This work has been funded by the Norwegian University of Life Sciences and the U.S. Fulbright program. The authors would like to thank Anthony Peltier and Lourdes Carreon G Tan at NMBU for fish facility maintenance and Guillaume Gourmelin from the ISC LPGP at INRAE (France) for providing fish and lab space to further test these methods.

All experimentation and animal handling were conducted in accordance with the recommendations on the experimental animal welfare at Norwegian University of Life Sciences (NMBU). The experiments were performed using adult (6-9-month-old) male Japanese medaka (Hd-rR strain) raised at NMBU (&#197;s, Norway). The methods were also briefly tested in 9-month-old male Japanese medaka (CAB strain) raised at the National Research Institute for Agriculture, Food, and the Environment (INRAE, Rennes, France).
1. Instrument and solution preparation
<ol>
	<li>Prepare anesthetic stock solution (0.6% Tricaine).
	<ol>
		<li>Dilute 0.6 g of Tricaine (MS-222) in 100 mL of 10x Phosphate Buffer Saline (PBS).</li>
		<li>Aliquot 2 mL of the anesthetic stock solution into 50 2 mL plastic tubes and store at -20 &#176;C until use for anesthesia or euthanasia.</li>
	</ol>
	</li>
	<li>Prepare recovery water (0.9% sodium chloride [NaCl] solution).
	<ol>
		<li>Add 27 g of NaCl into 3 L of aquarium water.</li>
		<li>Store the solution at room temperature (RT) until use.</li>
	</ol>
	</li>
	<li>Adjust the activation medium if necessary (Hank&#39;s balanced salt solution [HBSS]). 
	NOTE: HBSS can be purchased commercially or made in the laboratory (Table of Materials).
	<ol>
		<li>Measure the pH of the HBSS using a pH meter. Adjust the pH if necessary, using hydrochloric acid or sodium hydroxide, so the final pH is 7.1-7.3.</li>
		<li>Measure the osmolality of the HBSS using an osmometer for future reporting. 
		NOTE: The range on the commercial product is 266-294 mOsmol/kg; in the current study, the osmolality was 287 mOsmol/kg. It may be diluted with distilled water to reduce the osmolality if desired, but this is not necessary as there is not a large difference in the activation of medaka sperm in 150-300 mOsmol/kg HBSS.</li>
		<li>Store the solution at RT until use.</li>
	</ol>
	</li>
	<li>Prepare the holding sponge.
	<ol>
		<li>Cut a soft sponge to fit snugly in a Petri dish.</li>
		<li>Cut a straight line in the middle of the sponge that is long enough to receive the fish (3-4 cm) and about 1 cm deep (Figure 1A). This slit in the sponge will hold the fish ventral side up to expose the cloaca.</li>
	</ol>
	</li>
</ol>
2. Sperm collection
NOTE: Sperm collection can be achieved by two different methods: abdominal massage or testes dissection.
<ol>
	<li>Sperm collection by abdominal massage
	<ol>
		<li>Prepare 0.03% anesthetic solution by diluting one tube of Tricaine stock (0.6%) in 38 mL of aquarium water in a 100 mL glass container.</li>
		<li>Prepare the instruments, including blunt end smooth forceps and a 10 &#181;L disposable calibrated glass micropipette and aspirator tube assembly (Figure 1A). Dampen the holding sponge prepared in step 1.4 with the anesthetic solution.</li>
		<li>Prepare tubes with 36 &#181;L of the activating solution for immediate analysis. Preheat the activating solution in a water bath or incubator set to 27 &#176;C for at least 5 min. 
		NOTE: Although samples can be analyzed from individual fish, individual variation can be reduced by pooling samples from multiple males in the same activating solution. When pooling samples from multiple fish, use 36 &#181;L of activating solution per fish. This dilution may need to be adjusted depending on the strain or rearing conditions of the medaka used as these factors can impact the sperm concentration and volume. The CASA program will indicate whether the concentration is too high to identify sperm.</li>
		<li>Anesthetize the fish by putting it in anesthetic solution for 30-90 s. 
		NOTE: The anesthesia duration must be adapted as it varies according to the size of the fish. To ensure that the fish is fully anesthetized, gently pinch the caudal peduncle with forceps. If the fish does not react, the massage can be started.</li>
		<li>Take the fish out of the anesthetic solution and use a paper towel or gently wipe to dry the abdomen of the fish. Place the fish in the trough of the damp holding sponge ventral side up so its gills are exposed to the anesthetic solution in the sponge (Figure 1B).</li>
		<li>If the area around the cloaca is wet, gently dry the underside of the fish with a disposable tissue wipe.</li>
		<li>Place the fish in the holding sponge under a dissecting microscope and place the micropipette with an aspirator tube attached against the cloaca of the fish (Figure 1C).</li>
		<li>Massage the abdomen of the fish by gently squeezing with blunt end smooth forceps in a rostral to caudal motion while simultaneously sucking to collect the expelled milt into the pipette (Figure 1D).</li>
		<li>Release the fish from the sponge into the recovery water. Allow them to recover in the solution for at least 15 min before returning them to the aquarium system.</li>
		<li>Transfer the milt into a prepared tube with preheated activating solution and pipette up and down several times by sucking and blowing on the aspirator tube assembly.</li>
		<li>Homogenize the diluted sperm gently by flicking the tubes before analysis. 
		NOTE: For best results, analyze samples immediately (e.g., 5 s) after activation. In medaka, the analysis may be delayed, if necessary, as sperm remain motile for several hours, but the time should remain consistent between samples as motility decreases with time.</li>
	</ol>
	</li>
	<li>Sperm collection by testes dissection
	<ol>
		<li>Prepare 0.08% euthanasia solution by diluting two tubes of Tricaine stock (0.6%) in 26 mL of aquarium water in a 100 mL glass container.</li>
