Oocyte Collection
Amphibian oocytes are used for studies in molecular
biology, embryology and biochemistry. Stage I-VI oocytes
are obtained by surgical laparotomy. Multiple surgeries
on a single animal may be justified considering the
reduction in the total number of animals used over
the long term. However, the total number of animals
used must be considered relative to the pain or distress
experienced by an individual animal. 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 (sixth
surgery is terminal) per animal are acceptable. Adequate
recovery time must be allowed between laparotomies.
The investigator can alternate oocyte collection between
left and right ovaries and consider rotation of frogs
so that the interval between surgeries in any individual
is maximized. Recovery time of less than one month
should have prior IACUC.
Guidelines for xenopus oocyte collection
1. Surgery area
• Although a dedicated facility is not necessary,
the surgical area should be a dedicated space that
is easily sanitized, uncluttered and away from the
flow of traffic to minimize contamination from other
laboratory activities.
• The work surface must be thoroughly cleaned
with an approved hard surface disinfectant (e.g. Clidox®,
10% sodium hypochlorite,70% alcohol) prior to and after
each procedure.
2. Preparation of the surgical instruments
• Sterilization methods include steam, ethylene
oxide, chemical sterilants and dry heat. For a small
number of instruments a glass bead sterilizer can be
used.
• If 70% alcohol is used to disinfect instruments
between animals, they must be immersed for at least
30 minutes and thoroughly rinsed with sterile water
or air dried.
3. Preparation of the animal
• Frogs are anaesthetized by immersion in a 0.15%
solution of tricaine methane sulfonate (MS222).
• Sodium bicarbonate (NaHCO3) must be used to
buffer the solution to a neutral pH of 7.0 because
the unbuffered MS222 is acidic and poorly absorbed,
resulting in a prolonged induction time.
• A fresh solution of MS222 must be made daily.
4. Preparation of the surgical site
• Surgical scrub solutions are not appropriate
for use in amphibians due to the nature of their semi
permeable skin.
5. Preparation of the surgeon
• Wash hands prior to procedure with a surgical
scrub solution.
• Wear sterile gloves. Change gloves between
animals.
• Recommended attire is a clean, designated labcoat.
6. Surgical procedure
• Place the frog in dorsal recumbency.
• A 1-1.5 cm incision is made in the abdomen,
lateral to the midline.
• A piece of ovary containing several dozen oocytes
is resected.
• A single absorbable suture is used to appose
the ovarian tissue and control any haemorrhaging.
• The skin must be kept moist throughout the
surgery using sterile physiologic saline.
• After ensuring that there is no haemorrhaging
from the surgical site, the abdominal muscles and skin
layers can be sutured together as a single layer or
in two separate layers.
• Skin sutures must be non absorbable and removed
10-14 days post operatively.
• Recovery from the anaesthesia can take up to
an hour. To hasten the recovery process, rinse the
frog with fresh water, placing it in shallow water
as it will drown if unconscious. During this time the
frogs are checked for general appearance, muscle tone
and mobility every 15 minutes.
• Thereafter, the frogs are inspected twice a
week and any frogs with poorly healing incisions are
euthanized.
7.Intra-operative monitoring
• Anaesthesia will usually last between 10-20
minutes. Anaesthetized frogs lose muscle tone and become
inactive.
• Monitor the animal regularly, at least every
10 minutes during the procedure and until the animal
is fully ambulatory.
• Anaesthetic depth parameters:
Respiratory rate: regular and relaxed
Reflexes: absent withdrawal reflex
External stimuli: no response
• Analgesic depth parameter:
Withdrawal reflex: absent. Reassess every 10-15 minutes.
• For longer procedures, immerse the frog in
a weaker solution of MS222 or drip the dilute solution
directly onto the skin. Hypothermia alone is inadequate
for maintaining a surgical plane of anaesthesia.
Template
Frogs are anesthetized by immersion in .15% MS222
sodium bicarbonate buffered solution.The frog is placed
in dorsal recumbency. A 1-1.5 cm incision is made in
the abdomen, lateral to the midline. A piece of ovary
containing several dozen oocytes is resected. A single
absorbable suture is used to appose the ovarian tissue
and control any haemorrhaging. The skin is kept moist
throughout the surgery using sterile physiologic saline.
After ensuring that there is no haemorrhaging from
the surgical site, the abdominal muscles and skin layers
are sutured together as a single layer using non absorbable
sutures. Sutures are removed 10-14 days post operatively.
Animals are recovered in a separate tank. The frong
is rinsed with fresh water and placed in shallow water
to hasten the recovery process. During the recovery
period frogs are checked for general appearance, muscle
tone and mobility every 15 minutes.
References
Report of the Subcommittee on Amphibian Standards.
ILAR, National Research council. National Academy of
Sciences , Washington D.C., 1974.
Hitt, Nancy D.: "Basics of Amphibian Medicine". Presented
at 44th Annual AALAS Meeting, Nashville, TN., Nov.
18, 1993.
Goodwin, Bradford S.: "Amphibians in the Lab". Presented
at 44th Annual AALAS Meeting, Nashville, Tenn., Nov.
18, 1993.
McGill University Research Grants Office Standard
Operating Procedures: http://www.mcgill.ca/rgo/animal/procedures
Office of Animal Care and Use ARAC Guidelines: http://oacu.od.nih.gov/ARAC/index.htm
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