Work in the Jaramillo-Lambert lab is supported by the National Institutes of Health NIGMS R35GM142524. All C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health, P40 OD010440. Figure 1D was created using Biorender.com.

NOTE: See the Table of Materials for details about all materials used in this protocol.
1. Preparing experimental plates
<ol>
	<li>Prepare 35 mm diameter Petri dishes with Modified Youngren&#39;s, Only Bacto-peptone (MYOB) media. To make MYOB plates, add 20 g of Bacto agar, 2 g of NaCl, 0.55 g of Trizma-HCl, 0.24 g of Trizma-OH, 3.1 g of Bacto peptone, and 1.6 mL of cholesterol to 1 L of ddH2O and autoclave for 40 min at 121 &#176;C. Let it cool to 55 &#176;C and using sterile technique, pour 4 mL of the molten media into each 35 mm Petri dish. 
	NOTE: A liter of MYOB makes ~250 plates, which can be stored at 4 &#176;C for up to 6 months. We recommend a minimum of 10 individuals per replicate with three replicate sets for each strain.</li>
	<li>Using sterile technique, seed the plates with a small spot (approximately 50 &#181;L) of OP50 bacteria in the middle of the plate. 
	NOTE: Small lawns are especially important for assessing cross-fertilization as the smaller spot leads to increased encounters between males and hermaphrodites, thus increasing the chance of mating.</li>
</ol>
2. Embryonic viability assays (hermaphrodite self-fertilization)
<ol>
	<li>Day 1
	<ol>
		<li>Label the back of each plate. Be sure to keep track of each plate and their respective worms throughout the experiment. 
		NOTE: Preferred labeling technique-Day 1: worm 1, Day 1: worm 2, &#8230; etc.</li>
		<li>Transfer an individual L4 stage hermaphrodite onto each plate. Ensure no embryos or other worms are transferred onto the plate. Allow the worms to develop into adults and lay self-progeny for 24 h at the standard culturing temperature of 20 &#176;C. Score these plates on day 3. 
		NOTE: The temperature of experiments can be altered in the case of temperature-sensitive mutations. For temperature sensitive mutations, embryonic viability assays should be performed at both the permissive (15-16 &#176;C) and nonpermissive temperatures (24-26 &#176;C).</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Label a set of new plates-Day 2: worm 1, Day 2: worm 2, &#8230; etc.</li>
		<li>Transfer day 1 individual worms onto the new plates. 
		NOTE: These worms should have reached adulthood, and wild-type worms should have already started laying embryos.</li>
		<li>Allow the worms to lay embryos for 24 h at 20 &#176;C or other appropriate temperature. Score these plates on day 4.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Label a set of new plates. Day 3: worm 1, Day 3: worm 2, &#8230; etc.</li>
		<li>Transfer day 2 individual worms onto new plates. Allow them to lay progeny for 24 h at 20 &#176;C or other appropriate temperature. Score these plates on day 5. 
		NOTE: In cases of lower temperatures, hatching times will be increased. Adjust accordingly.</li>
		<li>Draw a grid pattern on a 35 mm lid using a fine marker. Place the gridded lid under the test plate for counting to keep track of worms previously counted (Figure 1A-B).
		<ol>
			<li>Using a differential cell counter, score the day 1 plates for the presence or absence of progeny. Count the live larvae and unhatched embryos. 
			NOTE: At this point, a sufficient time has lapsed such that any viable embryos should have hatched. Any unhatched embryos are presumed dead.</li>
			<li>Within an individual square, count the unhatched embryos and live larvae that are entirely within the square (Figure 1C-F).</li>
			<li>For worms that are on the square borders, count based on the location of the worm head. Count worms with heads touching the top and left edges of the square (do not count those touching the bottom or right edges; Figure 1D). Do not count unfertilized oocytes for this assay (Figure 1F).</li>
		</ol>
		</li>
		<li>Record the number of live larvae and unhatched embryos in a laboratory notebook.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Score the day 2 plates by counting the live larvae and unhatched embryos, as described in step 2.3.3.1. Record the number of live larvae and unhatched embryos in a laboratory notebook.</li>
	</ol>
	</li>
	<li>Day 5
	<ol>
