Name: measuring embryonic viability and brood size in caenorhabditis elegans
Uploaded: 2023-02-24T13:50:00.000+00:00
Duration: 6 min 24 s
Description: Watch this Scientific Journal Video about Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans at JoVE.com

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><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</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&amp;nbsp;</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>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;C. elegans &lt;/em&gt;wild type strain</td><td>Caenorhabditis Genetics Center</td><td>N2</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Caenorhabditis Genetics Center</td><td>OP50</td><td></td></tr><tr><td>&lt;em&gt;him-5(e1490)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center</td><td>DR466</td><td></td></tr><tr><td>&lt;em&gt;spo-11(ok79)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center</td><td>AV106</td><td></td></tr><tr><td>&lt;strong&gt;Equipment/software&lt;/strong&gt;</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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Research

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Genetics

2.5K 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

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 ...

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Biology

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Developmental Biology

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Neuroscience

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Medicine

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Lifespan Analysis: Measuring C. elegans Longevity

Source: Cornwell, A. B., et al. <a href="https://www.jove.com/video/57819/the-replica-set-method-high-throughput-approach-to-quantitatively">The Replica Set Method: A High-throughput Approach to Quantitatively Measure&#160;Caenorhabditis elegans&#160;Lifespan</a>.&#160;J. Vis. Exp.&#160; (2018).
This video describes a traditional, plate-based method of measuring the lifespan of C. elegans. The example protocol measures the lifespan of worms treated with RNAi.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Summary

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Chapters in this video

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

Measuring Caenorhabditis elegans Life Span on Solid Media

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Lifespan Analysis: Measuring C. elegans Longevity