We thank Lu Cong, Zhang Hongxia, Zhang Beiyue, and Hu Mi for their suggestions for the experiments.

All experiments were performed in compliance with the guidelines of the Principles of Laboratory Animal Care (NIH Publication No. 80-23, revised 1996) and under the approval and supervision of the Academy of Experimental Animal Centre of the Institute of Medicinal Plant Development (China).
1. Animal husbandry
<ol>
	<li>House female and male mice at 25 &#176;C for 12 h light/12 h dark cycles.</li>
	<li>Provide free access to water and a standard pelleted diet.</li>
	<li>Allow mice to acclimate to their environment for 7 days prior to the operation if transported from a different facility.</li>
</ol>
2. Ovariectomy in female mice
<ol>
	<li>Anesthetize the female (8 weeks postnatal, not less than 6-weeks-old) with isoflurane (~4&#8722;5% for induction, ~1&#8722;2% for maintenance) in 100% oxygen via a face cone mask.</li>
	<li>Check that the appropriate depth of aesthesia has been achieved by making sure that there are no voluntary movements for over 30 s, in combination with an appropriate respiratory rate (e.g., 1 breath per 2 s or longer). Alternatively, test the mouse&#39;s response to gentle pressure on the toes of the hind paws. 
	NOTE: The normal respiratory rate is ~180/min. A rate drop of 50% is acceptable during anesthesia17.
	<ol>
		<li>Use ophthalmic ointment to prevent corneal drying and eye trauma while under anesthesia.</li>
		<li>Keep the body temperature of the mouse at or above 36 &#176;C. Provide supplemental heat support during the period of anesthesia when necessary.</li>
	</ol>
	</li>
	<li>Sterilize and disinfect all surgical instruments and hard surfaces of the operating table with 75% ethanol prior to use.</li>
	<li>Place animal on a sterile drape.</li>
	<li>Shave fur bilaterally over the lumbar spine on the back of the mouse to expose the skin.</li>
	<li>Sterilize the exposed skin with 75% ethanol.</li>
	<li>Make a single midline incision (about 0.5 cm in length) on the back from the center of the two thigh roots toward the head of ~1 cm distance (position is shown in Figure 1).</li>
	<li>Use small scissors to penetrate the skin to gently free subcutaneous tissue from the underlying muscle in order to expose the muscle layer.</li>
	<li>Locate the ovary under the thin muscle layer and make a small incision (about 5 mm in length) to gain entry to the peritoneal cavity.
	<ol>
		<li>Use small tweezers to pull the tissue slightly on the left side of the abdominal cavity to show the left-hand ovary wrapping around the white adipose tissue (a translucent, irregular mass as seen by the naked eye, see Figure 1).</li>
	</ol>
	</li>
	<li>Retract the ovarian fat pad surrounding the ovary with blunt forceps to expose the oviduct.</li>
	<li>Perform a single ligature around the oviduct to prevent bleeding.</li>
	<li>Use small scissors to sever the oviduct gently and remove the ovary.</li>
	<li>Check the oviduct carefully to confirm that all ovarian tissues are removed. The ovary is about 5 mm &#215; 4 mm &#215; 3 mm with irregular nodules on the surface.</li>
	<li>Place the remaining part of the oviduct back into the abdominal cavity.</li>
	<li>Suture the muscle layer with absorbable sutures.</li>
	<li>Pull the skin to the right side to expose the muscle layer on the right-hand side and remove the right ovary by repeating steps 2.9&#8211;2.16.</li>
	<li>Close the skin incision by using absorbable sutures.
	<ol>
		<li>Inject each mouse intraperitoneally with penicillin sodium (10,000 units/10 g per mouse) to prevent infection.</li>
		<li>Inject lidocaine (4 mg/kg, 0.4 mL/kg of a 1% solution) beneath the skin along the site of the incision. Also provide ibuprofen (50&#8211;60 mg/kg/day; 10 mL of Children&#39;s Motrin in 500 mL of water) continuously in the drinking water for 3 days for pain treatment.</li>
	</ol>
	</li>
	<li>Place each mouse into a sterilized cage individually.</li>
	<li>Keep under close observation for approximately 1&#8211;2 h until fully recovered from anesthesia.
	<ol>
