This publication was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), the COST Office (COST Action FA 1205, AQUAGAMETE), and the Research Centre of Excellence - 1476-4/2016/FEKUT.

All procedures for working with European eel described in this protocol were approved by the Committee of Ethics of Animal Experimentation at the Universitat Polit&#232;cnica de Val&#232;ncia, following the Spanish laws and regulations controlling the experiments and procedures on live animals.
1. Fish Maintenance
<ol>
	<li>Bring the European eels to a research facility and put them in a 200 L aquaria with a recirculation system. Use thermostats and coolers to maintain a constant 20 &#176;C temperature.</li>
	<li>Keep the fish in dark conditions to avoid stress22 and with no food during the experiment.</li>
	<li>Keep the fish in fresh water during the first 3 days. Then change 1/3 of the water and refill with seawater every other day until reaching a salinity of 37.0 &#177; 0.3 g/L.</li>
</ol>
2. Hormonal Treatment
NOTE: The hormonal treatment consists of weekly injections of human chorionic gonadotropin (hCG) throughout the whole duration (nine weeks) of the experiment.
<ol>
	<li>Prepare the hCG hormone in advance at a concentration of 1 IU/&#181;L by diluting the hormone in saline solution (0.9% NaCl). 
	NOTE: The hormone can be preserved diluted at this concentration for over a week at -18 &#176;C.</li>
	<li>To anesthetize the fish, prepare a 40 L flexible bucket with 5 L of system water.
	<ol>
		<li>In a 250 mL flask, dilute 300 mg of benzocaine in 100 mL of 70% ethanol.</li>
		<li>Pour the diluted benzocaine in the bucket (final concentration 0.36 mM) and mix properly. This is for a final benzocaine concentration of 60 mg/L.</li>
		<li>Transfer the fish, individually, into the water with benzocaine and wait a few seconds until the benzocaine takes effect and the fish is properly anesthetized. 
		NOTE: To confirm that the fish is anesthetized, place the fish in a supine position and check that it stays still in that position.</li>
	</ol>
	</li>
	<li>To administrate the hormone, weigh the fish and prepare the hCG hormone at a dose of 1.5 IU/g of fish, in a 1.5 mL plastic tube.
	<ol>
		<li>Fill a 1 mL syringe with the hCG hormone from the plastic tube.</li>
		<li>Place the anesthetized fish in supine position and with the assistance or the syringe, inject the hormone carefully in the intraperitoneal area.</li>
	</ol>
	</li>
	<li>Return the fish to the aquaria and monitor it until fully recovered. 
	NOTE: The hormonal administration has to be conducted weekly throughout the experiment. Normally, European eels start spermating after 6-7 weeks of hormonal treatment.</li>
</ol>
3. Sperm Sampling
<ol>
	<li>To obtain the best quality samples, extract sperm 24 h after the hormonal administration6,23.</li>
	<li>Anesthetize the fish with benzocaine as described in the step 2.2.</li>
	<li>Place the anesthetized fish in supine position, clean the genital area with a squirt of distilled water and dry carefully with paper, to avoid feces contamination present in the genital area or accidental sperm activation by contact with seawater from the aquaria.</li>
	<li>Placing the fingers on both lateral sides of the fish, massage carefully pressing laterally from the pectoral fins to the genital area to force the sperm out.
	<ol>
		<li>Repeat the massage until no more sperm comes out from the genital opening.</li>
	</ol>
	</li>
	<li>Collect the sperm into 15 mL plastic tubes using a vacuum pump.</li>
	<li>Dilute the extracted sperm 1:10 in modified Tanaka&#180;s extender solution24 (137 mM NaCl, 76.2 mM NaHCO3, in distilled water) at 4 &#186;C. 
	NOTE: The sperm in the extender solution should be maintained at 4 &#186;C.</li>
</ol>
4. Sperm Quality Evaluation
<ol>
	<li>Prepare artificial seawater13 (in mM: NaCl 354.7, MgCl2 52.4, CaCl2 9.9, Na2SO4 28.2, KCl 9.4, in distilled water) with 2% bovine serum albumin (BSA) (w:v), adjust pH to 8.2, osmolality to 1100 mOsm/kg and maintain it at 4 &#186;C to avoid bacterial growth.</li>
	<li>Open the software for computer-assisted sperm analysis (CASA) and select the fish sperm module.
	<ol>
		<li>Click on Properties to open the system setup of the software. There, set the capture options at 60 images per second. Select negative phase optics, Makler chamber and 10X scale.</li>
		<li>Then on the analysis values, select Fish as species and Particles area bigger than 2 &#181;m2 and smaller than 20 &#181;m2.</li>
		<li>On the velocity parameters, set to slow when cells move between 10 and 45 &#181;m/s, set to medium when moving between 45 and 100 &#181;m/s and set to rapid when moving faster than 100 &#181;m/s. 
		NOTE: Spermatozoa with velocity slower than 10 &#181;m/s were considered immotile.</li>
		<li>Select the progressive values as 80% of straightness (STR) and save the properties.</li>
		<li>Click on Capture Field (a window with the live images from the camera will be opened). 