		<li>Prepare dissection tools, including blunt and fine forceps and small dissecting scissors (Figure 1E).</li>
		<li>Prepare a tube for each sample with 120 &#181;L of activating solution for immediate analysis. Preheat the activating solution in a water bath or incubator set to 27 &#176;C for at least 5 min. 
		NOTE: Although samples can be analyzed from individual fish, individual variation can be reduced by pooling samples from multiple males in the same activating solution. For pooling samples from multiple fish, use 120 &#181;L of activating solution per fish. This dilution may need to be adjusted depending on the strain or rearing conditions of the medaka used as these factors can impact the sperm concentration and volume. The CASA program will indicate whether the concentration is too high to identify sperm.</li>
		<li>Euthanize the fish by putting it into the 0.08% anesthetic solution for 30-90 s. 
		NOTE: The duration depends on the size of the fish. To ensure that the fish is euthanized, wait for operculum movements to cease. The fish should not react to the touch of forceps.</li>
		<li>Remove the fish from the euthanasia solution and gently dry the fish with a paper towel or gently wipe. 
		NOTE: At this step, the fish can be weighed to later calculate the gonadosomatic index (GSI, gonadal weight / body weight).</li>
		<li>Place the fish under a dissecting microscope with its left lateral side facing up (Figure 1F).</li>
		<li>Using small dissecting scissors, cut a flap dorsally from the cloaca, and then across the ribs to the gills to expose the internal organs (Figure 1G).</li>
		<li>Locate the testes, cut the attachment at both ends with fine forceps, and remove the testes (Figure 1H). 
		NOTE: To calculate the GSI, the testes can be weighed at this step. Work quickly to avoid drying of the tissue.</li>
		<li>Transfer the testes to a prepared tube with the preheated activating solution.</li>
		<li>Use forceps to crush the testes several times against the side of the tube to release the sperm. Sperm release can usually be visualized and will make the solution slightly cloudy.</li>
		<li>Homogenize the diluted sperm gently by flicking the tubes before analysis. 
		NOTE: For best results, analyze samples immediately (e.g., 5 s) after activation. In medaka, the analysis may be delayed, if necessary, as sperm remain motile for several hours, but the time should remain consistent between samples as motility decreases with time.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64326/64326fig01.jpg" /> 
Figure 1: Milt collection by abdominal massage (A-D) and testes dissection (E-H). (A) Instruments for abdominal massage: holding sponge, blunt smooth forceps, and 10 &#181;L disposable calibrated glass micropipette with aspirator tube assembly; (B) Position of fish in the holding sponge, with gills exposed to anesthesia in the sponge and cloaca facing up; (C) Position of blunt smooth forceps on abdomen and micropipette against cloaca; (D) Milt in micropipette after gentle massage and sucking. (E)&#160;Instruments for testes dissection: blunt forceps, fine forceps, and small dissecting scissors; (F) Position of fish for testes dissection; (G) Lateral view of internal organs; (H) Remove the testes by cutting the attachment at both ends with fine forceps. Scale bar: 2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/64326fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Sperm analysis with CASA system
<ol>
	<li>The CASA system (SCA Evolution) should be set up according to the manual with a microscope using a green filter and 10x objective with phase contrast.</li>
	<li>Prepare disposable 20 &#181;m counting chamber slides by preheating on a warming plate or in an incubator set to 27 &#176;C for at least 5 min.</li>
	<li>Open the sperm analysis software and select the motility module.</li>
	<li>Set the configuration for the medaka as shown in Figure 2B.</li>
	<li>Place a prewarmed disposable 20 &#181;m counting chamber slide under the microscope on a heated stage set to 27 &#176;C.</li>
	<li>Pipette the sample into the chamber on the slide until it fills the chamber without overfilling. Carefully wipe away excess samples from the entrance of the chamber with a cotton tip or gently wipe to prevent floating cells.</li>
	<li>Select Analyze to look at the sample under the microscope. 
	NOTE: If the microscope icon is red, the microscope lighting needs to be adjusted for the program to track sperm accurately. Adjust the brightness of the microscope, so the tail movement of the sperm is clearly seen. The icon should be blue.</li>
	<li>Ensure the microscope is focused and select Analyze again to record the sperm in the field. Move the slide so a new area of the sample is in the frame and repeat to capture for 3-5 different fields of view. Avoid fields with air bubbles, cell masses, or artifacts.</li>
	<li>Select Results to view the results. 
	NOTE: If the fields on the results page are outlined in red, follow the system&#39;s prompts to delete the fields that vary too much in concentration or motility.</li>
	<li>Double-click on a field to view the results for the individual field or to manually check for any mislabeled or untracked spermatozoa. Right-click on individual spermatozoa to relabel the motility, if necessary (Figure 2A).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64326/64326fig02.jpg" /> 
Figure 2: SCA Evolution software screenshot. (A) Sperm tracking results for one field. View field data on the right side and double-click spermatozoa to view individual data; (B) Results summary for all fields with configuration menu open. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/64326fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors have nothing to disclose.