		<li>Recover animals on paper towels in a clean cage without bedding. This step minimizes the risk of tracheal obstruction or pneumonia. Provide supplemental heat support during anesthetic recovery. Monitor the surgical site to prevent the rupture of the wound.</li>
	</ol>
	</li>
	<li>Following the recovery period (approximately 24 h after surgery), place mice back to their home cage.</li>
	<li>Do not perform the experiment for at least 2 weeks after surgery.</li>
</ol>
3. Hormone-induced estrus in females
<ol>
	<li>Determine the estrous stage of the females by performing a vaginal smear as described in McLean et al.18. No estrus cycle change indicates that the ovariectomy of the female was successful.</li>
	<li>Inject estradiol benzoate (20 &#956;g per mouse, dissolved in 0.1 mL of sterilized olive oil, intraperitoneally) 48 h prior to the sexual behavior test.</li>
	<li>Inject progesterone (500 &#956;g per mouse, dissolved in 0.1 mL of oil, intraperitoneally) 4 h prior to the sexual behavior test. 
	NOTE: The eligibility of an estrus female is determined by the female accepting genital insertion of a male mouse 3 or more times, when they cohabit with a sexually active and experienced male in one cage.</li>
</ol>
4. Preparation for the sexual behavior test
<ol>
	<li>Conduct the sexual behavior test of male mice in a rectangular and open field box (40 cm x 40 cm x 40 cm) with black Plexiglass walls, except for a transparent front wall that allows for the observation of mouse movement.</li>
	<li>Set the general room lighting to 650 lux. 
	NOTE: The mice should not be illuminated directly to avoid abnormal behavior patterns.</li>
	<li>Use a digital camera linked to a computer to videotape the movement and behavior of the mice.</li>
	<li>Perform the behavioral test on the male mice during the first hours of the dark cycle.</li>
</ol>
5. Habituation
<ol>
	<li>Keep the experiment room quiet.</li>
	<li>Place the mice to be tested in the center of the open field box, allowing them to explore the environment freely for 30 min.</li>
	<li>Habituate mice for 2 consecutive days prior to the testing day in the apparatus to prevent stress from the new environment. 
	NOTE: Mice should move and explore freely without feeling stress. Both male and female mice need to be habituated to the testing environment.</li>
</ol>
6. Behavioral assays
<ol>
	<li>Turn on the camera prior to the beginning of the test.</li>
	<li>Place the mouse to be tested in the center of the open field in the test box, allowing free exploration for 5 min to acclimate to the environment.</li>
	<li>Place a female in estrus into the test box.</li>
	<li>Record the social and mating behaviors and the interactions between the male and female mice for 30 min.</li>
	<li>Turn off the camera and confirm that the video is saved.</li>
	<li>Take the female out of the test box and record the formation of a vaginal plug. 
	NOTE: A female mouse that accepted mating cannot be employed in another sexual behavior test in one day.</li>
	<li>Place the female back to its home cage.</li>
	<li>Return the male to its home cage.</li>
	<li>Clean the urine, feces, and padding within the apparatus.</li>
	<li>Remove the smell of the tested mice with 75% ethanol.</li>
	<li>Start the test of the next male mouse by repeating steps 6.1&#8211;6.10.</li>
</ol>
7. Behavioral data extraction
<ol>
	<li>Play back the video recording and extract the behavioral parameters (see Figure 2).
	<ol>
		<li>Record the number of mounts in 30 min.</li>
		<li>Record the number of intromissions in 30 min. Count a pelvic thrust as an intromission.</li>
		<li>Record the time from the introduction of the female to the first mounting as mounting latency.</li>
		<li>Record the time from the introduction of the female to the first intromission as intromission latency.</li>
		<li>Record the time from first intromission to the first ejaculation as ejaculation latency.</li>
		<li>Record the time from ejaculation to the next mounting as post-ejaculatory mount latency.</li>
		<li>Record number of copulatory series in 30 min. A copulatory series is each sequence from mounting to ejaculation.</li>
		<li>Record the time of all copulatory series in 30 min as the duration of the copulatory series.</li>
	</ol>
	</li>
</ol>