		<li>Score the day 3 plates by counting the live larvae and unhatched embryos, as described in step 2.3.3.1. Record the number of live larvae and unhatched embryos in a laboratory notebook. Analyze the data obtained using the method described in the data analysis. 
		NOTE: Some genetic mutants may have either a delayed or expanded reproductive cycle. Monitor individual strains for the continued laying of embryos beyond the day 3 plates. If embryo production has not ceased by day 4, continue to transfer adults to new plates.</li>
	</ol>
	</li>
</ol>
3. Embryonic viability assays (male/hermaphrodite cross-fertilization)
<ol>
	<li>Day 1
	<ol>
		<li>Label the back of each plate. Be sure to keep track of each plate and their respective worms throughout the experiment.</li>
		<li>Transfer an individual L4 hermaphrodite worm onto each plate. Ensure no embryos or other worms are transferred onto the plate. 
		NOTE: Feminized strains, such as fog-2 loss-of-function mutants, which do not produce sperm, can be used in place of hermaphrodites.</li>
		<li>Transfer a single L4 male worm onto each labeled plate containing a L4 hermaphrodite. Ensure no embryos or other worms are transferred onto the plate. 
		NOTE: Strains such as plg-1 variant males can be used to identify whether mating has occurred, as these males deposit a copulatory plug on the hermaphrodite vulva after mating.</li>
		<li>Allow the worms to mate and lay progeny for 24 h at 20 &#176;C, or another appropriate temperature. Score these plates for live larvae versus unhatched embryos on day 3.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Label a set of new plates and transfer the worms, as in day 2 above. In this case, make sure to transfer both the hermaphrodite and male onto new plates. Ensure that the hermaphrodite has reached adulthood.</li>
		<li>Allow the hermaphrodites to lay progeny for 24 h at 20 &#176;C, or another appropriate temperature. Score these plates for live larvae versus unhatched embryos on day 4.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Label a set of new plates and transfer the worms, as in day 3 above. Make sure to transfer both the hermaphrodite and male onto new plates.</li>
		<li>Allow to lay progeny for 24 h at 20 &#176;C, or another appropriate temperature. Score these plates for live versus unhatched embryos on day 5.</li>
		<li>Using a differential cell counter, count the live larvae and unhatched embryos from the day 1 plates, as described in step 2.3.3.1. 
		NOTE: Do not discard the plates, as they are required for day 4.</li>
		<li>Record the number of live larvae and unhatched embryos in a laboratory notebook.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Count the live progeny and unhatched embryos from the day 2 plates, as described in step 2.3.3.1. Record the number of live larvae and unhatched embryos in a laboratory notebook.</li>
		<li>Check the day 1 plates for males. If mating has occurred, the expected genetic ratio of hermaphrodites to males should be 50:50. If day 1 plates do not contain any males, mating between the male and hermaphrodite did not occur. Discard this mating pair and record this observation in the laboratory notebook.</li>
	</ol>
	</li>
	<li>Day 5
	<ol>
		<li>Count the live larvae and unhatched embryos from the day 3 plates, as described in step 2.3.3.1. Record the number of live larvae and unhatched embryos in a laboratory notebook. Analyze the data obtained using the methods described in the data analysis.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Calculate the embryonic viability percentage of a given biological replicate of a strain using equation (1). 
	NOTE: The numbers of live progeny and unhatched embryos are obtained by totaling the daily count across the experimental period. 
	<img alt="Equation 1" src="/files/ftp_upload/65064/65064eq01.jpg" style="margin: auto;" />&#160; &#160; &#160; (1)</li>
	<li>Calculate the brood size per worm of a given strain by summing the number of progeny (unhatched embryos and larvae) produced by the parent hermaphrodite. Calculate the average brood size using equation (2). 
	<img alt="Equation 2" src="/files/ftp_upload/65064/65064eq02.jpg" style="margin: auto;" />&#160; &#160; &#160; (2)</li>
	<li>Report the embryonic viability percentage and average brood size with the average and standard deviation between biological replicates. Perform Student&#39;s t-test comparing the control and experimental data.</li>
</ol>

