Name: is my mouse pregnant? high-frequency ultrasound assessment
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All the steps in this protocol follow the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and have been approved by the Institutional Animal Care and Use Committee of New York University Grossman School of Medicine.
1. Mating of mice for timed pregnancies
<ol>
	<li>Pair the appropriate female mouse (usually a heterozygote) in a cage with the appropriate male mouse (usually a heterozygote) for overnight mating.</li>
	<li>Separate the mice the ne...

<ol>
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(128), e56207 (2017).</li><li>Greco, A., 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=High+frequency+ultrasound+for+in+vivo+pregnancy+diagnosis+and+staging+of+placental+and+fetal+development+in+mice.">High frequency ultrasound for in vivo pregnancy diagnosis and staging of placental and fetal development in mice.</a> PLoS One. 8 (10), 77205 (2013).</li><li>Flores, L. E., Hildebrandt, T. B., K&#252;hl, A. A., Drews, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+staging+of+spontaneous+embryo+resorption+by+ultrasound+biomicroscopy+in+murine+pregnancy.">Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.</a> Reproductive Biology and Endocrinology. 12, 38 (2014).</li><li>Norton, W. 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The most important first step in the imaging is to identify the vagina and then to determine the bifurcation of the uterine horn to the left and right. By following each uterine horn, the imager is less likely to mis-identify loops of the bowel as the uterus. Moreover, understanding the variations in the appearance of the bowel (with/without fecal matter) is important to distinguish these from the uterus; occasionally, fecal &#8220;balls&#8221; in bowel loops may mimic a gravid (pregnant) uterus. Although other authors h...

The mouse is the preferred mammalian model for many human diseases and biological processes1,2,3,4. Research in developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints5,6,7,8. Moreover, optimal and effective breeding of model mice requires an assessment of timed pregnancies, particularly when investigators are studying the effects of a gene mutation on development. Typically, investigators mate heterozygous mice overnight, look for a vaginal plug early the next morning, and hope that a pregnancy ensues9. Determining intrauterine loss typically starts with checking a newborn litter for Mendelian ratios of genotypes, then working backwards by sacrificing pregnant mice at various gestational stages, and recovering the embryos. Investigators may determine weight gain as a metric of a positive pregnancy10,11; however, especially with genetically-engineered mice, the litters may be very small and subsequently resorbed when there is intrauterine loss due to which the weight gain may not be obvious (particularly early in pregnancy, ~E6.5&#8211;8.5). A mouse may appear falsely pregnant due, for example, to a benign abdominal tumor. In essence, one works &#8220;blind&#8221;.
High-resolution ultrasound biomicroscopy allows for direct visualization of the gravid uterus and developing mouse embryos12,13,14,15,16. Although we had initially developed methods to assess embryonic mouse cardiovascular physiology16,17, we recognized the utility of this imaging modality to streamline our mouse breeding. Specifically, we no longer had to wait to &#8220;see&#8221; if a mouse were pregnant, based on either the obvious weight gain or delivery of a litter; we could determine the gravid state and re-mate mice quickly if the dam was not pregnant. Moreover, intrauterine losses could also be easily imaged, and a timeline of loss could be determined without sacrificing the mouse (see Figure 1 for a schematic). Time, valuable model mice, and funds can thus be saved.

<table>
	<tbody>
		<tr>
			<td>Depilatory cream</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 70%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fur clippers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze or KimWipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical oxygen (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse imaging system (including anesthesia set-up and imaging platform)</td>
			<td>Fujifilm Visual Sonics</td>
			<td>Various</td>
			<td>Any system with 40 MHz center-frequency ultrasound transducer probe</td>
		</tr>
		<tr>
			<td>Razor blade (not a safety razor)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scale (to weigh mouse)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

is my mouse pregnant? high-frequency ultrasound assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

This protocol will allow an investigator to determine confidently whether a mouse is pregnant, including during the early stages and to determine whether there are obvious prenatal embryonic or fetal losses without needing to sacrifice the pregnant dam. This protocol is especially useful when breeding genetically engineered mice; typically, heterozygous x heterozygous crosses to yield homozygous offspring leads to failure of proper development, which causes prenatal lethality. Figure 1 depic...

Watch this Scientific Journal Video about Is My Mouse Pregnant? High-Frequency Ultrasound Assessment at JoVE.com

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

High resolution ultrasound can help streamline experiments requiring timed pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. This protocol demonstrates how to assess mouse pregnancies and identify potential image artifacts that may mimic pregnancy. To begin, identify the bladder on the screen.Scanning caudally from the bladder, identify the vagina. When the ultrasound transducer is positioned midline and low on the abdomen, the bladder and vagina are visible. Then scanning slowly and smoothly in a cranial direction, identify the bifurcation of the vagina into the left and right uterine horns.Survey the left and right uterine horns. Up to mid-gestation, the mouse embryos will be positioned along the right and left peripheries. As they grow, the more distal portions of the uterus and their corresponding embryos will turn outwards and dorsally.This protocol is especially useful when breeding genetically engineered mice. Typically, heterozygous with heterozygous crosses to yield homozygous offspring lead to failure of proper development, causing prenatal lethality. Mouse embryos at various stages of development are shown here.Early stage mouse embryos, dead embryos, or resorbed embryos may resemble other organs in the abdomen or feces in the intestines, while intestinal loops may mimic the non-gravid uterus. Some investigators prefer to use a stage mounted transducer, such as shown in this image, but we prefer the handheld method of scanning, since this allows for fast and efficient imaging throughout the abdomen.

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