<ol><li>The staff of the Jackson laboratory. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Biology+of+the+Laboratory+Mouse%22">Biology of the Laboratory Mouse</a>. , Second Edition, Dover publications, inc. New York. Chapter 11 (2007).</li>
<li>Burns-Cusato, M., Scordalakes, E. M., Rissman, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Of+mice+and+missing+data%3A+what+we+know+%28and+need+to+learn%29+about+male+sexual+behavior%5BTitle%5D)+AND+%22Physiology+%26+Behavior%22%5BJournal%5D)">Of mice and missing data: what we know (and need to learn) about male sexual behavior.</a> Physiology & Behavior. 83 (2), 217-232 (2004).</li>
<li>Crawley, J. N. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCrawley%2C+JN+%22What%27s+Wrong+With+My+Mouse%3F%22">What's Wrong With My Mouse?</a>. , Second Edition, John Wiley & Sons, Inc. (2006).</li>
<li>Whishaw, I. Q., Haun, F., Kolb, B. Analysis of Behavior in Laboratory Rodents. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWhishaw%2C+IQ+%22Modern+Techniques+in+Neuroscience+Research%22">Modern Techniques in Neuroscience Research</a>. Windhorst, U., Johansson, H. , Springer. Berlin, Heidelberg. (1999).</li>
<li>Sachs, B., Barfield, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSachs%2C+B+%22Functional+Analysis+of+Masculine+Copulatory+Behavior+in+the+Rat%22">Functional Analysis of Masculine Copulatory Behavior in the Rat</a>. 7, (1976).</li>
<li>Hull, E. M., Dominguez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexual+behavior+in+male+rodents%5BTitle%5D)+AND+%22Hormones+and+Behavior%22%5BJournal%5D)">Sexual behavior in male rodents.</a> Hormones and Behavior. 52 (1), 45-55 (2007).</li>
<li>Mosig, D. W., Dewsbury, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+of+the+copulatory+behavior+of+house+mice+%28Mus+musculus%29%5BTitle%5D)+AND+%22Behavioral+Biology%22%5BJournal%5D)">Studies of the copulatory behavior of house mice (Mus musculus).</a> Behavioral Biology. 16 (4), 463-473 (1976).</li>
<li>Park, J. H. Assessment of Male Sexual Behavior in Mice. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPark%2C+JH+%22Mood+and+Anxiety+Related+Phenotypes+in+Mice%22">Mood and Anxiety Related Phenotypes in Mice</a>. Gould, T. 63, Humana Press. (2011).</li>
<li>Bonthuis, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Of+mice+and+rats%3A+key+species+variations+in+the+sexual+differentiation+of+brain+and+behavior%5BTitle%5D)+AND+%22Frontiers+in+Neuroendocrinology%22%5BJournal%5D)">Of mice and rats: key species variations in the sexual differentiation of brain and behavior.</a> Frontiers in Neuroendocrinology. 31 (3), 341-358 (2010).</li>
<li>Levine, L., Barsel, G. E., Diakow, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mating+behaviour+of+two+inbred+strains+of+mice%5BTitle%5D)+AND+%22Animal+Behavior%22%5BJournal%5D)">Mating behaviour of two inbred strains of mice.</a> Animal Behavior. 14 (1), 1-6 (1966).</li>
<li>McGill, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexual+Behavior+in+Three+Inbred+Strains+of+Mice%5BTitle%5D)+AND+%22Behaviour%22%5BJournal%5D)">Sexual Behavior in Three Inbred Strains of Mice.</a> Behaviour. 19 (4), 341(1962).</li>
<li>James, P. J., Nyby, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testosterone+rapidly+affects+the+expression+of+copulatory+behavior+in+house+mice+%28Mus+musculus%29%5BTitle%5D)+AND+%22Physiology+%26+Behavior%22%5BJournal%5D)">Testosterone rapidly affects the expression of copulatory behavior in house mice (Mus musculus).</a> Physiology & Behavior. 75 (3), 287-294 (2002).</li>