Osmolality is an important factor in the activation of fish sperm36,37. Generally, sperm are immotile in the testes and become motile in media that is hyperosmotic relative to seminal fluid for marine fishes, and hypo-osmotic relative to seminal fluid for freshwater fishes37. Similar to blood, seminal plasma in freshwater fishes is typically lower than that of marine fishes (about 300 mOsmol/kg compared to 400 mOsmol/kg)22,37. Thus, fish sperm is usually activated on contact with the water in which they live, and this water serves as the best and most biological activation medium for sperm analysis. However, medaka sperm sampled by abdominal massage and testes dissection were not motile in aquarium water (Figure 4A) in Hd-rR or CAB strain medaka raised in two different labs. Additionally, motility was higher in 287 mOsmol/kg HBSS than in 36 mOsmol/kg HBSS (Figure 4C), which is unusual for a freshwater fish.
Most previous studies involving sperm analysis in medaka used HBSS28,29,38&#160;or Yamamoto solution18,19 as activating mediums for medaka sperm without discussing osmolality. While HBSS is a good activating medium for medaka sperm, variation in osmolality can affect motility. It is, therefore, essential for sperm analysis studies to disclose the osmolality of their activating solution for comparison between experiments. One study investigating the ideal osmolality of activating medium found that medaka sperm is motile in deionized water (25 mOsmol/kg) and HBSS with osmolality values &#60; 686 mOsmol/kg, with the highest motility between 25 and 227 mOsmol/kg17. However, in the current study, sperm were not motile in deionized water either. As a euryhaline fish, medaka are very adaptable to different environments and can even live and reproduce in salt water39, so it&#39;s possible that different rearing conditions are responsible for this discrepancy. Interestingly, no other studies have reported testing medaka sperm motility in aquarium water (or similar, such as aged tap water), although it is the standard sperm activation medium for freshwater fish.
One possible explanation for this atypical sperm activation in medaka is interaction with the ovarian fluid. While fish sperm is usually activated in the surrounding water, ovarian fluid increases or extends the duration of sperm motility in many species40,41. Although medaka are externally fertilizing freshwater fish, their spawning behavior, which includes fin wrapping and quivering, puts them in much closer proximity than broadcast spawners23. As the osmolality of ovarian fluid in freshwater fish (about 300 mOsmol/kg) is close to HBSS40, it is possible that fluid released by the female activates the sperm, not the aquarium water. Since the osmolality of seminal and ovarian plasma are similar, it is possible that the ionic composition of medaka ovarian fluid plays an important role40,42. The role of ovarian fluid and ions in activating medaka sperm warrants further investigation.
In previous studies, medaka sperm has been collected only by crushing dissected testes17,28,29,30 or by abdominal massage to express the milt directly into activating medium18,19,20; no studies have reported data with sperm collected by abdominal massage into a capillary tube, although it is a common practice in zebrafish and other teleost fish33,43,44. This method is feasible in medaka as well. Milt is easily visualized in the capillary tube, and although the volume is too small to be accurately measured, this method enables confirmation of successful collection and analysis of color as a fast indicator of quality32. Avoidance of fecal contamination is also easier with collection in a capillary tube compared to in medium. Although sperm samples collected by testes dissection have better motility in medaka than samples collected by abdominal massage (Figure 5) and are thus preferable for experiments in which the fish may be sacrificed, abdominal massage is a minimally invasive procedure that does not require euthanasia and can be repeated in the same fish. Therefore, it is useful for experiments that follow the same fish over time. Also, samples collected with abdominal massage contain only mature cells released with seminal plasma44, while samples from testes dissection may include immature sperm cells and other debris. The CASA system discounts round cells and debris larger than the max area, but if this parameter is set too high, the motility results could be affected.
Due to the low amount of sperm collected with the abdominal massage technique in these medaka, it was impossible to accurately measure sperm volume in the capillary and thus to calculate sperm concentrations using regular approaches. However, for samples taken by testes dissection, by keeping the volume of activation medium consistent and weighing the testes, a relative concentration can be calculated based on sperm concentration given by the CASA system to compare between individuals or treatment groups. Besides quality analysis, relative concentration is also useful for determining optimal sample dilutions for cryopreservation. If the volume collected by abdominal massage is improved by rearing conditions or using a different strain, the volume can be calculated by measuring the height of the milt in the capillary tube and entered in the program to calculate the concentration. In these cases, in which several microliters can be obtained, motility may be higher by abdominal massage than testes dissection (Supplementary Table 2). When low volumes are collected, the samples are more prone to contamination, which may affect the motility, although these effects appear to be consistent when the volumes are similar, so comparisons can still be made between treatment groups. However, comparisons should not be made between samples that vary greatly in volume or coloration of milt.
The low volume of milt obtained from medaka using abdominal massage may be a limitation for experiments that require a large volume of milt. In such cases, testes dissection may be preferable, as shown in a study that collected milt from zebrafish and green swordtail by both dissection and massage, but only by dissection in medaka due to low volume30. Testes dissection is typically better for comparisons to other species for the same reason. The limited volume by abdominal massage compared to other, similar sized fish may be due to smaller testes (1.9 &#177; 0.6 mg in medaka compared to 7.0 &#177; 2.5 mg in zebrafish45) and less sperm produced (2.0 &#177; 0.4 x 106 sperm cells/mg testes in medaka compared to 7.7 &#177; 2.0 x 106 sperm cells/mg testes in zebrafish46), or to other unknown biological factors that limit the technique, as suggested in hatchery-raised African catfish47. The difference could be physiological, according to strain or environmental conditions, or anatomical, as unlike zebrafish, medaka testes are fused and located more medially, and therefore could be harder to access by abdominal massage, although there are other fish with fused testes as well.
For some species, such as zebrafish, it is best to sample sperm before the fish have a chance to spawn in the morning48 or to isolate males in individual tanks the night before to prevent spawning32. With Hd-rR medaka from this study, environmental conditions such as timing of sampling (before or after spawning) and housing conditions (with or without females for 1 month) did not have a significant effect on sperm motility (Figure 7). It is possible that a larger sample size or isolating males from females for a longer period of time could yield higher sperm quantity and/or motility. In CAB strain medaka from another facility, a similar trend was seen with better sperm quality and volume from males housed alone compared to males housed with females (too few fish were tested to draw statistical conclusions). It was also possible with these fish to collect relatively high volumes of milt by abdominal massage (Supplemental Table 2), although very small volumes of milt were obtained using Hd-rR strain medaka, and others have reported small volumes as well by the same technique in CAB strain medaka30. However, whether these differences are due to the strain (which is a commercial strain rather than inbred) or due to rearing differences (there are many between facilities), remains unclear. In these fish, several microliters of milt could be collected, which limits contamination and improves the quality. But, for experiments in which it is important to cohabitate both sexes, it does not seem necessary to separate them to yield results. It also does not seem necessary to sample males at a time before spawning.
