1. Generating embryos through natural mating
<ol> 
	<li>Culture embryos to 3 months of age, reproductive maturity.</li>
	<li>Segregate adult male and female fish from the desired strain into divided mating tanks on the evening before embryo collection, and add 2 males and 3 females to each tank. 
	NOTE: Use of the transgenic&#160;insulin2a:mCherry&#160;fluorescent reporter strain allowed the analysis of pancreatic &#946;-cells.</li>
	<li>Transfer fish to the mating tank with fresh system water and remove the divider immediately after the lights come on the following morning.</li>
	<li>Allow the fish to mate naturally until embryos are observed in bottom tank. Collect embryos in 30 min intervals until the desired amount is collected. Store each collected time point in separate Petri dishes in embryo media at 28.5 &#176;C.</li>
	<li>Perform microinjection of genetic material or placement in experimental culture media, if desired, and culture the embryos in fresh Hank&#39;s embryo medium&#160;in 10-cm Petri dishes at 28.5 &#176;C. 
	NOTE: For gene expression analysis in morpholino (MO) injected or mutant animals, note that each manipulation may have incidental impacts on gene expression. Mutants can exhibit genetic compensation at the level of transcription that is not observed by MO-based gene targeting.</li>
</ol>
2. Stage embryos
<ol>
	<li>Culture the embryos in groups of&#160;50&#8211;75 embryos&#160;per 10-cm Petri dish to promote consistent developmental timing of all embryos.</li>
	<li>Monitor the developmental morphology using dissecting microscope at blastomere, epiboly, and somite stages to ensure developmental progression. 
	NOTE: Remove any dying or malformed embryos to prevent developmental delay in the dish.</li>
	<li>Separate the embryos based on the developmental age. Stage the embryos and larvae using total body length after 24 hpf. 
	NOTE: The somites are the chevron-shaped mesodermal tissue present on the dorsal part of the embryo.</li>
</ol>

<table><tbody><tr><td>ins2a:mCherry</td><td>ZIRC&amp;nbsp;</td><td>ZL1483</td><td></td></tr><tr><td>Mating tanks 1.0L Crossing Tank Set&amp;nbsp;</td><td>Aquaneering&amp;nbsp;</td><td>ZHCT100</td><td></td></tr><tr><td>Dissecting Microscope&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

mating and egg staging: a method to generate embryos and sort them by developmental stage

Source: Hostelley T.L., et al. <a href="https://www.jove.com/v/56187/" target="_blank">Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish</a>. J. Vis. Exp. (2017)
The article describes a protocol to generate zebrafish embryos through natural mating and sorting their developmental stages. The protocol is used further for analysis of pancreatic &#946;-cells by using transgenic fishes.

Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage

Source: Hostelley T.L., et al. Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish. J. Vis. Exp. (2017)
The article ...

- Start with a mating tank, a tank with a removable insert that allows eggs to fall through. Add two male and three female adult fish to a divided mating tank in the evening before embryo collection to acclimate to each other overnight.
The next morning, transfer the fish into a new mating tank containing system water and remove the divider. Wait for 15 to 30 minutes to give the fish time to mate and spawn.
Now collect the embryos and store them in a Petri dish containing Hank&#39;s embryo medium. Grow them at 28.5 degrees Celsius throughout the protocol. Examine embryos&#39; morphology under a dissecting microscope.
You will observe several developmental features. Cleavage of the blastodisc gives rise to multiple cells. The blastoderm takes the shape of an inverted cup above the yolk cell. Paraxial mesoderm segments develop on the dorsal part of the embryo and so on.
Beyond 24 hours post-fertilization, simply sort embryos by total body length. In the example protocol, we will set up mating of transgenic mCherry reporter fish and sort the resulting embryos.
- To begin, culture embryos to three months of age, which is reproductive maturity. Segregate two adult male and three female fish from the desired strain into divided mating tanks of fresh system water on the evening before embryo collection. The following morning after the lights come on, remove the divider and allow the fish to mate naturally until embryos are observed in the bottom of the tank.
Collect embryos in 30-minute intervals and separate Petri dishes of embryo medium until the desired number are collected. To stage the embryos, culture them in groups of 50 to 75 per 10-centimeter Petri dish to promote consistent developmental timing of all embryos. Then keep the plates at 28.5 degrees Celsius.
Measure the embryo age using somite number after segmentation until approximately 24 hours post fertilization, or HPF, and separate the embryos based on developmental age.

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Research

JoVE Encyclopedia of Experiments

Biology

Danio rerio (zebrafish)

Embryo

2.5K Views. - Start with a mating tank, a tank with a removable insert that allows eggs to fall through. 

Abstract

Protocol

<table><tbody><tr><td>ins2a:mCherry</td><td>ZIRC&nbsp;</td><td>ZL1483</td><td></td></tr><tr><td>Mating tanks 1.0L Crossing Tank Set&nbsp;</td><td>Aquaneering&nbsp;</td><td>ZHCT100</td><td></td></tr><tr><td>Dissecting Microscope&nbsp;</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage

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ins2a:mCherry	ZIRC	ZL1483
Mating tanks 1.0L Crossing Tank Set	Aquaneering	ZHCT100
Dissecting Microscope	Zeiss