<li>Arteaga-Silva, M., Rodriguez-Dorantes, M., Baig, S., Morales-Montor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+castration+and+hormone+replacement+on+male+sexual+behavior+and+pattern+of+expression+in+the+brain+of+sex-steroid+receptors+in+BALB%2Fc+AnN+mice%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+-+Part+A%3A+Molecular+%26+Integrative+Physiology%22%5BJournal%5D)">Effects of castration and hormone replacement on male sexual behavior and pattern of expression in the brain of sex-steroid receptors in BALB/c AnN mice.</a> Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. 147 (3), 607-615 (2007).</li>
<li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletion+of+Type+3+Adenylyl+Cyclase+Perturbs+the+Postnatal+Maturation+of+Olfactory+Sensory+Neurons+and+Olfactory+Cilium+Ultrastructure+in+Mice%5BTitle%5D)+AND+%22Frontiers+in+Cellular+Neuroscience%22%5BJournal%5D)">Deletion of Type 3 Adenylyl Cyclase Perturbs the Postnatal Maturation of Olfactory Sensory Neurons and Olfactory Cilium Ultrastructure in Mice.</a> Frontiers in Cellular Neuroscience. 11, 1(2017).</li>
<li>Mandiyan, V. S., Coats, J. K., Shah, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deficits+in+sexual+and+aggressive+behaviors+in+Cnga2+mutant+mice%5BTitle%5D)+AND+%22Nature+Neurosciemce%22%5BJournal%5D)">Deficits in sexual and aggressive behaviors in Cnga2 mutant mice.</a> Nature Neurosciemce. 8 (12), 1660-1662 (2005).</li>
<li>Choi, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+deletion+of+floxed+genes+mediated+by+CaMKIIalpha-Cre+in+the+brain+and+in+male+germ+cells%3A+application+to+conditional+and+conventional+disruption+of+Goalpha%5BTitle%5D)+AND+%22Experimental+%26+Molecular+Medicine%22%5BJournal%5D)">Simultaneous deletion of floxed genes mediated by CaMKIIalpha-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goalpha.</a> Experimental & Molecular Medicine. 46, 93(2014).</li>
<li>Pelch, K. E., Sharpe-Timms, K. L., Nagel, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+model+of+surgically-induced+endometriosis+by+auto-transplantation+of+uterine+tissue%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue.</a> Journal of Visualized Experiments. (59), e3396(2012).</li>
<li>McLean, A. C., Valenzuela, N., Fai, S., Bennett, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Performing+vaginal+lavage%2C+crystal+violet+staining%2C+and+vaginal+cytological+evaluation+for+mouse+estrous+cycle+staging+identification%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification.</a> Journal of Visualized Experiments. (67), e4389(2012).</li>
<li>Liu, Z. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postweaning+Isolation+Rearing+Alters+the+Adult+Social%2C+Sexual+Preference+and+Mating+Behaviors+of+Male+CD-1+Mice%5BTitle%5D)+AND+%22Frontiers+in+Behavioral+Neuroscience%22%5BJournal%5D)">Postweaning Isolation Rearing Alters the Adult Social, Sexual Preference and Mating Behaviors of Male CD-1 Mice.</a> Frontiers in Behavioral Neuroscience. 13, 21(2019).</li>
<li>TIan, E. P., Long, T., Qin, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+and+applications+of+mating+model+in+male+rat%5BTitle%5D)+AND+%22Chinese+Journal+of+Andrology%22%5BJournal%5D)">Establishment and applications of mating model in male rat.</a> Chinese Journal of Andrology. 22 (1), 7-10 (2008).</li>
<li>University of Minnesota Academic Health Center Research Service. Anesthesia Guidelines: Mice. , Available from: 
 <a target="_blank" href="https://www.researchservices.umn.edu/services-name/research-animal-resources/research-support/guidelines/anesthesia-mice">https://www.researchservices.umn.edu/services-name/research-animal-resources/research-support/guidelines/anesthesia-mice</a> (2019).</li>
<li>Leypold, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+sexual+and+social+behaviors+in+trp2+mutant+mice%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Altered sexual and social behaviors in trp2 mutant mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (9), 6376-6381 (2002).</li>
</ol>

