Name: sperm collection and computer-assisted sperm analysis in the teleost model japanese medaka (oryzias latipes)
Uploaded: 2022-10-06T13:30:00
Description: Watch this Scientific Journal Video about Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes at JoVE.com

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).
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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)2...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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&#160;</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 (<strong ...

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 ...

This technique enables the sampling of milt from a small model fish, Japanese Madaka, and the investigation of its quality in a consistent manner. Specifically sperm collection by abdominal massage allows repeated sampling on the same fish as this approach is not invasive. Furthermore, this technique can be applied for Madaka sperm cryo preservation.Sperm quality is an essential indicator of fertilization success, and therefore an important parameter to analyze in ecological and whole productive physiology studies. To begin, prepare a soft holding sponge, by cutting it to fit smugly in a Petri dish. Cut a straight line in the middle of the sponge, that is long enough to hold the fish on its ventral side up.After anesthetizing the fish, dry the abdomen of the fish by gently wiping it with a paper towel. Place the fish in the trough of the damp holding sponge with the ventral side up, so its gills are exposed to the anesthetic solution in the sponge. If the area around the cloaca is wet, gently dry the underside of the abdomen with a disposable tissue wipe.Under a dissecting microscope, place the micro pipette with an attached aspirator tube against the cloaca of the fish. Now massage the abdomen of the fish by gently squeezing with a blunt end smooth forceps in a rostral to caudal motion. For recovery of the fish, release it from the sponge into the previously prepared recovery water before returning the fish to the aquarium system.Transfer the collected milt into a tube containing preheated activating solution and pipette solution up and down several times using the aspirator tube assembly. Homogenize the diluted sperm gently by flicking the tubes before analysis. After euthanizing the fish dry it as demonstrated previously and place the fish under a dissecting microscope with its left lateral side facing up.Using small dissecting scissors, cut a flap dorsally from the cloaca and then across the ribs to the gills to expose the internal organs. Locate the testes and cut the attachment at both ends with fine forceps. Remove the testes and transfer them to a prepared tube containing pre-heated activating solution.Use the forceps to crush the testes several times against the side of the tube to release the sperm, which can be visualized by the slightly cloudy appearance of the solution. To begin the analysis, open the sperm analysis software and select the motility module. Set the configurations in the software for all parameters.Place a pre-warmed disposable 20 micrometer counting chamber slide under the microscope on a heated stage, set to 27 degrees Celsius. Pipette the sample into the chamber on the slide until it fills, but without overfilling. Carefully wipe away excess samples from the entrance of the chamber to prevent drift movement of cells in the solution.Select analyze to look at the sample under the microscope. Ensure the microscope is focused and select analyze again to record the sperm in the field. Move the slide to a new area of the sample in the frame and repeat the procedure for three to five different fields of view, avoiding air bubbles, cell masses, or artifacts.To view the results, select results, and double click on a field to visualize the individual field or to manually check for any mislabeled or untracked spermatozoa. If necessary, right click on individual spermatozoa to relabel the motility. In the present study, computer assisted sperm analysis was used to record various velocity and movement parameters.The calculated values include the straightness index, linearity index, and wobble. The osmolarity of the activation solutions impacted the motility of the sperm from testees dissection. The sperm were immotile in aquarium water, but motile in HBSS with high and low osmolarity.However, motility is significantly reduced at 36 milli osmol per kilogram, compared to higher osmolality solutions. Motility was also affected by the sperm collection method, wherein the sperm are significantly more motile in the testis dissection method compared to abdominal massage. Also, a higher percentage of the cells were progressive and more cells were either medium or rapid moving.The percentage motility and progressivity are significantly higher in the sperm stored at four degrees Celsius or on ice, compared to 27 degrees Celsius storage. The environmental and housing conditions also slightly impacted sperm motility. No significant difference in sperm motility was seen in the sperm collected after and before spawning.Males housed without females for a month showed high motility, but the difference was insignificant. For sperm collection by abdominal massage, training is needed to learn the proper forceps pressure and movement and to avoid feced contamination. This technique will be very useful to investigate for instance, the effects of diverse pollutants on male fertility in ecotoxicologial studies, where Medaka is a commonly used model.

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Research

JoVE Journal

Biology

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

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

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

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

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

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

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 ...