Although further studies are required to determine exactly how medaka sperm differs according to population and environmental factors, the strain and rearing conditions should nevertheless be taken in consideration when designing the experimental setup, and caution should be used in comparing between populations that produce different amounts of sperm. If larger volumes of milt are obtained, the dilution of activating solution should be adjusted (e.g., 1:60, although the preferred concentration may vary with the CASA system). Similarly, if the dissected testes are much larger than typical (2 mg) due to the strain or rearing conditions, the dilution will need to be increased so the CASA program can accurately label all sperm cells.
Methods of sperm analysis vary widely for medaka and are often subjective, making results difficult to compare between studies. A study comparing subjective and objective methods used by technicians with varying levels of expertise found that highly experienced technicians can estimate fish sperm motility within 10 percentage points of the data provided by a CASA motility program, while medium and low experience technicians overestimate the CASA motility values with amplitudes up to 30 percentage points21. However, a lack of standardization for the parameters that determine motility in medaka can also cause variation between those using more objective methods. For instance, one study that recorded sperm at 33 frames per second (fps), analyzed 30 frames, and considered sperm moving faster than 2 &#181;m/s motile had an average velocity of about 60 &#181;m/s and motility of about 70% for their control fish18. Another study using the same protocol had an average velocity of 40 &#181;m/s for control sperm and a percent motility over 80%19. Another group, that analyzed 200 frames at 47 fps, had an average VCL over 100 um/s for control fish, but an average motility below 50%. They did not disclose what parameters determined motility. So, in this protocol, computer-assisted sperm analysis software is used to analyze sperm objectively, quickly, and reliably based on a set of parameters that have been customized for the characteristics of medaka sperm. As it is critical for motility parameters to be consistent for comparisons across labs, the complete configuration used in this study is available (Figure 2B), so this protocol can be reliably replicated in a different lab by different researchers.
Teleost fish show a wide diversity of sperm characteristics, so although this protocol was initially tested using the recommended zebrafish parameters for the SCA Evolution software, it was apparent that the parameters would need to be adjusted for the lower-velocity, greater-longevity medaka sperm. So, zebrafish parameters were adapted to medaka using literature from medaka and other species that reported similar sperm characteristics19,34,35&#160;and selected the thresholds that best fit the distribution of 17,580 analyzed sperm tracks from 18 individuals. Motility is based on curvilinear velocity (VCL) with immotile &#60; 10 &#181;m/s &#8804; slow &#60; 20 &#181;m/s &#8804; medium &#8804; 40 &#181;m/s &#60; rapid. Sperm are considered progressive if straightness index (STR) is &#62; 68%. The threshold defining motility was kept at 10 &#181;m/s rather than 2 &#181;m/s, as used in some literature18,19, as many sperm lacking flagellar movement were mislabeled as motile with this setting. Other fish species with similar sized sperm heads (~2 &#181;m) also used 10 &#181;m/s to define motile sperm35,43. The maximum area was decreased from the zebrafish setting of 90 &#181;m2 to 20 &#181;m2 so large cellular debris from testes dissection would be ignored.
Duration of motility appears to be dependent on the amount of ATP stored prior to activation, as flagellar movement requires a rapid consumption of energy. Likely due to this rapid exhaustion, there is a correlation between high initial velocity sperm and a shorter duration of motility49. While an osmotic shock is typically required to activate fish sperm, it can also lead to membrane damage during the motility period, which can also impact the longevity. This effect is more critical in freshwater species49, which may explain why marine sperm are capable of longer motility duration (about 550 s on average compared to about 150 s for freshwater species), despite exhibiting a similar average velocity and motility as freshwater sperm14,22. Motility duration longer than 30 min is not common, but it has been reported in several marine species22,49. Despite being a freshwater fish, results with medaka fit the profile of sperm that have a lower velocity and longer duration from marine species. This may be related to the activation in medium similar to seminal plasma-without an osmotic shock, medaka sperm likely does not suffer membrane damage similar to other freshwater fish.
A non-activating solution and very quick, consistently timed analysis after activation is often necessary for analysis of sperm that are only motile for minutes. However, medaka sperm motility naturally lasts several hours and higher motility may be preserved by storing at 4 &#176;C or on ice (Figure 6). Thus, for experiments in which immediate analysis is not possible, the protocol may be modified so, for instance, testes are stored in activating solution on ice for an hour, as long as the collection time is recorded so the analysis can remain consistent. However, for optimal results, immediate analysis is still the best option. While motility does still decrease, because it is gradual, the results are less impacted by minor differences in time of analysis following activation. Still, it is important to report both the storage temperature of samples and the time of analysis after activation so data can be compared and reproduced.
It is also typically very important in fish to avoid contamination of milt with urine while sampling, as this can change the osmolality and prematurely activate the sperm16,50. However, this is less of a concern in medaka (due to the longer duration of motility) and with this protocol, as the sample is placed in activation medium immediately. Low volumes of milt are prone to urine contamination, so some discoloration may be expected with abdominal massage from medaka when low volumes are procured. However, if this is consistent in the population, then comparisons can still be made between treatment groups. Caution should be used in comparing populations of medaka that vary in the volume or coloration of milt.
As sperm analysis results are very dependent on the methods used, detailed and reliable methods that can be easily repeated in different labs are beneficial. It is also vital for papers to disclose specifics about their methods to enable repeatability. With medaka used increasingly as a model for reproduction research, the body of information regarding the assessment of sperm quality is lacking. The two distinct methods described in this paper for sperm sampling may be beneficial for different experiments. Testes dissection typically yields higher motility sperm and allows for relative concentration calculations, while abdominal massage can be done repeatedly on the same fish and is a more pure, biological representation of a spawning event. This manuscript, therefore, provides the parameters for CASA, which is a reliable, objective technique that provides ample data about sperm movement, including motility, progressivity, velocity, and other kinematic parameters. These protocols will thus be useful for a variety of studies in medaka, including toxicology, ecology, reproduction, and physiology.

