Fish Sperm Assessment Using Software and Cooling Devices

Summary

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.

Abstract

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.

Introduction

The efficacy of reproduction depends on the quality of both gametes (eggs and sperm)^1,2. This is the major factor that contributes to successful fertilization, allowing the development of viable offspring^3,4. The convenient evaluation of gamete quality is the best tool for defining the fertility potential of a specimen.

Mixing sperm from multiple males is a common practice in the production of many aquatic commercial species⁴. However, the sperm variability between males can lead to sperm competition and, consequently, not all the males are equally contributing to the gene pool⁵. In this sense, the correct evaluation of individual ejaculate/spermatozoa features, such as motility, is fundamental to obtain discriminatory information regarding individual male fertility potential. Direct observation of sperm motility can produce inaccurate and subjective data as it requires time and experience, which leads to a lack of consistency and incompatibility of results^6,7. However, there are many innovative, rapid and quantitative techniques that can provide a reliable sperm quality analysis^2,4.

Computer-assisted sperm analysis was developed to offer accurate data about sperm quality⁸. This technology includes the development of software associated with a phase contrast microscope that allows the assessment of sperm motility. However, a limiting factor of motility parameter is the frame rate of the video camera. Individual spermatozoa trajectories are based on spermatozoa head centroid position in consecutive frames of video recordings, which is correlated with the flagellar movement patterns^3,9,10,11. The main kinetic parameters measured are the straight-line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP). VSL is the distance between the start and end-point taken by the spermatozoa divided by time. VCL is the real velocity along the precise trajectory taken by the spermatozoa. VAP is the velocity along a derived smoothed path of trajectory. These parameters allow additional kinetic information, including linearity (LIN), straightness (STR), wobble (WOB) and beating measurements like amplitude of lateral head movement (ALH) and beat-cross frequency (BCF)^4,10.

The sperm analysis system was originally used for mammalian species, and one of the requirements for the system is to operate at the body temperature of the donor (about 37 °C). This software could also be used for fish species; although, it is necessary to make some adaptations to reduce the error of sperm analysis results. In some fish species, such as salmonids and eel^8,12, fertilization occurs at low temperature (around 4 °C)^2,4. Thus, cooling devices should be developed to avoid uncomfortable working conditions. In addition, fish spermatozoa are immotile in seminal fluid and require an osmotic shock to activate motility. For freshwater species, the activator medium should have hypotonic osmolality, while for marine species the medium should be hypertonic. However, for some species, as salmonids, the ion concentration could also be important^3,4,9. After activation, fish sperm is characterized by a rapid decrease of motility (less than 2 min)^13,14 and high velocity, being vital to determine the optimal frame rate to obtain reliable data¹⁵.

The objectives of this study are to design and apply refrigeration systems for fish sperm samples. In addition, this protocol defines how to determine the optimal frame rates for the establishment of standard protocols depending on the species. The use of this protocol opens new doors in the context of fish seminal evaluation, using the European eel as a model.

Protocol

Procedures involving animal subjects have been approved (2015/VSC/PEA/00064) by the General Direction of Agricultural Production and Livestock at the Universitat Politècnica de València.

1. Collect Sperm from Mature European Eels in Captivity

NOTE: Use European eel males maintained in tanks with seawater and a recirculation system at constant temperature (20 °C). Treat with hormones through weekly intraperitoneal injection (human chorionic gonadotropin (hCG); 1.5 IU per g of fish body weight). Acclimatize fish gradually starting with 3 days in freshwater followed by replacement with seawater (1/3 of the total water in the tank) every 2 days until reaching a salinity of 37.0 g/mL.

1. Anesthetize the eel 24 h after hormone injection to obtain samples with better sperm quality.
   1. Prepare the anaesthesia beforehand: add 300 mg of benzocaine to 100 mL of 70% ethanol. Mix well and store at 4 °C.
   2. Dilute benzocaine in a flexible bucket with 5 L of system water to get a final concentration of 60 mg/L and mix properly.
   3. Transfer the fish to the bucket with system water and benzocaine. Wait a few minutes until the fish calm down.
2. Clear the genital area with water and dry with absorbent paper to avoid contamination of sperm samples by faeces, urine or seawater.
3. Apply gentle pressure in the abdominal area and collect the sperm samples into 15 mL plastic tubes using the vacuum pump. Sperm volume up to 6-7 mL, depending on the male.
4. Keep sperm samples at 4 °C for at least 1 h until motility analysis is carried out.

2. Refrigerate and Dilute the Sperm Samples

1. Prepare the diluter solution beforehand.
   NOTE: The sperm samples should be diluted in the extender solution specific for each species.
   1. Prepare the non-activator medium for eel sperm samples (P1 medium). Add 0.42 g of sodium bicarbonate, 1.828 g of sodium chloride, 0.127 g of magnesium chloride, 0.56 g of potassium chloride and 0.037 g of calcium chloride to 250 mL of distilled water. Mix well to dissolve. Store at 4 °C.
2. Set the cooler block at 4 °C to maintain samples at a constant temperature. Wait until the temperature stabilizes.
3. Dilute the sperm samples using the diluter solution specific for each species.
   NOTE: The dilution ratio is species specific and must be defined to standardize the protocol. First, estimate sperm concentration based on the concentration obtained in the pre-analysis of motility using software, as explained in steps 3 and 4. With this analysis, it is also possible to select the best sperm samples based on the percentage of motility. After this, the researcher can start to collect data for the experiment using the best samples with the correct concentration.
   1. Dilute eel sperm samples in the P1 medium with a ratio of 1:50. To prepare 500 µL of eel diluted sperm, add 50 µL of the fresh sperm sample and 450 µL of P1. Mix well.