<ol>
	<li>Hillers, K., Jantsch, V., Martinez-Perez, E., Yanowitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis.">Meiosis.</a> WormBook: the Online Review of C. elegans Biology. , (2017).</li><li>O'Connor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+abnormalities:+aneuploidies.">Chromosomal abnormalities: aneuploidies.</a> Nature Education. 1 (1), 172 (2008).</li><li>Marcello, M. R., Singaravelu, G., Fertilization Singson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization.">Fertilization.</a> Advances in Experimental Medicine and Biology. 757, 321-350 (2013).</li><li>Marcello, M. R., Singson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+and+the+oocyte-to-embryo+transition+in+C.+elegans.">Fertilization and the oocyte-to-embryo transition in C. elegans.</a> BMB Reports. 43 (6), 389-399 (2010).</li><li>Robertson, S., Lin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oocyte-to-embryo+transition.">The oocyte-to-embryo transition.</a> Advances in Experimental Medicine and Biology. 757, 351-372 (2013).</li><li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transparent+window+into+biology:+A+primer+on+Caenorhabditis+elegans.">A transparent window into biology: A primer on Caenorhabditis elegans.</a> WormBook: the Online Review of C. elegans Biology. , 1-31 (2015).</li><li>Hodgkin, J., Horvitz, H., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondisjunction+mutants+of+the+nematode+CAENORHABDITIS+ELEGANS.">Nondisjunction mutants of the nematode CAENORHABDITIS ELEGANS.</a> Genetics. 91 (1), 67-94 (1979).</li><li>Chu, D. S., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis.">Spermatogenesis.</a> Advances in Experimental Medicine and Biology. 757, 171-203 (2013).</li><li>Dernburg, A. F., 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=Meiotic+recombination+in+C.+elegans+initiates+by+a+conserved+mechanism+and+is+dispensable+for+homologous+chromosome+synapsis.">Meiotic recombination in C. elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis.</a> Cell. 94 (3), 387-398 (1998).</li><li>Bhandari, N., 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=Identification+of+suppressors+of+top-2+embryonic+lethality+in+Caenorhabditis+elegans.">Identification of suppressors of top-2 embryonic lethality in Caenorhabditis elegans.</a> G3. 3 (4), 1183-1191 (2020).</li><li>Jaramillo-Lambert, A., Fabritius, A. S., Hansen, T. J., Smith, H. E., Golden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+a+novel+mutant+allele+of+topoisomerase+II+in+Caenorhabditis+elegans+reveals+a+unique+role+in+chromosome+segregation+during+spermatogenesis.">The identification of a novel mutant allele of topoisomerase II in Caenorhabditis elegans reveals a unique role in chromosome segregation during spermatogenesis.</a> Genetics. 204 (4), 1407-1422 (2016).</li><li>Jaramillo-Lambert, A., Golden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C-terminus+of+SPE-11+is+required+for+proper+embryonic+development+in+C.+elegans.">The C-terminus of SPE-11 is required for proper embryonic development in C. elegans.</a> microPublication Biology. , (2020).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: the Online Review of C. elegans Biology. , 1-11 (2006).</li></ol>

The authors have no conflicts of interest to declare.

