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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of the Laboratory Mouse. , (2007).</li><li>Burns-Cusato, M., Scordalakes, E. M., Rissman, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+missing+data:+what+we+know+(and+need+to+learn)+about+male+sexual+behavior.">Of mice and missing data: what we know (and need to learn) about male sexual behavior.</a> Physiology &amp; Behavior. 83 (2), 217-232 (2004).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's Wrong With My Mouse?. , (2006).</li><li>Whishaw, I. Q., Haun, F., Kolb, B., Windhorst, U., Johansson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Behavior+in+Laboratory+Rodents.">Analysis of Behavior in Laboratory Rodents.</a> Modern Techniques in Neuroscience Research. , (1999).</li><li>Sachs, B., Barfield, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional Analysis of Masculine Copulatory Behavior in the Rat. 7, (1976).</li><li>Hull, E. M., Dominguez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+behavior+in+male+rodents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+copulatory+behavior+of+house+mice+(Mus+musculus).">Studies of the copulatory behavior of house mice (Mus musculus).</a> Behavioral Biology. 16 (4), 463-473 (1976).</li><li>Park, J. H., Gould, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Male+Sexual+Behavior+in+Mice.">Assessment of Male Sexual Behavior in Mice.</a> Mood and Anxiety Related Phenotypes in Mice. 63, (2011).</li><li>Bonthuis, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+rats:+key+species+variations+in+the+sexual+differentiation+of+brain+and+behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+behaviour+of+two+inbred+strains+of+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+Behavior+in+Three+Inbred+Strains+of+Mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testosterone+rapidly+affects+the+expression+of+copulatory+behavior+in+house+mice+(Mus+musculus).">Testosterone rapidly affects the expression of copulatory behavior in house mice (Mus musculus).</a> Physiology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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 &amp; Integrative Physiology. 147 (3), 607-615 (2007).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Type+3+Adenylyl+Cyclase+Perturbs+the+Postnatal+Maturation+of+Olfactory+Sensory+Neurons+and+Olfactory+Cilium+Ultrastructure+in+Mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+sexual+and+aggressive+behaviors+in+Cnga2+mutant+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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 &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+surgically-induced+endometriosis+by+auto-transplantation+of+uterine+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+vaginal+lavage,+crystal+violet+staining,+and+vaginal+cytological+evaluation+for+mouse+estrous+cycle+staging+identification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postweaning+Isolation+Rearing+Alters+the+Adult+Social,+Sexual+Preference+and+Mating+Behaviors+of+Male+CD-1+Mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+applications+of+mating+model+in+male+rat.">Establishment and applications of mating model in male rat.</a> Chinese Journal of Andrology. 22 (1), 7-10 (2008).</li><li>. 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+sexual+and+social+behaviors+in+trp2+mutant+mice.">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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Choral hydrate</td>
			<td>Sinopharm Chenmical Reagent Co., Ltd.</td>
			<td>20160225</td>
			<td></td>
		</tr>
		<tr>
			<td>Coated VICRYL Plus Sutures</td>
			<td>Ethicon, Inc.</td>
			<td>missing</td>
			<td></td>
		</tr>
		<tr>
			<td>Estradiol benzoate</td>
			<td>J&#38;K Scientific, Ltd.</td>
			<td>L930Q170</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Beijing Chemical Works Co., Ltd.</td>
			<td>20160715</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen (Children&#39;s Motrin)</td>
			<td>Shanghai Johnson &#38; Johnson Co., Ltd.</td>
			<td>160629478</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co., Ltd.</td>
			<td>217180501</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>HebeI Tiancheng Pharmacreutical Co., Ltd.</td>
			<td>1170506107</td>
			<td></td>
		</tr>
		<tr>
			<td>Male and female CD-1 mice</td>
			<td>Vital River Beijing</td>
			<td>SCXK(<img src="/files/ftp_upload/60154/SCXK.jpg" />)2013-0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Olive oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin sodium</td>
			<td>North China Pharmaceutical Co., Ltd.</td>
			<td>F5126420</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>J&#38;K Scientific, Ltd.</td>
			<td>LR50Q07</td>
			<td></td>
		</tr>
		<tr>
			<td>Sony digital camera</td>
			<td>Sony Corporation</td>
			<td>HDR-CX290E</td>
			<td></td>
		</tr>
		<tr>
			<td>Test box</td>
			<td></td>
			<td></td>
			<td>DIY</td>
		</tr>
		<tr>
			<td>ThinkStation Computer</td>
			<td>Lenovo</td>
			<td>S/N PCOGLQKG</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer for Isoflurane</td>
			<td>RWD Life Science Co., Ltd.</td>
			<td>E05904-009M</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

assessment-of-sexual-behavior-of-male-mice

Research

JoVE Journal

Behavior

20.0K Views.  Hunan Key Laboratory of Psychiatry and Mental Health. This article describes how to perform sexual behavior tests in male mice.

Video: Assessment of Sexual Behavior of Male Mice

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

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 ...

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

Analysis of Sugar-Induced Foraging Behavior in Drosophila

Tracking Sugar-Elicited Local Searching Behavior in Drosophila

Foraging behavior is essential for the survival of organisms as it enables them to locate and acquire essential food resources. In Drosophila, hunger triggers a distinct search behavior following the consumption of small quantities of a sugar solution. This report presents a simple experimental setup to study sugar-elicited search behavior with the aim of uncovering the underlying mechanisms. Minute quantities of concentrated sugar solution elicit sustained searching behavior in flies. The involvement of path integration in this behavior has been established, as flies utilize their trajectory to return to the sugar location. The most recent findings provide evidence of temporal modulation in the initiation and intensity of the search behavior after sugar intake. We have also used this setup for artificial activation of specific taste-receptor neurons in the pharynx, which elicits the search behavior. The Drosophila neurogenetic toolkit offers a diverse array of tools and techniques that can be combined with the sugar-elicited search behavior paradigm to study the neural and genetic mechanisms underlying foraging. Understanding the neural basis of hunger-driven searching behavior in flies contributes to the field of neurobiology as a whole, offering insights into the regulatory mechanisms that govern feeding behaviors not only in other organisms but also in humans.

Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication

This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms.

Foraging behavior is essential for the survival of organisms as it enables them to locate and acquire essential food resources. In Drosophila, hunger ...