3. Evaluate the Sperm Motility Parameters

1. Set-up the Motility Module of the Software
   1. Set the cooler stage at 4 °C and wait until it stabilizes.
   2. Open the software and select Motility Module. Create username and password if necessary.
   3. Select Properties and choose the desired parameters before starting the sperm analysis.
      1. Click on Species | Fish.
      2. Select the Frames Per Second and Number of Images, depending on the best technical conditions for each species. Set both options at 120 images per second.
      3. Choose negative contrast. It is mandatory for accurate reconstruction of the spermatozoa trajectories¹⁶.
      4. Select the corresponding Counting Chamber and Scale of the video camera. Calibrate the video camera for the magnification lens used in the experiment. Set SpermTrack10 as counting chamber and 10X scale.
         NOTE: In general, a depth of 10 µm in the counting chamber and a magnification lens of 10X are the recommended conditions since they provide better focusing of all spermatozoa and capture the highest number of cells. However, it is recommended to make a previous study of the optimal conditions for motility analysis of each species.
      5. Adjust the Particle Area and Connectivity for each species. Set minimum particle area at 2 µm² and connectivity at 7 µm.
         NOTE: For fish, the minimum value of particles area ranges between 2-5 µm, and connectivity depends on the spermatozoa velocity and the frame rate.
      6. Save the set-up.
   4. Select Capture.
2. Analyze European Eel Sperm with Software
   1. Prepare the activator solution (artificial seawater) by adding 0.946 g of commercial salt to 25 mL of distilled water with 2% BSA.
   2. Take 500 mL of activator solution (artificial seawater) and place in the cooler block.
      NOTE: In general, fish sperm is activated by an osmotic shock event, although for some species the ion concentration could also be important. For freshwater species, the activator medium should have hypotonic osmolality, while for marine species the medium should be hypertonic.
   3. Put the counting chamber under the cooling stage and wait until the temperature stabilizes to 4 °C.
   4. Mix the activator and diluted sperm to activate spermatozoa and start the motility video recording between 5-10 s after activation.
      1. Take 4 µL of activator solution and place in the counting chamber.
      2. Gently homogenize the diluted sperm by shaking the microcentrifuge 3 times to avoid damage in spermatozoa cells.
      3. Take 0.5 µL of diluted sperm, mix with the activator, and put the cover in the counting chamber quickly.
         NOTE: This step should not take more than 5 s to start the analysis as soon as possible.
      4. Focus on the spermatozoa cells and find the best visual area, which is defined by a low number of spermatozoa (150 to 200 cells) to avoid interception between cells (Supplemental Figure 1A). Select Capture Video to obtain the spermatozoa tracks, which are separated according to speed.
      5. Select Capture to obtain 3 to 7 fields, which are the optimal interval to obtain a lower variability in the results, and proceed as before. Record videos until 120 s post-activation in order to avoid condensation on the cover of the counting chamber.
      6. Select Exit to have a general view of all the fields.

4. Obtain Motility Data

1. Select Fields | Partial | Save and choose a file name. Click Save.
   NOTE: The spreadsheet provides mean values of partial data, charts and images of spermatozoa tracks.
2. Select General Data | File Name | Save.
   NOTE: The data provide kinetic values for individual spermatozoa of all fields.

Results

Analysis of the time effect on sperm motility

In the case of the European eel, the percentage of static spermatozoa increased from 15 s to 120 s after activation (from 24.4% to 40.7%), and the percentage of mobile progressive spermatozoa decreased (from 36.9% to 20.9%) (Figure 1A and 1B). Based on speed, spermatozoa cells showed a decrease in velocity over time...

Discussion

The sperm analysis software used in this protocol has been used by researchers worldwide for different species, including fish. However, fish have some specific features that can affect the sperm assessment. Fish spermatozoa showed high speed in the moment of activation which declines quickly and leads to a short time of motility after activation. Besides, the temperature of reproduction is species-dependent and, in some cases, could be around 4 °C^2,4,<...

Materials

Name	Company	Catalog Number	Comments
Human Chorionic Gonadotropin	Argent Chemical Laboratories	hCG	Hormone
Benzocaine	Merck	E1501 Sigma	Anesthesia
sodium bicarbonate	Merck	S5761 Sigma	P1 medium
sodium chloride	Merck	1.06406 EMD Millipore	P1 medium
magnesium chloride	Merck	1374248 USP	P1 medium
potassium chloride	Merck	P3911-500G	P1 medium
calcium chloride	Merck	C7902-500G	P1 medium
commercial salt	Aqua Medic	Meersalz	Activator solution
BSA	Merck	05470 Sigma	Activator solution
Falcon tubes 15 ml	Merck	T1943-1000EA
Falcon tubes support	Merck	R5651-5EA
Eppendorfs	Merck	T9661-1000EA
Micropipet 20 µl	Gilson	PIPETMAN® Classic
Micropipet 10 µl	Merck	Z683787-1EA
Tips for micropipets 20 µl	Merck	Z740030-1000EA
Tips for micropipets 10 µl	Merck	Z740028-2000EA
Spermtrack	PROiSER	Counting chamber
TruMorph	PROiSER	TruMorph
Microscope UB 200i Serie	PROiSER	Microscope
Cooler plate	PROiSER		Prototype
Cooler block	PROiSER		Prototype
ISAS v1	PROiSER	ISAS	Software