as well as
the life span of the animal and the duration of egg production.
Up to five recovery surgeries (the 6 th would be terminal)
per animal are acceptable. Additional survival surgeries must have
approval of the IACUC.
Surgeries
should be performed by trained personnel using an appropriate anesthetic
such as tricaine methane sulfonate (MS 222). Recommended concentrations
of MS222 are 0.5 - 2 g/L, buffered with sodium bicarbonate at 0.42
- 1.05 g/L. A solution of 1 g/L MS222 buffered with 0.7 g/L sodium
bicarbonate provides ample anesthesia for oocyte retrieval. Surgeries
should be done as aseptically as possible including the use of sterilized
instruments, gloves and mask. Harvests should take less than 30
minutes.
Upon
completion of the surgery, remove the animal from anesthetic, rinse
in dechlorinated water, and place it in a shallow container of dechlorinated
water. To reduce stress, the water for induction and recovery from
anesthetic can be from the container that originally housed the
animal. To prevent drowning, monitor the frog during induction and
recovery from anesthetic.
Analgesics
are permissible, but the animal must be monitored to verify the
analgesic does not impair the animals motor functions required for
swimming. Useful analgesics include morphine (10 mg/Kg intracoelomically
every 12 hr), flunixin meglumine (25 mg/Kg intracoelomically every
4 hr), butorphanol (25 mg/Kg intracoelomically every 12 hr) and
xylazine ( 10 mg/kg intracoelomically every 12 - 24 hr).
Single
housing or small group housing for several days after surgery should
be considered as part of the post surgical care of laparotomized
animals. Frogs should be monitored daily during this period for
appetite as well as any complications such as dehiscence or infection.
Such adverse events would be reasons for euthanasia.
Adequate
recovery time should be allowed between laparotomies. At least a
month should pass between surgeries on frogs. Alternating oocyte
collection between left and right ovaries and rotation of frogs
so the time between surgeries is maximized should be considered.
Recovery time of less than one month should have approval of the
IACUC.
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