All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia.
1. Washing using Traditional Centrifugation
<ol>
	<li>Prepare phosphate buffer solution (PBS). Add 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 to 800 mL of distilled water (dH2O). Adjust the pH to 7.4 using 0.1 N HCl and bring the solution to 1 L using dH2O.</li>
	<li>Prepare motility buffer. Add 6.5 g of NaCl, 4.5 g of glucose, 0.444 g of CaCl2 and 11.5 g of N-tris-[hydroxymethyl] methyl-2-amino-ethanesulfonic acid (TES) to 800 mL of dH2O. Adjust the pH to 7.4 using 1 M NaOH and bring the solution to 1 L using dH2O.</li>
	<li>Pipette 0.5 mL of semen into a polypropylene microcentrifuge tube. Add 1.0 mL of PBS and mix gently.</li>
	<li>Centrifuge at 1,500 x g for 10 min at room temperature (RT) and discard the supernatant. Resuspend the sperm pellet with PBS up to 1.5 mL.</li>
	<li>Centrifuge at 1,500 x g for 10 min at RT. Resuspend the sperm pellet with motility buffer up to 0.5 mL.</li>
</ol>
2. Performing the PDGC technique
NOTE: Perform the entire process of PDGC at room temperature.
<ol>
	<li>Make 3.0 mL of 1.08 g/mL and 1.07 g/mL Percoll solutions in two separate tubes.
	<ol>
		<li>In a clean test tube, add 1.712 mL of the 1.13 g/mL original Percoll to 0.3 mL of 1.5 M NaCl solution. Add 0.988 mL of dH2O and mix by gentle inversion to make 3.0 mL of a 1.08 g/mL density solution.</li>
		<li>In a clean test tube, add 1.482 mL of the 1.13 g/mL original Percoll to 0.3 mL of 1.5 M NaCl solution. Add 1.218 mL of dH2O and mix by gentle inversion to make 3.0 mL of a 1.07 g/mL density solution.</li>
	</ol>
	</li>
	<li>In a clean test tube, dilute 1.0 mL of semen sample 1:2 with 2.0 mL of PBS. Mix gently by pipetting.</li>
	<li>Pipet 3.0 mL of the 1.07 g/mL density solution into a sterile 15 mL conical tube. Carefully pipet 3.0 mL of the 1.08 g/mL density solution beneath the 1.07 g/mL density solution. Ensure that the two layers do not mix. A long-form (9 in) Pasteur pipette can make this step easier.</li>
	<li>Pipet 3.0 mL of diluted semen sample overtop the PDG. To ensure that the semen sample does not mix with the PDG, gently tilt the conical tube containing the PDG at a 45&#176; angle. Pipet the sample along the wall of the tube and allow it to flow down the tube and over the PDG.</li>
	<li>Prepare a blank tube to match the mass of the PDG with overlaid sample. Centrifuge both tubes at 1500 x g for 20 min. Be careful to maintain the discontinuous gradient while transferring the tubes from the bench to the balance and then to the centrifuge. 
	NOTE: Do not use the brake at the end of centrifugation.</li>
	<li>Observe the results. Ensure that three distinct semen layers have formed in the tube, as seen in Figure 1.</li>
	<li>Aspirate isolated semen layers with a pipette. Collect the top layer of semen first, the middle layer second, and last the hard pellet at the bottom of the tube. Transfer each to a clean and sterile polypropylenemicrocentrifuge tube.</li>
	<li>Dilute each sample to 1.5 mL with PBS. Centrifuge at 1500 x g for 10 min.</li>
	<li>Pour off the supernatant. Reconstitute sperm pellet with motility buffer by gentle pipetting. 
