Chapter 29

Description of Ovariectomy Protocol in Mice

Vanessa R. Souza, Eduardo Mendes, Mateus Casaro,
Ana Tada F. B. Antiorio, Fernando A. Oliveira, and Caroline M. Ferreira

Abstract
Estrogen and ovarian function decline are relevant characteristics of menopause period. Numerous physio-
logical, metabolic and immunological alterations in the female body occur in the menopause period and
some of these changes remain uncertain. The animal model that mimics menopause phase is an important
approach to better comprehend the biological process involved in this period of women life. Ovariectomy is
a procedure where ovaries are surgically excised and have been a valuable tool for understanding estrogen
deficiency through animal experiments. Despite the diversity of ovariectomy protocols, the aim of this
chapter is to provide a comprehensive guideline in performing ovariectomy in mice. Furthermore, isoflur-
ane anesthesia system, postoperative care and surgery success evaluation will be described. We highlight that
all procedures must be carried out by a qualified and trained professional, respecting ethical and safety
principles.

Key words Ovariectomy, Menopause, Ovaries, Female reproductive tract

1 Introduction

The female reproductive tract has two major components: (1) the
ovaries which produce the mature ovum and secrete hormones such
as estrogens, and (2) the ductal system which transports the ovule
[1]. Menopause is the reduction of ovarian function and is asso-
ciated with a decrease in estrogen secretion, cessation of the men-
strual period and loss of reproductive function. Women’s health is
usually affected by the natural decline of estrogen levels in meno-
pause presenting several physiological [2], metabolic, and immu-
nological changes [3]. Menopause is a later life associated
condition. By the year 2030, more than 1.2 billion women will be
50 years or older worldwide [4]. Animal models are pivotal to
elucidate menopausal issues and consequently to improve quality
of life.

Vanessa R. Souza and Eduardo Mendes contributed equally to this work.

Paul C. Guest (ed.), Pre-Clinical Models: Techniques and Protocols, Methods in Molecular Biology, vol. 1916,
https://doi.org/10.1007/978-1-4939-8994-2_29, © Springer Science+Business Media, LLC, part of Springer Nature 2019

303
304 Vanessa R. Souza et al.

Fig. 1 Female mice reproductive tract major components: ovaries and ductal system

Ovariectomy, that is surgical excision of the ovaries, is a model

of menopause which has improved the knowledge of ovarian hor-
mone action in many organs systems and menopause-associated
events including osteoporosis [5], cardiometabolic disorders [6],
and inflammatory diseases [7]. The mouse ovaries are located at the
posterolateral poles of the kidneys, each attached by the mesovar-
ium to the dorsal body wall of the abdominal cavity [1] (Fig. 1). An
ovary in 8-week-old mice that weigh approximately 5 to 7 mg but
can change according to the cycle [8]. Ovariectomy can be per-
formed accessing ovaries by (1) a single surgical incision, (2) double
dorsolateral incisions, and (3) a single ventral transverse incision on
the middle part of the abdomen [5]. Double dorsolateral incisions
present some advantages such as facilitated access to ovaries and
rapid recovery after surgical procedures, according to our research
group experience.
The method presented here provides a detailed description of
the ovariectomy surgical procedure (double dorsolateral incisions)
in 8-week-old mice. For the surgery procedures, the isoflurane
anesthesia system was chosen due to the rapid animal recovery
from anesthesia and it will be described later.

2 Materials

2.1 Equipment 1. Veterinary anesthesia system (Fig. 2).

2. Sterile surgery material (scissors, curved tip scissors, tweezers,
tweezers hemostatic, and needle holder).
 Ovariectomy Protocol 305

Fig. 2 Veterinary anesthesia system. (1) Digital flowmeter for O2. (2) Isoflurane
concentrator: this part of the device lets the liquid isoflurane evaporate in the air.
(3) Valve to open and close isoflurane flow. (4) The connection of nose cone

3. Needle wire 4/0 45 cm, 17.5 mm.

4. Gauze.
5. Analytical balance.
6. Pasteur pipette.
7. Sterile line.
8. Electric razor.
9. Heating pad.
10. Glass slide.
11. Microscope slide cover.
12. Micropipette.
13. 10 μL sterile tip.
14. Optical microscope.

2.2 Reagents 1. Isoflurane (100%; 1 mg/mL) inhalant solution.

2. Acetaminophen solution (100 mg/mL).
3. Chlorhexidine solution (1%).
4. Tramadol (5 mg/kg).
5. Sterile phosphate buffered saline (PBS).
6. Alcohol 70%.
7. Crystal violet stain solution (1 mg/mL) in 30% acetic acid.
306 Vanessa R. Souza et al.

