Materials

1. Phosphate-buffered saline (PBS): 20 mM sodium phosphate buffer, pH 7.4, containing 0.8% NaCl or 0.9% NaCl alone.

2. 1-mL disposable syringe (without needle).

3. Petri dish.

4. Light microscope with 40x magnification (and 400x magnification if checking for the presence of sperm).

5. Female rats and mice (preferably experienced mothers).

Table 1

Identification of Ovulating Mice

Stage of the oesterous cycle Vaginal characteristics

Dioestrous Opening small, tissues small and moist

Prooestrous Opening gaping, tissues red-pink and moist

Dorsal and ventral folds Oestrous (ovulating) Opening gaping, tissues pink and moist

Pronounced folds

Metoestrous Tissues pale and dry, white cell debris

12.3. Method

1. To screen for potential surrogate mothers, use female rats that have given birth to at least one litter and weaned for more than a week.

2. Mark rats on tail for identification.

3. Fill a 1-mL syringe with 0.2-0.3 mL PBS or 0.9% saline.

4. Expose the rat's vagina by lifting its tail.

5. Gently insert the syringe into the vaginal opening until resistance is felt. Expel the PBS or 0.9% NaCl into the vagina, and then draw the fluid back into the syringe to collect vaginal cells into the fluid.

6. Transfer the contents onto a suitably labeled Petri dish. Repeat with several other rats.

7. Examine the cells under a light microscope with 40x magnification, and identify ovulating rats from the characteristics detailed below.

Stage of estrous cycle Cell characteristic

Diestrous Few epithelial cells and leukocytes

Proestrous (pair rats at this stage) Many nucleated epithelial cells, yellow appearance Estrous Many large cornified epithelial cells, brownish appearance Metestrous Fewer cornified epithelial cells, many leukocytes

8. If checking for the presence of sperm, examine the vaginal smear under 400x magnification.

12.4. Notes

1. Although virgin female rats can make suitable surrogate mothers, they tend to consume a part of their litters. Experienced mothers tolerate the traumas associated with the experimental procedures better to care for the unusually large or small litters that may eventually be produced.

2. Some female rats are anestrous, remaining always in a particular phase of the estrous cycle. These are not suitable for mating.

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