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 acclima...

<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>

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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			<td>Choral hydrate</td>
			<td>Sinopharm Chenmical Reagent Co., Ltd.</td>
			<td>20160225</td>
			<td></td>
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			<td>Coated VICRYL Plus Sutures</td>
			<td>Ethicon, Inc.</td>
			<td>missing</td>
			<td></td>
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			<td>Estradiol benzoate</td>
			<td>J&#38;K Scientific, Ltd.</td>
			<td>L930Q170</td>
			<td></td>
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			<td>Ethanol absolute</td>
			<td>Beijing Chemical Works Co., Ltd.</td>
			<td>20160715</td>
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			<td>Shanghai Johnson &#38; Johnson Co., Ltd.</td>
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			<td>Isoflurane</td>
			<td>RWD Life Science Co., Ltd.</td>
			<td>217180501</td>
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			<td>Lidocaine</td>
			<td>HebeI Tiancheng Pharmacreutical Co., Ltd.</td>
			<td>1170506107</td>
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		<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>
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			<td>Olive oil</td>
			<td></td>
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			<td></td>
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			<td>Penicillin sodium</td>
			<td>North China Pharmaceutical Co., Ltd.</td>
			<td>F5126420</td>
			<td></td>
		</tr>
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			<td>Progesterone</td>
			<td>J&#38;K Scientific, Ltd.</td>
			<td>LR50Q07</td>
			<td></td>
		</tr>
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			<td>Sony digital camera</td>
			<td>Sony Corporation</td>
			<td>HDR-CX290E</td>
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			<td>Test box</td>
			<td></td>
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			<td>DIY</td>
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			<td>ThinkStation Computer</td>
			<td>Lenovo</td>
			<td>S/N PCOGLQKG</td>
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			<td>Vaporizer for Isoflurane</td>
			<td>RWD Life Science Co., Ltd.</td>
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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 ...

Sexual behavior is a dynamic and a complex process that's influenced by biologic, social, and the environment factors. The study of mouse sexual behavior can provide more information about this interactions. This procedure facilitates the recording of the number and the duration of male mice sexual behaviors to allow the identification and the characterization of mouse sexual behavior interactions and the patterns.48 hours before the sexual behavior test, inject 20 micrograms of Estradiol benzoate in 100 microliters of sterilized olive oil intraperitoneally into a six to eight week old female mouse to induce estrus. On the same day, place the male mice to be tested in the center of a 40 by 40 by 40 centimeter square open field box with three black plexiglass walls and one transparent front wall. Allow the animals to explore the environment freely for 30 minutes per day for the next two days.On the day of the test, four hours before the experiment inject 500 micrograms of progesterone in 100 microliters of sterile olive oil intraperitoneally into the female mice and set the room lighting to 650 lux. Then set up a digital camera linked to a computer to videotape the movement and behavior of the mice. To conduct the behavioral assay, during the first hours of the dark cycle turn on the camera and place the male mouse to be tested in the center of the open field in the test box for five minutes.When the mouse has acclimated to the test environment, place a female mouse in hormone induced estrus into the test box and record the social and mating behaviors and interactions between the male and female mice for 30 minutes. At the end of the test period, confirm that the video has been saved and turn off the camera. Return the mice to their individual home cages and clean any urine, feces, and padding within the apparatus.Then remove the smell of the tested animals with 75%ethanol and begin the test for the next male mouse as just demonstrated. To analyze the behavioral data, when all of the mice have been tested play back the video recording and record the number of mounts and the number of intromissions that occurred during the 30 minute experimental period. Record the time from the introduction of the female to the first mounting as the mounting latency, the time from the introduction of the female to the first intromission as the intromission latency, and the time from the first intromission to the first ejaculation as the ejaculation latency.Then record the number and the time of all the copulatory series in 30 minutes. The success rate of copulation tends to be lower in isolation reared groups than in group housed groups, likely due, at least in part, to the longer mounting latency observed in isolation reared groups compared to the group housed groups. The intromission latency is also longer in isolation reared groups indicating that these animals require a longer time to perform the insertion of the penis into the female's vagina.No statistically significant difference between the two groups is observed in terms of ejaculation latency or post-ejaculatory mount latency. The duration of the copulatory series is shorter in the isolation housed group than in the group housed group. No statistically significant differences between the two groups are observed in terms of the number of mounts, number of intromissions, or number of copulatory series.Sexual behavior is a refraction of neural and the neurocrine function. Understanding mouse sexual behavior patterns can help further our understanding of human sexual behaviors and their interactions.

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JoVE Journal

Behavior

19.6K visualizações.  Hunan Key Laboratory of Psychiatry and Mental Health. O comportamento sexual é um processo dinâmico e complexo que é influenciado por fatores biológicos, sociais e ambientais. O estudo do comportamento sexual do rato pode fornecer mais informações sobre essas interações. Este procedimento facilita o registro do número e a duração dos comportamentos sexuais dos camundongos masculinos para permitir a identificação e a caracterização das interações de comportamento sexual do rato e os padrões.48 horas antes do teste de comp...