Japanese medaka is a small, egg-laying freshwater teleost fish native to East Asia. Medaka has become an excellent vertebrate model system for ecotoxicology, developmental genetics, genomics, and evolutionary biology and physiology studies1,2. Similar to the popular zebrafish, they are relatively easy to breed and highly resistant to many common fish diseases1,2. There are several advantages of using medaka as a model, including a short generation time, transparent embryos1,2, and a sequenced genome3. Unlike zebrafish, medaka has a sex-determining gene4&#160;as well as a high temperature (from 4-40 &#176;C)&#160;and salinity (euryhaline species) tolerance5. Also, many genetic and anatomical tools, as well as protocols6,7,8,9,10,11,12, have been developed in medaka to facilitate the study of its biology.
Reproduction is an essential physiological function as it allows a species to perpetuate. Vertebrate reproduction requires a myriad of precisely orchestrated events, including the production of oocytes in females and the production of sperm in males. Sperm are unique cells, produced through the complex process of spermatogenesis, in which there are a number of checkpoints in place to guarantee delivery of a high-quality product13. Gamete quality has become a focus in aquaculture and fish population studies due to its impact on fertilization success and larval survival. Sperm quality is, therefore, an important indicator of male fertility in vertebrates.
Three useful factors for assessing fish sperm quality are motility, progressivity, and longevity. Percent motility and progressive motility are common indicators of sperm quality as progressive motion is necessary for and correlates strongly with fertilization success14,15. Duration of movement is also an important indicator in fish as sperm remain fully motile for less than 2 min in most teleost species and the trajectory of sperm is generally less linear than in mammals15. However, many studies assessing sperm motility in the past relied on subjective or semi-quantitative methods of analyzing sperm15,16. For instance, sperm motility in medaka has been estimated in the past visually under a microscope17. It has also been estimated by recording sperm movement and using imaging software to merge frames and measure swimming path and velocity18,19,20. Such approaches often lack robustness, providing different results according to the person performing the analysis15,21.
Computer-assisted sperm analysis (CASA) was initially developed for mammals. CASA is a fast quantitative method to assess sperm quality by recording and measuring velocity and trajectory in an automated manner15. In fishes, it has been used in different species to monitor the effects of several water pollutants on sperm quality, for identifying interesting progenitors to improve broodstock, to improve the efficiency of cryopreservation and storage, and to optimize conditions for fertilization15. Therefore, it is a powerful tool for reliably assessing sperm quality in different vertebrate species. However, due to the important diversity in reproductive strategies between fishes, the sperm of teleost fish differs from that of mammals and from one fish species to another. Teleost fish, which primarily fertilize eggs externally by releasing gametes into water, have highly concentrated sperm that are relatively simple in structure with no acrosome, unlike mammals, which fertilize internally and therefore do not have to compensate for dilution in water, but do have to withstand more viscous fluids14. Additionally, sperm from most fish move rapidly but are fully motile for less than 2 min after activation, although there are several exceptions15,22. Because motility can decrease rapidly in most fish, extreme care should be taken with the timing of analysis after activation when determining a sperm analysis protocol for fish.
Reproduction is one of the fields in biology in which teleosts and medaka have been extensively used as model organisms. Indeed, medaka males show interesting reproductive and social behaviors, such as mate guarding23,24. In addition, several transgenic lines exist to study the neuroendocrine control of reproduction in this species25,26,27. Sperm sampling, a procedure that is relatively simple in larger fish, can be more complicated in small model fish as they produce less sperm and are more delicate. For this reason, most studies involving sperm sampling in medaka extract milt (fish semen) by crushing dissected testes17,28,29,30. A few studies also use a modified abdominal massage to express the milt directly into activating medium18,19,20; however, with this method it is difficult to visualize the amount and color of milt extracted. In zebrafish, abdominal massage is commonly used to express milt, which is immediately collected in a capillary tube31,32,33. This method enables estimation of the volume of milt, as well as observation of ejaculate color, which is a quick and simple indicator of sperm quality32,33. Therefore, a clear and well described protocol for sperm collection and analysis is lacking for medaka.
This article therefore describes two methods of sperm collection in the small model fish Japanese medaka: testes dissection and abdominal massage with capillary tubes. It demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recovers from the procedure. It also describes a protocol for computer-assisted sperm analysis in medaka to reliably measure several important indicators of medaka sperm quality (motility, progressivity, longevity, and relative sperm concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