		NOTE: The computer-assisted sperm analysis system is formed by a microscope, a camera and an image analysis software.</li>
	</ol>
	</li>
	<li>On the counting chamber, put 4 &#181;L of artificial seawater and add 0.5 &#181;L of sperm solution (sperm diluted in extender solution).
	<ol>
		<li>Focus the image with the microscope at 10X magnification in the negative phase. 15 s after activation, click on Capture - from video in the computer-assisted sperm analysis software.</li>
		<li>Analyze every sample in triplicate and select samples with a motility higher than 70% for cryopreservation. 
		NOTE: It is very important to select only high-quality sperm for the success of this cryopreservation protocol.</li>
	</ol>
	</li>
</ol>
5. Sperm Freezing Method
<ol>
	<li>Prepare in advance liquid nitrogen (approximately 2.5 L) in a 34 cm x 34 cm x 30 cm and 5 cm thick Styrofoam box. 
	NOTE: Maintain a level of liquid nitrogen of 4-5 cm height at all times.
	<ol>
		<li>Build a floating structure to pre-freeze the sperm. Use 2 pieces of polystyrene (20 cm x 5 cm), bind them with 2 plastic tubes, and place the structure on the liquid nitrogen. This structure needs to float over the liquid nitrogen at an approximate height of 3 cm over the surface (Figure 1).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg" /> 
Figure 1. Schematic drawing of the floating structure used for pre-freezing over liquid nitrogen. The structure consists of two pieces of low density Styrofoam of 20 cm x 4 cm x 5 cm connected with plastic tubes of 14 cm. The straws are placed over the plastic tubes at 3 cm over the liquid nitrogen. <a href="//cloudfront.jove.com/files/ftp_upload/56835/56835fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Prepare the cryopreservation solution by mixing sperm, extender solution and methanol at ratio of 1:8:1 in 1.5 mL plastic tubes and stir gently.</li>
	<li>With the help of a pipette, add 480 &#181;L of the cryopreservation solution into the 500 &#181;L straws and if necessary, mark them by closing one end with modelling clay.</li>
	<li>Put the straw on the floating device at a height of 3 cm over the liquid nitrogen for 3 min. Then, place the straw into the liquid nitrogen.</li>
	<li>After 10-15 min, transfer the straws into a liquid nitrogen storage tank using long forceps and keep them submerged in liquid nitrogen at all times. Here, the sperm can be stored indefinitely.</li>
</ol>
6. Thawing Method
<ol>
	<li>Prepare a Styrofoam box with liquid nitrogen as described in step 5.1, and prepare a water bath using a 3 L beaker with tap water at 40 &#186;C.</li>
	<li>Transfer the straws with frozen sperm from the storage tank into the Styrofoam box with liquid nitrogen using long forceps.
	<ol>
		<li>Put each straw into a water bath at 40 &#186;C for 13 s.</li>
		<li>Pour the sperm into a 1.5 mL plastic tube by cutting the closed ends of the straw with scissors.</li>
	</ol>
	</li>
	<li>Analyze sperm motility using computer-assisted sperm analysis system as explained in the step 4.1.</li>
	<li>Keep 100 &#181;L of sperm to analyze the viability of spermatozoa with a flow cytometer.</li>
</ol>
7. Flow Cytometry
<ol>
	<li>Use a fluorescent kit containing propidium iodide (PI), which is a red fluorescent compound that stains the nuclei of dead cells, and a membrane-permeant nuclear fluorescent compound, that green stains the nuclei of living cells.
	<ol>
		<li>Prepare the green fluorescent staining solution by diluting it from the stock solution (1 mM) 1:10 in Tanaka&#180;s medium to a working solution of 100 &#181;M.</li>
		<li>Do not dilute the PI solution. The stock solution is at 2.4 mM.</li>
	</ol>
	</li>
	<li>For each sample, take 50 &#181;L of fresh or thawed sperm and add 0.5 &#181;L of green fluorescent staining working solution (final concentration 1 &#181;M) and 2 &#181;L of PI solution (final concentration 100 &#181;M).</li>
	<li>Incubate the samples containing the dyes (PI and green fluorescent staining) in the dark for 5 min.
	<ol>
		<li>Dilute the samples in 500 &#181;L of Tanaka&#180;s extender solution and analyze with the flow cytometer.</li>
	</ol>
	</li>
	<li>Turn on the flow cytometer and create a new protocol containing at least 2 plots: SS log vs FS log and FL1 vs FL3. 
	NOTE: Both, green fluorescent staining and propidium iodide can be excited with visible-wavelength light. When bound to DNA, the maximum fluorescence emission of these dyes are 516 nm and 617 nm, respectively.
	<ol>
		<li>Adjust the voltages of the different lasers: SS= 199; FS= 199; FL1= 377; FL3= 372</li>
		<li>Set up the acquisition settings accord to maximum events = 5000 or 15 s (the final concentration of the sample should be around 1 million of cells/mL). Read the sample using a LOW flow.</li>
		<li>Select the reading mode Single tube fixed position mode.</li>
		<li>Put the sample in the right number of the carousel</li>
		<li>Read the sample (pressing F9) and save the data collected to an excel file (pressing F7) for further analysis.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