The authors have nothing to disclose.

There are a few critical steps in the presented protocol. Regarding the ovariectomy of females, the surgery incision opening from the back is less detrimental than that from the abdomen. Given that the position of the ovary is deep, pulling other organs when the incision is cut open from the abdomen often leads to bleeding and results in unclear surgical vision20. We performed the incision on the back to reach the ovary easily and shorten the surgical time, as well as to ensure the safety of the s...

Sexual behavior in mature male mice results from the interaction of a series of related and interdependent hormonal systems and neural systems in different brain circuits1. It also requires developmental experiences, learning, context, and an appropriate partner. Behavioral analysis is an important reflection on neural or neurocrine function. Hence, sexual behavior study on animal models has been widely used in behavioral neuroscience and other related research2. The ethogram of sexual behaviors in rodents has been explained in many articles and books1,3,4. For instance, Scahs and Barfield&#39;s description of sexual behavior in the rat5 has helped understand a similar behavioral pattern in mice5. The mouse is one of the most commonly used subjects for behavioral studies. Hull et al.6 gave a detailed introduction of male mouse sexual behaviors: When a male mouse encounters a female, it starts to investigate the anogenital region of the female. Then, the male presses his front paws against the female&#39;s flanks to mount the female from the rear. The female exhibits a characteristic sexually receptive posture, bending its spine down into a bow and moving its tail to one side of the body, exposing an opening introitus for the sexual penetration of the male (i.e., lordosis). Following the mounting, the male makes rapid, shallow pelvic thrusts, followed by slow and deep vaginal thrusts. After numerous intromissions, a long-lasting thrust results in the ejaculation of semen, during which the male mouse may freeze for about 25 s before dismounting or falling off from the female6. At ejaculation the male mouse accessory glands may produce a mixture containing semen that hardens to form the copulatory plug. Finally, following ejaculation, the male begins genital grooming and displays a lack of interest in the female. In brief, the basic sequence of male sexual behavior consists of sniffing, following, mounting, intromission, ejaculation, and post-ejaculation grooming. Mouse sexual behavior exhibits strain differences. For instance, ejaculation latencies range from 594 to 6943 s, and the numbers of intromissions range from 5 to more than 100. Post-ejaculation latencies range from 17 to 60 min. However, the introduction of a novel female can decrease this time interval. In some cases, the male ejaculates on the first intromission with the new female7.
The major events for the evaluation of sexual behavior are mounting, intromission, and ejaculation. Behavioral scientists have recommended the measurement of not only the frequency of each action, but also its latency and time interval5,8. Some major measurement indicators in past studies include: number of mounts, number of intromissions, mount latency, intromission latency, ejaculation latency, post-ejaculatory mount latency (or post-ejaculatory intervals), post-ejaculatory intromission latency, number of copulatory series, and duration of copulatory series. Park et al.8 and Sachs et al.5 described how to identify each action of mounting, intromission, and ejaculation of rodents. Mounting is defined as the male mounting the female from the rear, palpating her flanks with his forelegs, and thrusting his penis rapidly and repeatedly without penile insertion. Intromission, also known as penile insertion, is identified by one or more of the following acts: a long, deep thrust after rapid shallow thrusts, a rapid kick with one hindleg, and a marked lateral withdrawal of the male from the female. Ejaculation is identified by a terminal pelvic thrust that is slower and deeper than that of an intromission and a reduction in the elevation of the hindleg. A copulatory series is identified by each sequence from mounting to ejaculation. The definitions of behavioral parameters used in the present study are listed as follows: 1) Mounting latency: the time from the introduction of the female to the first mounting of the male; 2) Intromission latency: the time from the introduction of the female to first intromission; 3) Ejaculation latency: the time from the first intromission to first ejaculation (generally following the last pelvic thrust); 4) Post-ejaculatory mount latency: the time from ejaculation to the next mounting; 5) Post-ejaculatory intromission latency: the time from ejaculation and the next intromission; 6) Number of mounts: the number of mounting times before first ejaculation; 7) Number of intromissions: the number of intromissions before the first ejaculation; 8) Number of copulatory series: the number of copulatory series during the observation period; 9) Duration of copulatory series: the time of all copulatory series during the observation period.