<table><tbody><tr><td>1.5 mL tubes</td><td>Axygen</td><td>MCT-150-C</td><td>Any standard brand can be used</td></tr><tr><td>10 &amp;micro;L disposable calibrated glass micropipette and aspirator tube assembly</td><td>Drummond</td><td>2-000-010</td><td></td></tr><tr><td>10x objective with phase contrast</td><td>Nikon</td><td>MRP90100</td><td></td></tr><tr><td>2 mL tubes</td><td>Axygen</td><td>MCT-200-c-s</td><td>Any standard brand can be used</td></tr><tr><td>Blunt forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Blunt smooth forceps</td><td>Millipore</td><td>XX6200006P</td><td></td></tr><tr><td>Disposable 20 micron counting chamber slide</td><td>Microptic</td><td>20.2.25&amp;nbsp;</td><td>Leja 2 chamber slides</td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZX7</td><td>Any standard brand can be used</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11253-20</td><td></td></tr><tr><td>HBSS</td><td>Sigmaaldrich</td><td>H8264-1L</td><td></td></tr><tr><td>Holding sponge</td><td>self-made</td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse Ts2R</td><td></td></tr><tr><td>SCA Evolution</td><td>Microptic</td><td></td><td></td></tr><tr><td>Small dissecting scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigmaaldrich</td><td>S9888</td><td></td></tr><tr><td>Tabletop vortex</td><td>Labnet</td><td>C1301B</td><td></td></tr><tr><td>Tricaine</td><td>Sigmaaldrich</td><td>A5040</td><td></td></tr></tbody></table>

