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

Lauren Closs; Amin Sayyari; Romain Fontaine

doi:10.3791/64326

Biology

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

Published: October 6, 2022 doi: 10.3791/64326

Lauren Closs¹, Amin Sayyari², Romain Fontaine¹

¹Department of Preclinical Sciences and Pathology, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, ²Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences

Summary

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).

Abstract

Japanese medaka (Oryzias latipes) is a teleost fish and 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.

Introduction

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 as well as a high temperature (from 4-40 °C) 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.

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Protocol

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 (Ås, 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

Prepare anesthetic stock solution (0.6% Tricaine).
1. Dilute 0.6 g of Tricaine (MS-222) in 100 mL of 10x Phosphate Buffer Saline (PBS).
2. Aliquot 2 mL of the anesthetic stock solution into 50 2 mL plastic tubes and store at -20 °C until use for anesthesia or euthanasia.
Prepare recovery water (0.9% sodium chloride [NaCl] solution).
1. Add 27 g of NaCl into 3 L of aquarium water.
2. Store the solution at room temperature (RT) until use.
Adjust the activation medium if necessary (Hank's balanced salt solution [HBSS]).
NOTE: HBSS can be purchased commercially or made in the laboratory (Table of Materials).
1. 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.
2. 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.
3. Store the solution at RT until use.
Prepare the holding sponge.
1. Cut a soft sponge to fit snugly in a Petri dish.
2. 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.

2. Sperm collection

NOTE: Sperm collection can be achieved by two different methods: abdominal massage or testes dissection.

Sperm collection by abdominal massage
1. 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.
2. Prepare the instruments, including blunt end smooth forceps and a 10 µL disposable calibrated glass micropipette and aspirator tube assembly (Figure 1A). Dampen the holding sponge prepared in step 1.4 with the anesthetic solution.
3. Prepare tubes with 36 µL of the activating solution for immediate analysis. Preheat the activating solution in a water bath or incubator set to 27 °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 µL 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.
4. 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.
5. 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).
6. If the area around the cloaca is wet, gently dry the underside of the fish with a disposable tissue wipe.
7. 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).
8. 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).
9. 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.
10. 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.
11. 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.
Sperm collection by testes dissection
1. 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.
2. Prepare dissection tools, including blunt and fine forceps and small dissecting scissors (Figure 1E).
3. Prepare a tube for each sample with 120 µL of activating solution for immediate analysis. Preheat the activating solution in a water bath or incubator set to 27 °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 µL 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.
4. 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.
5. 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).
6. Place the fish under a dissecting microscope with its left lateral side facing up (Figure 1F).
7. 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).
8. 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.
9. Transfer the testes to a prepared tube with the preheated activating solution.
10. 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.
11. 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.

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 µL 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) 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. Please click here to view a larger version of this figure.

3. Sperm analysis with CASA system

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.
Prepare disposable 20 µm counting chamber slides by preheating on a warming plate or in an incubator set to 27 °C for at least 5 min.
Open the sperm analysis software and select the motility module.
Set the configuration for the medaka as shown in Figure 2B.
Place a prewarmed disposable 20 µm counting chamber slide under the microscope on a heated stage set to 27 °C.
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.
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.
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.
Select Results to view the results.
NOTE: If the fields on the results page are outlined in red, follow the system's prompts to delete the fields that vary too much in concentration or motility.
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).

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. Please click here to view a larger version of this figure.

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Representative Results

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 < 10 µm/s ≤ slow < 20 µm/s ≤ medium ≤ 40 µm/s < rapid. Sperm are considered progressive if the straightness index (STR) is > 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.

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). Please click here to view a larger version of this figure.

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).

Figure 4: Motility by osmolality of activating solution. Sperm sampled by testes dissection in (A) aquarium water (16 mOsmol/kg) and (B) 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 µm. (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 ± SEM. Please click here to view a larger version of this figure.

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).

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 ± SEM, n = 9. Please click here to view a larger version of this figure.

Motility duration according to storage condition
Sperm sampled by testes dissection and maintained at 27 °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 °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 °C and 33% at 4 °C (Figure 6B). Both on ice and at 4 °C, some sperm (<3%) were still moving 42 h after activation.

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 °C, 4 °C, or on ice. The data points are mean ± SEM, n = ≥4. Please click here to view a larger version of this figure.

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 µL 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 µL, and the average motility was 46.2% from three males (Supplemental Table 2).

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 ± SEM with circles representing individual fish. Please click here to view a larger version of this figure.

Supplemental Table 1: Mean velocity and movement calculations for sperm sampled by abdominal massage and testes dissection (n = 9). Please click here to download this File.

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. Please click here to download this File.

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Discussion

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 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 < 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'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 ± 0.6 mg in medaka compared to 7.0 ± 2.5 mg in zebrafish⁴⁵) and less sperm produced (2.0 ± 0.4 x 10⁶ sperm cells/mg testes in medaka compared to 7.7 ± 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 µm/s motile had an average velocity of about 60 µm/s and motility of about 70% for their control fish¹⁸. Another study using the same protocol had an average velocity of 40 µm/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 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 < 10 µm/s ≤ slow < 20 µm/s ≤ medium ≤ 40 µm/s < rapid. Sperm are considered progressive if straightness index (STR) is > 68%. The threshold defining motility was kept at 10 µm/s rather than 2 µm/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 µm) also used 10 µm/s to define motile sperm³⁵^,⁴³. The maximum area was decreased from the zebrafish setting of 90 µm² to 20 µm² 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 °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.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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.

Materials

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

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