Sexual behavior and related behavior can be conducted in either the male&#39;s home cage or in an enclosed arena, among which an apparatus called Rissman&#39;s &#34;No Secrets&#34; mirrored box is introduced to observe the mating behavior3. A video camera is placed in front of the box to simultaneously record the action of the mice from a lateral view and through an inclined mirror from a ventral view. However, this method requires bright lights, which inevitably leads to longer habituation in order to eliminate environmental stress in mice. As for the measuring method, video-based behavioral analysis is recommended to record and quantify behavior4. A video recorder that has a frame-by-frame video advance option with recommended shutter speeds greater than 1/1000 s can be used to record rapid mouse movements. The high resolution infrared camera is necessary when recording in a dark environment. To analyze the film, a computer with a frame grabber to allow the individual frames of behavior to be captured for computer manipulation is needed. Mice are extremely versatile and can display compensatory behavior after almost any treatment. Ambiguity can exist about every moving body part4. Hence, the analysis of some behaviors may require still greater resolution and higher speed cameras.
Male sexual behaviors in mice are affected by many factors, including strain differences, hormone changes, and gene mutantions1,3,9,10. McGill and Blight11 illustrated the strain differences in mouse mating behaviors. For example, C57BL/6 males typically gain intromission quickly and ejaculate in about 20 min11. DBA/2 males are slow to gain intromission but ejaculate rapidly. BALB/c males are slow to achieve ejaculation (average latency of 1 h) due to a long period of courtship11. Testosterone facilitates and maintains male sexual behavior2, and changes in testosterone levels can alter sexual behavior performance12. Both surgical castration and antiandrogen treatment can reduce the level of testosterone and result in a rapid decline of sexual behaviors and even sexual motivation and sexual arousal13. Administered testosterone can restore precopulatory and copulatory behaviors in castrated mice. Lastly, knockout and knockdown mice display differences in facets of sexual behaviors compared to wild type mice. For example, male mice with targeted mutations of Adcy3, Cnga2, and Gnao exhibit a reduced ability to detect pheromones, whereas Trpc2 knockout mice show altered partner preference14,15,16. Other effects of transgenics and knockouts on the sexual behavior of mice are explained by Crawley3.
Here, one of the most common procedures to assess sexual behavior in the pairing of a male mouse with an ovariectomized female that has been hormonally primed to be receptive is described. An experimental protocol is presented for conducting sexual behavior experiments in mice. In addition, an example of changing sexual behavior patterns resulting from social isolation in CD-1 mice is shown.

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assessment of sexual behavior of male mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

A comparison of sexual behavior between CD-1 mice reared in isolation and group-housed CD-1 mice is shown. Male CD-1 mice were randomly assigned into an isolation-reared group (IS, one mouse per cage, n = 30) and a group-housed group (GH, five mice per cage, n = 15). The mice underwent isolation rearing from postnatal day 23 to day 93. Then, both groups of mice were assessed for sexual behavior. Our study found that the success rate of copulation tended to be lower in the IS group than in the GH group (IS: 80.0%, GH: 86....

Watch this Scientific Journal Video about Assessment of Sexual Behavior of Male Mice at JoVE.com

Assessment of Sexual Behavior of Male Mice

This article describes how to perform sexual behavior tests in male mice.

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

20.4K Views.  Hunan Key Laboratory of Psychiatry and Mental Health. This article describes how to perform sexual behavior tests in male mice.

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Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Neuroscience

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval1, juvenile interactions2, opposite and same sex interactions3,4,5, and territorial interactions6. For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders7,8,9, and more recently to understand mechanisms and evolution of vocal communication among vertebrates10. Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds11. The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal&#39;s preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

Developmental Biology

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

Medicine

Adolescent Social Stress Paradigm in C57BL&#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...