Name: a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization
Uploaded: 2009-07-06T19:03:00
Duration: 11 min 1 s
Description: Watch this Scientific Journal Video about A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization at JoVE.com

This work was supported by NIH R01 HG002995 "TILLING the Zebrafish Genome: a Reverse Genetics Approach" to CBM.

Part A: Sperm Freezing Solutions
1. Ginsberg Fish Ringers (Make fresh every 3 days ;Ginsburg, 1963)
NOTE: ORDER OF ADDITION IS IMPORTANT TO PREVENT PRECIPITATION
In 450 ml sterile ddH2O dissolve:
<ul>
<li>NaCl 3.25 g</li>
<li>KCl 0.125 g</li>
<li>CaCl2&bull;2 H2O 0.175 g 
 Then add:</li>
<li>NaHCO3 0.10 g</li>
</ul>
Bring final volume to 500 ml with sterile ddH2O and store at 4&deg;C.
2. Freezing Medium (Make fresh daily)
<ol style="list-style-type:lower-roman">
<li>WITHOUT Methanol
<ul><li>Ginsburg Fish Ringers 10 ml (room temp.)</li>
<li>Powdered Skim Milk 1.5 gm</li>
</ul></li>
<li>WITH Methanol
 NOTE: ORDER OF ADDITION IS IMPORTANT TO PREVENT PRECIPITATION 
<ul>
<li>Ginsburg Fish Ringers 9 ml (room temp.)</li>
<li>Methanol 1 ml</li>
<li>Powdered Skim Milk 1.5 g
</li></ul>
 After assembling freezing media, mix well for 20 min. on orbital shaker or rocker. Before using, aliquot into 1 ml eppendorf tubes, avoiding surface bubbles. 
</li></ol>
Part B: Materials needed for sperm freezing
Materials needed for sperm freezing
<ol>
 <li>10 &mu;l disposable pipettes (Fisherbrand cat# 22-358697) </li>
 <li>250ml beakers for fish water containing MESAB/Tricaine </li>
 <li>watch glasses (Pyrex cat# 9985-75) </li>
 <li>P20 pipetman (or equivalent) and tips </li>
 <li>Sponge fish holder (to hold male while squeezing) </li>
 <li>Dissecting microscope with above stage lighting </li>
 <li>Flat forceps (e.g. Millipore type) </li>
 <li>2.0ml cryogenic vials (Corning cat# 430488) </li>
 <li>10X10 cryoboxes (Nalgene cat#03-337-7AA) </li>
 <li>Styrofoam container filled with ~ 15 cm of finely crushed dry ice1 </li>
 <li>15 ml conical tubes (Falcon # 352099) </li>
 <li>Dewar flask containing liquid nitrogen </li>
 <li>Long tongs (e.g. CMS Fisher Health Care cat# 10-316B) </li>
 <li>Cryogloves </li>
 <li>Recovery tank for males </li>
 <li>One eppendorf tube filled with freezing media without methanol </li>
 <li>One eppendorf tube filled with freezing media with methanol</li>
 <li>Cryofreezer (e.g. Taylor-Warton K-series)</li>
</ol>
1 A simple method for making finely crushed dry ice is to wrap it in a towel and pulverize it with a hammer. A more efficient method is to use a dry ice grinder, e.g. Clawson model RE-2.
Part C: Sperm Freezing Method
<ol>
<li>MARK CAPILLARY TUBES: Prior to beginning, mark the 10&mu;l capillary tubes with a lab pen at the level of 3.33 &mu;l (i.e. 16.7 mm from bottom; see Fig. 1.1).</li>
<li>ANESTHETIZE MALE: Place male(s) in beaker containing MESAB/tricaine diluted in fish water (recipe in "The Zebrafish Book")</li>
<li>DRY FISH: Once anesthetized, lift fish out of anesthetic using a plastic spoon and gently blot fish dry on a paper towel, paying special attention to drying the ventral side. Water activates sperm so it is important to thoroughly dry around the cloaca. Position fish on sponge holder, ventral side up (Fig. 1.2). If region around anal fin is still moist, gently dry with a kimwipe. Take care to not squeeze sperm prematurely!</li>
<li>Place marked capillary tube in rubber mouth pipette adapter that is included with capillary tubes. The adaptor is a rubber hose that has a mouthpiece on one end and a capillary holder on the other. Alternatively, the capillary tube can be connected to a 50&mu;l Hamilton syringe via a short piece of tygon tubing of the appropriate diameter.</li>
<li>Place male under the scope and expose the urogenital opening by carefully spreading the pelvic fins apart using the end of the capillary tube.</li>
<li>COLLECT SPERM: Expel sperm by gently stroking the sides of the fish with smooth forceps (e.g. Millipore), or by gently squeezing the sides of the fish between your index finger and thumb. Collect sperm in capillary tube as it is expelled using gentle suction (Fig. 1.2). Avoid feces that may be expelled with sperm.</li>
<li>NORMALIZE SPERM VOLUME TO 3.3 &mu;l: If the volume of sperm reaches, or exceeds pen mark on capillary tube (i.e. 16.7 mm or greater), then proceed to step 8. If sperm volume does not reach target volume, then normalize volume to pen mark using Freeze Medium WITHOUT Methanol (Fig. 1.3). Minimum amount of sperm that is acceptable varies with quality of sperm. Good sperm is white and opaque. Poor sperm looks watery. In general, we accept as minimums: 1 &mu;l good sperm (or 1/3 target) and 2 &mu;l poor sperm (or 2/3 target). Volume is now normalized to 3.3 &mu;l.</li>
<li>ADD CRYOPROTECTANT TO SPERM : Suck up Freezing Medium WITH Methanol to the orange mark (approx. total volume is now 20 &mu;l; Fig. 1.4). Expel sperm and cryoprotectant mixture onto clean area of a watch glass, paying special attention to not introduce bubbles. Gently mix by pipetting up and down ~ 4 times (avoid introducing bubbles).</li>
<li>PLACE 10&mu;l SPERM INTO EACH OF 2 CRYOVIALS: Pipette 10 &mu;l of the sperm:cryoprotectant solution into the bottom of two separate cryovials that have been labeled with relevant information (Fig. 1.5). Moving quickly, cap vials and drop them into the bottom of room temperature 15 ml Falcon tubes- one cryovial/tube. Cap Falcon tube and insert the pair of cryovial-containing Falcon tubes into crushed dry ice.</li>
<li>FREEZE SPERM FOR 20 MIN. ON DRY ICE: Dry ice should be in fine powder form, not pellets. The tubes should be inserted into the dry ice deep enough that only their caps show (Fig. 1.6). In order to keep track of tubes in dry ice, give each pair of Falcon tubes a number from 1-20 (written on the caps) and record the time they go into the dry ice.</li>