Propagation of sexual reproducing species requires the formation of haploid gametes (i.e., eggs and sperm) through meiosis, which are then unified at fertilization, restoring the diploid chromosome number and initiating embryonic development. Errors in any of these processes can lead to infertility, embryonic lethality, and/or birth defects. C. elegans is a powerful model system to study sexual reproduction. The effects of gene mutations or gene expression knockdown (e.g., RNA interference) can be assessed relatively quickly and easily using the embryonic viability and brood sizing assays described above. We have used these methods for the initial characterization of genes involved in meiotic chromosome segregation and fertilization/egg activation10,11,12. An observed reduction in embryonic viability or brood size indicates a disruption in meiosis, gametogenesis, fertilization, or embryogenesis.
As embryonic viability and brood sizing are assessed relatively easily through counting progeny and a simple mathematical calculation, these are optimal introductory experiments for research novices either in the laboratory or the classroom. The ease of C. elegans husbandry and economic advantages make them particularly suited to experimental biology classes. The students gain valuable research experience through C. elegans husbandry, learn to use dissecting microscopes, and can ask biological questions in a developmental system that can be answered in a relatively short amount of time (approximately 5 days with the protocol described in this paper).
The timing of progeny counts is very important for embryonic viability assays. At 20 &#176;C, embryogenesis takes approximately 16 h, and reproductively mature adults begin laying eggs approximately 60 h after hatching as L1 larvae. As the life cycle is rapid, it is important to count progeny within the appropriate window, allowing sufficient time for embryos to hatch but before the progeny themselves begin to lay eggs. It is also important to note that growth periods vary depending on temperature. Growth is approximately 2.1 times faster at 24-25 &#176;C than 15-16 &#176;C, and approximately 1.3 times faster at 20 &#176;C than 15-16 &#176;C13. In this protocol, we recommend that counts occur 48 h after the adults are placed on a fresh plate. This timeframe ensures that all embryos with wild-type development have sufficient time to hatch (&#62;16 h), but do not age to the point of reproductive capability. Assays performed at temperatures lower than 20 &#176;C may need to be extended (transfer animals for 4 days) for embryos to hatch and for hatched progeny to reach larval stages that are easier to observe among the bacteria on the MYOB plates (L3-L4 stages).
A limitation to embryonic viability assays and brood sizing is that the specific developmental process that is perturbed is not readily apparent. However, these initial assays can be followed up with cytological techniques to determine which process is affected. For example, dissection of the adult worms to release the gonad followed by 4&#39;,6-diamidino-2-phenylindole (DAPI) staining and careful analysis of DNA morphology within the germline can reveal whether meiotic processes are disrupted. In addition, DAPI staining of embryos can reveal at which stage embryogenesis arrests.
In conclusion, we have described a protocol for assaying the number of embryos produced (brood) and the percentage of embryos that are viable for various C. elegans mutants. This assay can be used for both self-fertilizing hermaphrodites and for male/hermaphrodite crosses. With the short life cycle of C. elegans, this protocol can be completed in less than 1 week. Embryonic viability assays and brood sizes can be used as first analyses of genes involved in meiosis, fertilization, or embryonic development, and are appropriate protocols for more advanced researchers and research novices (undergraduate and first-year graduate students) alike.