This protocol describes the complete process for European eel maturation, handling and sperm cryopreservation. The husbandry conditions described here are optimal for fast maturation and production of high volumes of high-quality sperm in this species 6,7,25. The success of this cryopreservation protocol and its potential use for fertilization after thawing depend greatly on the quality of fresh sperm 26....

Over the last 25 years, the number of European eels (Anguilla anguilla) arriving at the European coast have decreased steadily by 90% 1,2,3. There are several factors that explain this drastic drop including pollution, infections, overfishing and habitat destruction. All of this has had a profound effect on this species, leading to the inclusion of the European eel on the International Union for Conservation of Nature (IUCN) list as critical endangered 4. Consequently, the development of techniques and protocols for reproduction in captivity are necessary.
The maturation of the European eel in captivity is acheived by hormonal treatment 5,6,7 but the production of gametes in both sexes is difficult to synchronize 8. Even though the development of new androgen implants has shown to accelerate oogenesis in eels 9,10, the timing of final maturation in females is still highly variable and difficult to control 11. Therefore, short-term storage of sperm 12,13,14 and cryopreservation techniques are necessary for reproduction management, making gamete synchronization unnecessary 8.
Cryopreservation of European eel sperm has been developed since 2003 15,16. Several researchers designed successful protocols using either dimethyl sulfoxide (DMSO) or methanol as cryoprotectants 16,17,18,19,20. Although both protocols have been successfully used, the obtained cell viability of thawed sperm cryopreserved with DMSO is lower than with methanol 20,21. Moreover, eel sperm is activated on contact with DMSO and requires more tedious sperm manipulation 19, therefore methanol is a more suitable cryoprotectant for European eel sperm than DMSO.
Here, the protocol for optimal handling and hormonal treatment of the European eel will be described below. In addition to this, the best European eel sperm cryopreservation protocol using methanol as a cryoprotectant and a protocol for the assessment of sperm quality in this species will also be described.