sperm collection and computer-assisted sperm analysis in the teleost model japanese medaka (oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka is also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and, thus, reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including teleost fish. Collecting sperm is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article, therefore, describes two methods of sperm collection in the small model fish, Japanese medaka: testes dissection and abdominal massage. This paper demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. This article also describes a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Type of data obtained 
Sperm motility analysis from the SCA Evolution software provides data on motility (percentage of motile and immotile sperm), as well as progressivity (percentage of progressive and non-progressive sperm), and velocity (percentage of rapid, medium, and slow-moving sperm). It also combines progressivity and velocity (rapid progressive, medium progressive, non-progressive). These labels are based on measurements (Figure 3A) and calculations (Figure 3B) of spermatozoon movement, which is provided by the program (Supplemental Table 1). For medaka, the following thresholds were adapted from the recommended zebrafish parameters based on previous literature19,34,35 and the distribution of medaka data from 18 individuals. Motility is based on curvilinear velocity (VCL) with immotile &#60; 10 &#181;m/s &#8804; slow &#60; 20 &#181;m/s &#8804; medium &#8804; 40 &#181;m/s &#60; rapid. Sperm are considered progressive if the straightness index (STR) is &#62; 68%.
SCA Evolution also calculates the concentration of sperm if provided the volume of the sample and the dilution. While the abdominal massage method is helpful for visualizing the coloration and confirming the presence of the milt, the volume collected is too small to be accurately measured. If the milt volume is improved by rearing conditions or using a different strain and can be measured, then the program can be used to calculate the concentration. However, relative concentration can also be calculated to compare fish or treatment groups for samples taken by testes dissection, as long as the whole testis is dissected and diluted in the same volume of liquid.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64326/64326fig03.jpg" /> 
Figure 3: Analysis of spermatozoon movement. (A) Data recorded by the CASA system include curvilinear velocity (VCL, velocity calculated using the distance along the actual path), average path velocity (VAP, velocity calculated using the distance of the average path), straight line velocity (VSL, velocity calculated using the distance between the start and end of the sperm track), amplitude of lateral head displacement (ALH, magnitude of lateral displacement of a sperm head about its average path), beat cross frequency (BCF, rate at which the curvilinear path crosses the average path); (B) Calculated values from the CASA system include straightness index (STR, linearity of the average path), linearity index (LIN, linearity of the curvilinear path), and wobble (WOB, oscillation of the actual path about the average path). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/64326fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sperm motility evaluation: Different activating solutions 
Surprisingly, sperm sampled by both abdominal massage and testes dissection were immotile in aquarium water (16 mOsmol/kg) (Figure 4A) and in aquarium water adjusted with NaCl solution to 34 mOsmol/kg. There was also no motility in deionized water (-1 mOsmol/kg), or deionized water adjusted to 23 mOsmol/kg. Sperm were motile in HBSS (287 mOsmol/kg) (Figure 4B), as well as in HBSS diluted with deionized water to 36 mOsmol/kg and 113 mOsmol/kg, although the percent motility was significantly reduced at 36 mOsmol/kg (Figure 4C).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64326/64326fig04.jpg" /> 
Figure 4: Motility by osmolality of activating solution. Sperm sampled by testes dissection in (A) aquarium water (16 mOsmol/kg) and (B)&#160;unadjusted HBSS (287 mOsmol/kg). Yellow circles label immotile sperm and colored lines indicate sperm movement: red (rapid progressive), green (medium progressive), blue (non-progressive). Scale bar: 100 &#181;m. (C)Sperm motility activated with HBSS at 36, 113, and 287 mOsmol/kg H2O. For 36 and 113, n = 4 with two fish pooled per sample. For 287, n = 9 with two fish pooled per sample. Statistical analyses were performed using an ANOVA with Tukey post-hoc test and significant differences are indicated by different letters. The data are represented as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/64326fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sampling methods: Abdominal massage versus testes dissection 
Sperm sampled by testes dissection is significantly more motile compared to abdominal massage samples from the same fish (Figure 5A). Of the motile sperm, a higher percentage are also progressive and medium or rapid-moving. In sperm sampled by abdominal massage, more motile sperm are slow-moving and non-progressive (Figure 5B-C). However, different results may be obtained by using a different strain of medaka (CAB) or alternative rearing conditions (Supplemental Table 2).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64326/64326fig05.jpg" /> 
