On 2014 Aug 16, Melissa A Farmer commented:

Dear Dennis,

Thank you for your interest in our methods, and we are pleased to address your questions.

The first question raised is related to the amount of solution injected. For estradiol and progesterone, the mice received injections of 0.1 mL of each solution under the scruff of the neck. We found that the mice could easily tolerate this volume (no scratching or visual discomfort), provided that injections were limited to once per week (thereby limiting frequency of mating). We chose this volume due to the sensitivity of our scale, which constrained the concentration of stock solutions we could make. Lesser volumes with appropriately adjusted dilutions would work, as well.

The second issue is related to optimizing copulation/female receptivity in novel couples. On one hand, the probability of a novel couple mating can be enhanced by providing the male with prior sexual experience with other receptive females (“stimulus” females). This prior learning will increase the frequency with which he approaches the novel female, which in turn increases probability that copulation will occur.

A complementary strategy is to optimize hormonal and environmental factors that can facilitate female receptivity in virgin mice, independent of the actions of the male. Sexually mature female mice should be used (> 6 weeks age). Based on our experience, the timing of the progesterone injection is the most important factor that optimizes a female’s behavioral response during testing. Unfortunately the mouse mating literature is older and authors infrequently reported the timing of progesterone administration in females (hence the Jones et al. reference). In pilot experiments, we varied progesterone injections from 4-6 hours pre-test, and in our hands, 5.5 hours produced the most consistent behavioral results. To further optimize behavioral receptivity, we scheduled testing towards the beginning of the dark cycle when mice would be most physically active. Even though mice were maintained in an artificially-lit vivarium, we noticed seasonal variation in the onset of dark cycle physical activity, and so we shifted the timing of testing (and therefore progesterone injections) accordingly, with a later testing time during winter. Also note that more complicated mating environments, like a paced mating boxes, require repeated habituation to reduce novelty effects.

On 2014 Aug 14, Dennis Eckmeier commented:

Dear authors,

A really interesting paper!

I have a question according to methods:

You write "Sexual receptivity was induced in ovariectomized females with subcutaneous (s.c.) injections of estradiol benzoate (5 μg/0.1 ml in sesame oil; Sigma-Aldrich) given 48 h pretest, and progesterone (500 μg/0.1 ml in sesame oil) 5.5 h pretest. These parameters are known to induce a state of optimal sexual receptivity, comparable to the estrus phase (Jones et al., 2013)."

What amount of drug solutions did you administer?

You determined that some pairs of treated ovariectomized females and males would indeed mate, so no doubt it worked for your study. However, a study I am planning doesn't allow previous encounters between the stimulus female and the male. The paper you cite (Jones et al., 2013) is about rats. Did you somehow determine whether your treatment induces optimal sexual receptivity in mice?

On 2014 Aug 14, Dennis Eckmeier commented:

Dear authors,

A really interesting paper!

I have a question according to methods:

You write "Sexual receptivity was induced in ovariectomized females with subcutaneous (s.c.) injections of estradiol benzoate (5 μg/0.1 ml in sesame oil; Sigma-Aldrich) given 48 h pretest, and progesterone (500 μg/0.1 ml in sesame oil) 5.5 h pretest. These parameters are known to induce a state of optimal sexual receptivity, comparable to the estrus phase (Jones et al., 2013)."

What amount of drug solutions did you administer?

You determined that some pairs of treated ovariectomized females and males would indeed mate, so no doubt it worked for your study. However, a study I am planning doesn't allow previous encounters between the stimulus female and the male. The paper you cite (Jones et al., 2013) is about rats. Did you somehow determine whether your treatment induces optimal sexual receptivity in mice?

On 2014 Aug 16, Melissa A Farmer commented:

Dear Dennis,

Thank you for your interest in our methods, and we are pleased to address your questions.

The first question raised is related to the amount of solution injected. For estradiol and progesterone, the mice received injections of 0.1 mL of each solution under the scruff of the neck. We found that the mice could easily tolerate this volume (no scratching or visual discomfort), provided that injections were limited to once per week (thereby limiting frequency of mating). We chose this volume due to the sensitivity of our scale, which constrained the concentration of stock solutions we could make. Lesser volumes with appropriately adjusted dilutions would work, as well.

The second issue is related to optimizing copulation/female receptivity in novel couples. On one hand, the probability of a novel couple mating can be enhanced by providing the male with prior sexual experience with other receptive females (“stimulus” females). This prior learning will increase the frequency with which he approaches the novel female, which in turn increases probability that copulation will occur.

A complementary strategy is to optimize hormonal and environmental factors that can facilitate female receptivity in virgin mice, independent of the actions of the male. Sexually mature female mice should be used (> 6 weeks age). Based on our experience, the timing of the progesterone injection is the most important factor that optimizes a female’s behavioral response during testing. Unfortunately the mouse mating literature is older and authors infrequently reported the timing of progesterone administration in females (hence the Jones et al. reference). In pilot experiments, we varied progesterone injections from 4-6 hours pre-test, and in our hands, 5.5 hours produced the most consistent behavioral results. To further optimize behavioral receptivity, we scheduled testing towards the beginning of the dark cycle when mice would be most physically active. Even though mice were maintained in an artificially-lit vivarium, we noticed seasonal variation in the onset of dark cycle physical activity, and so we shifted the timing of testing (and therefore progesterone injections) accordingly, with a later testing time during winter. Also note that more complicated mating environments, like a paced mating boxes, require repeated habituation to reduce novelty effects.