<li>PLACE CRYOVIALS INTO LIQUID NITROGEN: After the sperm has been frozen on dry ice for 20 min., transfer them to a liquid nitrogen- containing dewar flask. Sperm is stored here until time to place into liquid nitrogen freezer. When placing in the freezer boxes, place freezer box in a bath of liquid nitrogen so that samples do not heat up. Use long metal tongs to recover vials from dewar flask and handle with cryogloves. Alternatively, cryovials can be transferred directly from dry ice into the freezer box if the freezer box is kept immersed in liquid nitrogen in a Styrofoam container.</li>
<li>Store cryovials long term in a cryogenic liquid nitrogen freezer. To maintain viability of sperm, it is important that vials are stored immersed in liquid nitrogen. In our experience, vials stored in the vapor phase lose viability over time. Speed is important! The time between adding the cryoprotectant and placing sperm vials in dry ice (i.e. steps 6-10) should be no more than 30 sec. </li></ol>
Part D: In Vitro Fertilization Method Using Cryopreserved Sperm
We find that this works best with two people. One person squeezes eggs from females while the other person thaws the sperm.
<ol><li>Set a water bath to 33&deg;C.</li>
<li>Remove cryovial from liquid nitrogen freezer and transfer to liquid nitrogen-containing dewar flask until ready to thaw.</li>
<li>Squeeze eggs from females into 35mm plastic petri dish. If possible, try to obtain 3 clutches of eggs and combine into one dish. If you cannot get three good clutches within 1 min of your first clutch, then proceed to step 4 (one good clutch is sufficient). Definition of good clutch: &gt;150 uniformly nice looking eggs (i.e. yellowish, with no white debris indicative of degradation). Keep dish covered while sperm is thawed.</li>
<li>Remove vial from liquid nitrogen and remove cap. Quickly immerse vial ~1/2 way into 33&deg;C water bath for 8-10 sec.</li>
<li>Quickly add 70 &mu;l room temperature Hanks to vial and mix by pipetting up and down. Immediately add to eggs and mix gently with pipette tip.</li>
<li>Without delay, activate sperm and eggs by adding 750 &mu;l fish water. Swirl to mix. Incubate 5 min at room temperature.</li>
<li>After 5 min, fill dish with fish water and incubate dish at 28&deg;C. After 2-3 hrs, count and transfer fertile embryos to 100mm dishes (50/dish). Count infertile eggs so that fertilization frequency can be determined.</li>
<li>Take good care of the larvae! For first 5 days, change water daily and remove dead embryos. Put only larvae that have swim bladders into nursery on day 5.</li>
</ol>
Part E: Representative Results
We used this crypreservation methodology to make a library of 8600 frozen sperm samples for TILLING between the years 2001 and 2003. We have thawed 106 of these samples and determined their fertility. Fertility is the ratio of viable embryos produced in an in vitro fertilization divided by the total number of eggs that were fertilized with the frozen sperm. Fresh (non cryporeserved) sperm typically has &gt;95% fertility; cryopreserved sperm has much lower fertility: an average of 29% (n=106 vials). The vial-to-vial variability in fertility is large, ranging from less than 1% to as high as 62%. This variability is represented by the large error bars representing standard deviation in Fig. 2. However in 106 sperm samples thawed to date we have only once experienced 0% fertility in both sperm samples and failed to recover the corresponding TILLING mutation.
By thawing sperm vials from our library over the course of several years (2001-2008) we have found that the average fertility of sperm samples frozen by this method remains stable over time (Fig. 2). The average fertility of frozen sperm samples thawed in 2001 was 23.5% (based on 11 sperm samples) and in 2008 was 25% (based on 21 sperm samples).
A number of other investigators are now using this sperm freezing protocol in their labs. Some have reported good success with fertility rates and sample-to-sample variability similar to what we have observed. Others have reportedly failed to make this protocol work in their hands. We believe that likely sources of problems are:
<ol>
<li>Failure to use the exact cryovials or conical tubes we have suggested in the protocol, leading to a different rate of cooling when in dry ice;</li>
<li>Failure to use powdered dry ice, leading to a sub-optimal cooling rate;</li>
<li>Using male fish with poor fertility: you should get at least 1 &mu;l of sperm from a single male. You can also test male fertility by doing an in vitro fertilization with fresh sperm, however this can be misleading because it takes very little fresh sperm to get near 100% fertility.</li>
<li>Using sub-standard quality eggs in the in vitro fertilization. Using anything but perfect clutches always leads to near 0% fertility, even from sperm samples that actually have good fertility (based on the fertility of the duplicate vial).</li>
</ol>
<img src="/files/ftp_upload/1395/1395fig1.jpg" alt="Figure 1" /> Figure 1. Schematic of sperm freezing methodology.
<img src="/files/ftp_upload/1395/1395fig2.jpg" alt="Figure 2" /> Figure 2. Cryopreserved sperm fertility remains stable over time. Percent fertility is the number of viable embryos produced in an in vitro fertilization divided by the total number of eggs that were fertilized with the frozen sperm. "n" refers to the number of cryopreserved sperm vials whose fertility was tested in the corresponding year. Error bars represent standard deviation. Since each sperm vial is from a different male, vial-to-vial variability is observed.