Figure 5: Abdominal massage versus testes dissection. Percentage of (A) immotile and motile sperm sampled by abdominal massage or testes dissection. Percentage of motile sperm that are (B) non-progressive and progressive sperm, and (C) slow, medium, and rapid-moving. All sperm samples were activated in HBSS (287 mOsmol/kg H2O). Statistical analyses were performed using a Mann-Whitney U test and significant differences are indicated by asterisks. The data are represented as mean &#177; SEM, n = 9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/64326fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Motility duration according to storage condition 
Sperm sampled by testes dissection and maintained at 27 &#176;C had a 50% reduction in motility in the first 30 min after activation. After 2.5 h, less than 5% of sperm were motile. When stored at 4 &#176;C, motility was reduced by only 14% in the first 30 min, and it took 5 h to see a 50% reduction from the initial motility. Ice had a similar extending effect, but was less effective, with a 26% motility reduction in the first 30 min (Figure 6A). Progressivity also dropped 52% in the first 30 min on ice, compared to 65% at 27 &#176;C and 33% at 4 &#176;C (Figure 6B). Both on ice and at 4 &#176;C, some sperm (&#60;3%) were still moving 42 h after activation.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64326/64326fig06.jpg" /> 
Figure 6: Sperm longevity according to storage condition. Percent (A) motility and (B) progressivity over time for samples activated with HBSS (287 mOsmol/kg H2O) and stored at 27 &#176;C, 4 &#176;C, or on ice. The data points are mean &#177; SEM, n = &#8805;4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/64326fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Importance of the environmental and housing conditions 
Males co-housed with females were sampled by testes dissection in the morning before having an opportunity to spawn (before lights turned on) or after the lights turned on and females in the tank had eggs. There was no significant difference in sperm motility. Males housed without females for a month were also sampled, and while there was a trend of higher motility, the difference was also not significant (Figure 7).
However, when CAB strain medaka was raised in a different facility with males separated from females for at least one month, the fish were larger and 5-7 &#181;L of milt was collected by abdominal massage. Samples collected from five fish by abdominal massage had motility of 68.34% when activated with 300 mOsmol/kg HBSS. When held in the same conditions but with females, the volume of milt collected was about 2 &#181;L, and the average motility was 46.2% from three males (Supplemental Table 2).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64326/64326fig07.jpg" /> 
Figure 7. Effect of environmental conditions on sperm motility. Percent motility of sperm sampled before (n = 5) and after (n = 4) spawning from males that were co-housed with females, and sperm from males housed without females (n = 6). All the samples were sampled by testes dissection and activated with HBSS (287 mOsmol/kg H2O). Statistical analyses were performed using t-tests and the differences were not significant. The data are shown as mean &#177; SEM with circles representing individual fish. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/64326fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table 1: Mean velocity and movement calculations for sperm sampled by abdominal massage and testes dissection (n = 9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/Supplemental Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental Table 2: Sperm analysis data from CAB strain (also called Carbio) medaka reared at INRAE in Rennes, France. All the samples were activated in 300 mOsmol/kg HBSS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64326/Supplemental Table 2.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes at JoVE.com

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

This article describes two quick and efficient methods for collecting sperm from the small model fish medaka (Oryzias latipes), as well as a protocol for reliably assessing sperm quality using computer-assisted sperm analysis (CASA).

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology ...

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Nanyang Technological University

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JoVE Journal

Biology

3.9K Views.  Norwegian University of Life Sciences. Deze techniek maakt het mogelijk om milt te bemonsteren van een kleine modelvis, Japanse Madaka, en de kwaliteit ervan op een consistente manier te onderzoeken. Specifiek spermaverzameling door buikmassage maakt herhaalde bemonstering op dezelfde vis mogelijk, omdat deze aanpak niet invasief is. Bovendien kan deze techniek worden toegepast voor Madaka sperma cryo conservering.Spermakwaliteit is een essentiële indicator voor bevruchtingssucces en daarom een belangrijke parameter om te ...

Video: Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Developmental Biology

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

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.

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.

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

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

Genetics

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

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