Superovulation Protocols for Mice

To increase the numbers of embryos available for an experiment and to reduce the numbers of animals that are required, it is common to induce multiple ovulations in mice by administering hormones (Table 2.1). Superovulation protocols also assist in the synchronization of embryo timing between females. Best results from superovulation protocols are achieved by using prepubertal females (3-5 weeks old). Two hormones are administered by intraperitoneal injection 48 h apart. The first injection of pregnant mare's serum gonadotrophin (PMSG) provides FSH that recruits follicles for ovulation. This is

Table 2.2. Formulations of Media for Manipulation of Mouse Embryos

Component

Handling Medium (mM)

Mouse Fertilization Medium (mM)

NaCl

90.5

100.5

KCl

5.5

5.5

NaHPO4

0.5

0.5

MgSÖ4

1.0

1.0

NaHCO3

2.0

25.0

CaCl2

1.8

1.8

Lactate

10.5

10.5

Pyruvate

0.35

0.35

Glucose

0.5

3.15

Alanyl-glutamine

0.5

0.5

Alanine

0.1

0.1

Aspartate

0.1

0.1

Asparagine

0.1

0.1

Glutamate

0.1

0.1

Glycine

0.1

0.1

Proline

0.1

0.1

Serine

0.1

0.1

Taurine

0.1

0.1

MOPS/HEPES

23.0

Glutathione

1.0 g/l

Phenol red

0.005g/l

0.005 g/l

For protocols on how to prepare media, see instructions in chapter 3.

For protocols on how to prepare media, see instructions in chapter 3.

Table 2.3. Formulations of Media used in Bovine IVM/IVF

Component

Modified Fert-Talp

Sperm-Talp

HEPES/MOPS-Fert-Talp

NaCl

114.0

100.0

114.0

KCl

3.2

3.1

3.2

NaHPO4

0.4

0.3

0.4

MgSÖ4

0.5

1.2

0.5

NaHCO3

25.0

25.0

5.0

CaCl2

2.6

2.6

2.6

Lactate

10.0

21.0

10.0

Pyruvate

0.25

1.0

0.25

Glucose

Alanine

0.1

0.1

Aspartate

0.1

0.1

Asparagine

0.1

0.1

Glutamate

0.1

0.1

Glycine

0.1

0.1

Proline

0.1

0.1

Serine

0.1

0.1

MOPS/HEPES

10.0

20.0

Phenol red

0.001

0.001

0.001

followed 48 h later by an injection of human choronic gonadotrophin (hCG), which mimics the LH surge and results in ovulation. The injection interval should be kept between 46 and 48 h. It is essential that the hCG injection is given before the endogenous LH surge, which occurs around 15-20 h after the middle of the second dark cycle. Females are then placed with males and allowed to remain overnight. Mating can be detected by the presence of a vaginal plug the next morning. Fertilized embryos can then be collected (see below). Response to superovulation procedures vary significantly between strains of mice. We typically give a dose of 5 IU per mouse for mice 3-4 weeks old.

Hormone solutions can be prepared by reconstituting lyophilized powder in sterile saline at a concentration of 50 IU/ml. For most prepubertal mice this results in a injection of 0.1 ml per mouse. Hormones can be stored for 2-3 weeks at -20°C or at -80°C for 2-3 months. There are significant differences in the ability of different batches of hormones, so each new lot should be prescreened before use.

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