<ol>
	<li>Ginsburg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm-egg+association+and+its+relationship+to+activation+of+the+egg+in+salmonid+fishes.">Sperm-egg association and its relationship to activation of the egg in salmonid fishes.</a> J. Embryol. Exp. Morphol. 11, 13-33 (1963).</li><li>Harvey, B., Kelley, R. N., Ashwood-Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+zebra+fish+spermatozoa+using+methanol.">Cryopreservation of zebra fish spermatozoa using methanol.</a> Can. J. Zoology. 60, 1867-1870 (1982).</li></ol>

A number of other investigators are now using this sperm freezing protocol in their labs. Some have reported similar fertility and the same sample-to-sample variability that we have observed. Others have reportedly failed to make this protocol work in their hands. We believe that likely sources of problems are: 
<ol>
<li>Failure to use the exact cryovials or conical tubes we have suggested in the protocol, leading to a different rate of cooling when in dry ice;</li>
<li>Failure to use powdered dry ice, leading to a sub-optimal cooling rate;</li>
<li>Using male fish with poor fertility: you should get at least 1 &mu;l of sperm from a single male. You can also test male fertility by doing an in vitro fertilization with fresh sperm, however this can be misleading because it takes very little fresh sperm to get near 100% fertility. Larger, more robustly colored males typically give more higher quality sperm.</li>
<li>Using sub-standard quality eggs in the in vitro fertilization. Using anything but perfect clutches always leads to near 0% fertility, even from sperm samples that actually have good fertility (based on the fertility of the duplicate vial). </li>
</ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>10 &mu;l disposable pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-358697</td>
<td>&nbsp;</td>
<tr>
<td>Watch glasses</td>
<td>&nbsp;</td>
<td>Pyrex</td>
<td>9985-75</td>
<td>&nbsp;</td>
<tr>
<td>2 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>10 x 10 cryoboxes</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>03-337-7AA</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>Tongs</td>
<td>&nbsp;</td>
<td>Fisher Health Care</td>
<td>10-316B</td>
<td>&nbsp;</td>
<tr>
<td>Cryofreezer</td>
<td>&nbsp;</td>
<td>Taylor-Warton</td>
<td>K-series</td>
<td>For example</td>
</tr>		</tbody>
		</table>
		</div>

a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

Watch this Scientific Journal Video about A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization at JoVE.com

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

a-high-throughput-method-for-zebrafish-sperm-cryopreservation-vitro

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High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

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The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...