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			<td>BD Plastipak</td>
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			<td height="19" style="height:19px;">FC500</td>
			<td>Beckman Coulter</td>
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			<td>Falcon</td>
			<td>175747</td>
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			<td height="19" style="height:19px;">Flexible bucket</td>
			<td>Fiel</td>
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			<td>40 L, Black color.</td>
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			<td>Guinama SL</td>
			<td>Mg96270</td>
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			<td height="20" style="height:20px;">Cyopreservation straws&#160;</td>
			<td>IMV Technologies</td>
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			<td>500 &#181;L straws</td>
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			<td height="19" style="height:19px;">Live/Dead Sperm Viability Kit</td>
			<td>Invitrogen</td>
			<td>L7011</td>
			<td>Cytometer Kit</td>
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			<td height="19" style="height:19px;">Modelling clay</td>
			<td>JOVI</td>
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			<td>KERN &#38; Sohn GmbH</td>
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			<td height="19" style="height:19px;">Saline solution Vitulia 0.9%</td>
			<td>Laboratorios Ern, S.A.</td>
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			<td>Levantina de Lab. SL</td>
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			<td>Levantina de Lab. SL</td>
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			<td>Merck SL</td>
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			<td>Human chorionic gonadotropin</td>
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			<td height="19" style="height:19px;">Nikon Eclipse 80i</td>
			<td>Nikon</td>
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			<td>Panreac Quimica SAU</td>
			<td>2112211210</td>
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			<td height="19" style="height:19px;">Na2SO4</td>
			<td>Panreac Quimica SAU</td>
			<td>3257091611</td>
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			<td height="19" style="height:19px;">NaHCO3</td>
			<td>Panreac Quimica SAU</td>
			<td>1416381210</td>
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			<td height="19" style="height:19px;">782M Camera</td>
			<td>Proiser</td>
			<td>782M</td>
			<td>Camera for CASA</td>
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		<tr height="19">
			<td height="19" style="height:19px;">ISAS v1</td>
			<td>Proiser</td>
			<td>ISAS v1</td>
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			<td height="19" style="height:19px;">SpermTrack-10</td>
			<td>Proiser</td>
			<td>SpermTrack-10</td>
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			<td height="19" style="height:19px;">Beaker Pyrex 3L</td>
			<td>PYREX</td>
			<td>2110668</td>
			<td>Glass beaker 3L</td>
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			<td height="19" style="height:19px;">Flask Pyrex 250 GL-45</td>
			<td>PYREX</td>
			<td>21801365</td>
			<td>Glass flask 250 mL</td>
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			<td height="19" style="height:19px;">KCl</td>
			<td>Scharlab SL</td>
			<td>PO02000500</td>
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			<td>Scharlab SL</td>
			<td>ME03061000</td>
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		<tr height="19">
			<td height="19" style="height:19px;">MgCl2</td>
			<td>Scharlab SL</td>
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			<td height="19" style="height:19px;">BSA</td>
			<td>Sigma Aldrich</td>
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			<td height="19" style="height:19px;">Styrofoam box</td>
			<td></td>
			<td></td>
			<td>34x34x30 cm and 5cm thick</td>
		</tr>
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handling and treatment of male european eels (anguilla anguilla) for hormonal maturation and sperm cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Sperm from 18 eels with a sperm motility of 70% or higher, was selected for this study. The results showed a reduction in all quality parameters after thawing compared to those from fresh sperm (Table 1 and Figure 2). The motility results (mean &#177; S.E.M., n=18) showed a higher total motility and a progressive motility in fresh sperm than the total motility and progressive motility found in the post-thaw sperm samples.
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Watch this Scientific Journal Video about Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation at JoVE.com

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

Hi, my name is Juan Asturiano from the Polytechnic University of Valencia, and we are with some of our colleagues from the Szent Istv

handling-treatment-male-european-eels-anguilla-anguilla-for-hormonal

Research

JoVE Journal

Developmental Biology

7.8K Views.  Universitat Politècnica de València. Hi, my name is Juan Asturiano from the Polytechnic University of Valencia, and we are with some of our colleagues from the Szent Istv

Video: Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

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 ...