Sexual reproduction in eukaryotic organisms requires the production of functional gametes that merge to form an embryo through the process of fertilization. Maternal and paternal gametes, ovum (eggs), and sperm are created through the specialized cell division and differentiation processes of meiosis and gametogenesis1. Meiosis starts with a single diploid cell and ends with the formation of daughter cells that contain half the number of chromosomes of the original parental cell. From the reduction of ploidy to the shuffling of genetic material via independent assortment and crossover recombination, meiosis serves multiple important functions1. Errors within meiosis can result in aneuploidy, in which there are too many or too few chromosomes within a gamete. Incidences of aneuploidy have tremendous impacts on human health, as chromosomal imbalances are a major cause of miscarriages and developmental disorders such as Down syndrome and Edwards syndrome2.
Fertilization is the process by which the maternal and paternal gametes fuse to generate a new organism3. Gamete-gamete recognition is facilitated by proteins on the gamete surface3. Errors with gamete compatibility lead to infertility as sperm and egg fusion is unable to proceed. The fusion of sperm with an oocyte triggers a host of events that lead to the proper formation of an active embryo that can begin the developmental journey from a single-cell embryo to a fully functional multicellular organism via mitotic divisions4. Throughout embryogenesis, molecular events that regulate development must be tightly regulated and precisely timed to enable proper growth of the organism5. Proper cellular differentiation during early development is crucial as the organism transitions from a pluripotent embryo to a fully-fledged organism. Due to the complexities of these events, disruptions can lead to developmental defects that result in embryonic lethality.
Caenorhabditis elegans is an excellent model organism to study meiosis, fertilization, and embryonic development. C. elegans is a transparent nematode that has two sexes, males and hermaphrodites. C. elegans hermaphrodites, which are capable of self-fertilization, are the predominant sex6,7. The hermaphrodite gonad first produces sperm during the fourth larval (L4) stage, which are stored in the spermatheca. At the L4 to adulthood transition, the germ line switches to producing oocytes, which are then fertilized via the stored sperm. Males, which arise in hermaphrodites at a rate of less than 0.2%, only produce sperm and can mate with hermaphrodites. Upon cross-fertilization, male sperm outcompete hermaphrodite sperm in the fertilization of oocytes8. This allows for the relatively easy maintenance of homozygous mutants through self-fertilizing stocks and for genetic manipulations through genetic crosses.&#160;The two sexes allow for studies exploring differences between meiosis in male and female germ lines. Further, due to the transparent nature of C. elegans and its eggs, the processes of meiosis, gametogenesis, fertilization, and embryogenesis can be studied in live, intact animals using fluorescence imaging techniques.
When analyzing new mutations in genes that may play a role in meiosis, fertilization, and/or embryonic development in C. elegans, a crucial first step is determining embryonic viability and brood size since errors in these processes often lead to a failure or reduction in the production of viable progeny. This paper describes a protocol for assessing fertility, embryonic viability, and brood size from either self-fertilizing hermaphrodites or crosses between hermaphrodites and males. While this classic assay has been used in many C. elegans studies, we provide a standardized protocol for setup and accurate quantification. In this protocol, individual worms or male/hermaphrodite pairs are isolated to allow mating and progeny production. Progeny production and viability are observed over a series of days to determine the number of viable progeny and non-viable embryos. At the conclusion of the experiment, individual broods are analyzed to calculate the embryonic viability percentage and total brood size.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Tritech research</td>
			<td>T3501</td>
			<td>Semi-stackable, non-vented.</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Becton, Dickinson and Company</td>
			<td>214010</td>
			<td>For MYOB</td>
		</tr>
		<tr>
			<td>Bacto-Peptone</td>
			<td>Gibco</td>
			<td>211677</td>
			<td>For MYOB</td>
		</tr>
		<tr>
			<td>Cholestrol</td>
			<td>Sigma&#160;</td>
			<td>C8503</td>
			<td>For MYOB</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>J.T. Baker</td>
			<td>FW 58.440</td>
			<td>For MYOB</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>For MYOB</td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td>For MYOB</td>
		</tr>
		<tr>
			<td>Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans wild type strain</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N2</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td></td>
		</tr>
		<tr>
			<td>him-5(e1490)</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>DR466</td>
			<td></td>
		</tr>
		<tr>
			<td>spo-11(ok79)</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>AV106</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment/software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Differential cell counter</td>
			<td>Fischer Scientific</td>
			<td>02-670-12</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroSoft Excel or Prism</td>
			<td>MicroSoft or GraphPad</td>
			<td></td>
			<td>For recording and creating graphical representations of data.</td>
		</tr>
		<tr>
			<td>platinum wire</td>
			<td>Tritech research</td>
			<td>PT9901</td>
			<td>For making worm picks. 99.5% Platinum, 0.5% Iridium. This comes as 3 ft/pack, which is sufficient for making 36 worm picks (~1 inch platinum wire per pick).</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ-745</td>
			<td>Diascopic base with focus mount, integrated LED module, power cord, 6.7x to 50x Zoom range [WD 115 mm], Widefield Eyepiece C-15x/17 (Note: equivalent stereomicroscopes are available from other manufacturers.)</td>
		</tr>
		<tr>
			<td>worm pick handle</td>
			<td>Tritech research</td>
			<td>TWPH1</td>
			<td>For making worm picks. Mount ~1 in platinum wire into worm pick handle. Alternatively, worm picks can be made by mounting platinum wire in a glass Pasteur pipette.</td>
		</tr>
	</tbody>
</table>

measuring embryonic viability and brood size in caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