3 Methods (See Note 1)

3.1 Anesthesia 1. Open the isoflurane valve (see Note 2).

2. Place the animal inside the isoflurane chamber (Fig. 3) until it is
immobilized (see Note 3).
3. After immobilization, place the animal in a prone position
(Fig. 4), connected with isoflurane nose cone (see Note 4).
4. Apply eye lubricant (sterilized PBS) frequently as needed with a
Pasteur pipette in order to prevent corneal drying and damage.

3.2 Surgical 1. Shave hair off the flank area (between the last rib and above the
Procedure pelvis).
2. Disinfect skin with chlorhexidine solution.
3. Work in an aseptic area (see Note 5).
4. Make an incision in the skin on the right side (Fig. 5).
5. Separate the musculature using curved tip scissors.
6. Carefully pull the ovarian fat pad out of the incision (see Note
6).
7. Using the tweezers hemostatic, tightly clamp the region below
the ovary.
8. Using a sterile thread, make two knots in order to delimitate
the area to be removed (Fig. 6).
9. Remove the ovary (see Note 7).

Fig. 3 Isoflurane chamber. Isoflurane is administered in conjunction with air

and/or pure oxygen: 3.5–4.5% for anesthesia induction in mice
 Ovariectomy Protocol 307

Fig. 4 Isoflurane nose cone. Isoflurane is administered in conjunction with air

and/or pure oxygen: 1.5–3% for maintenance of anesthesia in mice

Fig. 5 Incision sites. Access into the abdominal area by a double dorsolateral
incision in the area between the last rib and hips

Fig. 6 Ovaries excision. X denotes ‘removal’

10. Repeat steps 3–9 on the left side (Fig. 5).

11. To conclude, inject tramadol subcutaneously.
12. Place the animal on a heating pad until it is recovered (see
Note 8).
13. Add 1.4 mL acetaminophen solution to 300 mL water (final
concentration, 0.47 mg/mL) and maintain ad libitum for
3 days.
14. Observe the animal daily for any inflammatory signs. Inspect
the surgical wound and look for any behavior that
suggests pain.
308 Vanessa R. Souza et al.

15. Ten days after surgery, evaluation of ovariectomy efficacy can

be performed through vaginal cytology [9] and by uterus
weight, to check for uterus atrophy [10] (see Note 9).

4 Notes

1. All procedures should be approved by institutional ethical

committees and performed accordingly to international and
local principles of care and use of laboratory animals.
2. Adequate infrastructure and equipment are required to work
with isoflurane. Pregnant women should not work with or
expose themselves to isoflurane. Personal protective equipment
(PPE) such as lab coats, gloves, masks, and goggles are neces-
sary to avoid health complications. Check all the connections
to prevent contamination.
3. Carefully observe the animal’s breathing. Anesthesia equip-
ment allows for oxygen increases in order to favor the recovery
of the animal if necessary.
4. Check to ensure that the animal is properly connected to the
nose cone. Anesthesia induced by inhalant agents is rapidly
reversed simply by discontinuing the administration of
anesthetics.
5. Every surgical implement has to be sterilized and hygienized
with the chlorhexidine solution.
6. The same surgical procedure is followed for sham operations,
but the ovaries are left intact.
7. Avoid manipulating the animal’s tissue too much. The muscu-
lature does not need to be sutured. We suggest simple suture
pattern by needle wire for animal skin, using a tweezers and a
needle holder. After this procedure, clean the surgical wound.
8. Animals should be monitored until full recovery.
9. Uterus atrophy is checked by first obtaining animal body
weights for both OVX and sham groups. The uterus has to be
removed after euthanasia. Dry the uterus at 37–40
C for 24 h
and weight the dried uterus. Obtain the ratio (R), normalizing
dried uterus weight according to body weight:
R ¼ ðdried uterus weightÞ=ðbody weightÞ
To confirm surgery success statistical difference has to be
verified between OVX and sham groups by applying the appro-
priate statistical test.
 Ovariectomy Protocol 309

Acknowledgments

This study was funded by São Paulo Research Foundation

(FAPESP), project number 2012/50410-8 to CMF; 2012/
50336-2 and 2016/50484-2 to FAO. CNPq (The National Coun-
cil for Scientific and Technological Development), project number
486037/2012-6 to CMF. Also, the authors would like to acknowl-
edge for the fellowships provided by the Coordenação de Aperfei-
çoamento de Pessoal de Nı́vel Superior - Brasil (CAPES) -Finance
Code 001 to VRS and EM; São Paulo Research Foundation
(FAPESP), project number 2016/13496-2 to MCC.

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