We performed embryonic viability and brood sizing assays on N2 (wild type) and on two strains harboring mutations in genes involved in meiosis, him-5(e1490) and spo-11(ok79). As both him-5 and spo-11 play a role in meiotic crossover formation, mutations in these two genes result in the formation of aneuploid gametes. This embryonic viability assay for N2 yielded a viability percentage of 98.9%, while both him-5(e1490) and spo-11(ok79) showed a reduction in progeny viability with a percentage of 74.9% and 0.8%, respectively (Figure 2A; p &#60; 0.0005). These results are consistent with previously published results7,9. The average brood size of N2, him-5(e1490), and spo-11(ok79) were determined to be 217, 105, and 219, respectively (Figure 2B). Consistent with previous publications, him-5(e1490) has a significantly reduced brood size compared to wild type, whereas spo-11(ok79) does not7,9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65064/65064fig01.jpg" /> 
Figure 1: Demonstration of counting setup and embryo morphology on plates. (A) Image of a lid with a cross-hatched grid pattern drawn using a fine sharpie. (B) A 35 mm MYOB plate with the patterned lid underneath for counting. (C) Image demonstrating a low magnification (10x) field of view showing multiple boxes of the grid. White arrowheads point out several larvae within this field of view. Counting should be done at a higher magnification (only one square in field) to observe both larvae and embryos. Scale bar = 1,000 &#181;m. (D) Cartoon of the lid with the grid pattern placed under a 35 mm plate. The inset denotes the proper technique to determine which larvae are counted in a given square. Count from top to bottom, left to right. Count any worms that are entirely within the square. Worms touching the boundary should be counted based on the position of the worm head, not the tail. Count worms facing the current grid or grids that have been previously counted (i.e., top and left edges). Do not count worms with heads touching the bottom or right edges. From the inset, blue worms will be counted while red worms will not. (E) Representative image of healthy N2 L1 hatched larvae (white arrowhead). (F)&#160;Representative image of an unfertilized oocyte (black arrowhead) and an unhatched embryo (red arrowhead). Unfertilized oocytes should not be counted for this assay. Scale bars (E,F) = (100 &#181;m). Note: Images in C, E, and F were taken with a Zeiss AxioZoom microscope, with a camera attached for the purpose of demonstrating the appearance of larvae, embryos, and oocytes on worm plates. For embryonic viability assays, counts are done while observing the plates using a stereomicroscope (no camera). <a href="https://www.jove.com/files/ftp_upload/65064/65064fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65064/65064fig02.jpg" /> 
Figure 2: Representative graphs depicting the results from embryonic viability assays and brood sizes. (A) Embryonic viability percentage of N2, him-5(e1490), and spo-11(ok79) at 20 &#176;C. (B) Brood sizes of N2, him-5(e1490), and spo-11(ok79) at 20 &#176;C. The progeny of at least 28 hermaphrodites were scored for each strain. The total number of unhatched embryos plus larvae scored were N2 = 6302, him-5(e1490) = 2,945, and spo-11(ok79) = 7,230. Error bars indicate the standard deviation across three separate individual replicates. Statistics calculated using Student&#39;s t-test, n.s. = not significant, *p &#60; 0.0005. <a href="https://www.jove.com/files/ftp_upload/65064/65064fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

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2.4K Views.  University of Delaware. Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Video: Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

Assisted Selection of Biomarkers by Linear Discriminant Analysis Effect Size (LEfSe) in Microbiome Data

There is growing attention toward closed biological genomes in the environment and in health. To explore and reveal the intergroup differences among different samples or environments, it is crucial to discover biomarkers with statistical differences among groups. The application of Linear discriminant analysis Effect Size (LEfSe) can help find good biomarkers. Based on the original genome data, quality control, and quantification of different sequences based on taxa or genes are carried out. First, the Kruskal-Wallis rank test was used to distinguish between specific differences among statistical and biological groups. Then, the Wilcoxon rank test was performed between the two groups obtained in the previous step to assess whether the differences were consistent. Finally, a linear discriminant analysis (LDA) was conducted to evaluate the influence of biomarkers on significantly different groups based on LDA scores. To sum up, LEfSe provided the convenience for identifying genomic biomarkers that characterize statistical differences among biological groups.

Assisted Selection of Biomarkers by Linear Discriminant Analysis Effect Size LEfSe in Microbiome Data

LEfSe (LDA Effect Size) is a tool for high-dimensional biomarker mining to identify genomic features (such as genes, pathways, and taxonomies) that significantly characterize two or more groups in microbiome data.

There is growing attention toward closed biological genomes in the environment and in health. To explore and reveal the intergroup differences among ...

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.