	NOTE: Alternative densities may be used to suit investigator needs. Determine amounts of ingredients used with the following equation, where v0 is volume of stock density solution used, v is final volume of solution desired, p is density of final density solution desired and p0 is the density of the stock density solution: 
	<img alt="Equation 1" src="/files/ftp_upload/58833/58833eq1.jpg" /> 
	Always use 0.3 mL of 1.5 M NaCl in preparation of density solutions to match the NaCl concentration of physiological saline.</li>
</ol>
3. Determining Sperm Quality
<ol>
	<li>Calculate the sperm concentration as previously described22.</li>
	<li>Perform eosin-nigrosin vital staining as previously described12 with the following modifications:
	<ol>
		<li>Prepare 100 &#181;L of sperm solution at a concentration of 1 x 108 cells/mL.</li>
		<li>Pipet 50 &#181;L of sperm solution in a polypropylene microcentrifuge tube containing an equal volume of eosin-nigrosin stain. Incubate the mixture for 5 min at room temperature.</li>
		<li>Place a 20 &#181;L drop of stained sperm sample at one end of a glass slide and smear uniformly in a manner similar to that used for blood smears. Air-dry the smeared slides at room temperature for 3-5 min.</li>
		<li>Observe the smear under microscope. Count the number of live sperm (no stain) and dead sperm (stained pink) and calculate the percentage of live sperm.</li>
	</ol>
	</li>
	<li>Perform the Accudenz assay, as previously validated for the chicken sperm, to objectively assess the sperm mobility8 with the following modifications:
	<ol>
		<li>Pipet 1.0 mL of 6% assay solution into polystyrene cuvettes, as illustrated in Figure 2. Incubate to 41 &#176;C. 
		NOTE: 41 &#176;C is used to match the internal temperature of a hen. The incubation temperature should match that of the female reproductive tract of the species being investigated.</li>
		<li>Overlay the preheated assay solution with 100 &#181;L of semen sample at a concentration of 5 x 108 cells/mL.</li>
		<li>Place the cuvette containing overlaid sperm sample in the spectrophotometer. Record the absorbance value at 550 nm.</li>
	</ol>
	</li>
	<li>Perform IPVL-penetration assay as previously described11 with the following modifications:
	<ol>
		<li>Cut a piece (0.5 cm x 0.5 cm) of non-germinal disc region of intact IPVL.</li>
		<li>Adjust sperm concentration to 4 x 106 cells/mL</li>
		<li>Incubate sperm in motility buffer with IPVL in a small glass vial for 15 min at 37 &#176;C, as illustrated in Figure 3.</li>
		<li>Immerse the IPVL piece in 3% NaCl to stop the interaction between the IPVL and sperm.</li>
		<li>Mount the IPVL piece on a microscope slide and stain with Schiff&#39;s reagent for 10 min following fixation with 10% formalin for 20 s.</li>
		<li>Observe the IPVL under a microscope for successful sperm penetration holes and count the number of all visible holes per 0.25 mm2 at 40X magnification.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Fertility not only determines the profitability of animal production but also acts as a means of natural selection of species for existence. The ultimate function of a sperm cell is to fertilize an ovum. The oviduct of a female selects only those fittest sperm in order to ensure fertilization of the ovum23,24. In vitro studies have also revealed a close correlation between qualitative sperm traits and fertilization success4,11,23,24. Reduced fertility is associated with poor quality of sperm4,11,25. As such, it necessitates processing semen for improvement of sperm quality. In ART, PDGC has been applied to collect only the high-quality sperm to supplement fertility of humans and agriculturally-important animals13,16,20,26. Using this technique, we, however, demonstrated a gentle, non-invasive technique of separating low-, medium- and high-quality sperm in the domestic chicken model.
The presented protocol of PDGC, when performed correctly, allows separation of not only high-quality sperm, but also effective separation of low- and medium-quality sperm. The degree of success of PDGC is reliant on careful preparation of the discontinuous gradient as well as equally careful collection of isolated layers. Diameter of the conical tube used also influences success of PDGC. We have observed that a larger tube diameter results in decreased efficiency of the technique. It is important that PDGC is performed at room temperature in order to maintain integrity of the PDG. Any previously refrigerated samples should be adjusted to room temperature before being placed on the PDG. The most common issue that could be observed after performing PDGC is inefficient separation; the isolated layers will not be distinct from one another. If this issue occurs, in addition to addressing the above critical steps, it may be corrected by reducing sample dilution, increasing the volumes of density solutions used and ensuring the volumes of solutions used are equal.
PDGC is very adaptable. Any range of densities between 1.00 and 1.13 g/mL may be used for preparation of the gradient. We have found that 1.07 and 1.08 g/mL density solutions work well for separation of chicken sperm by quality, but these densities may need adjustment for other sample types or experimental purposes. The number of density solutions used in the gradient may also be adjusted to suit the needs of the experimenter. The number of quality groups isolated will always be one more than the number of density solutions used.
PDGC is much more effective than alternative methods of sample separation in situations where large quantities of sample are needed13,16,20,26. Migration assays effectively separate the highest quality sperm from a sample18,19, but they cannot be used to resolve the sample any further. Migration assays are also incapable of preparing large volumes of sample18,20. Adherence column assays effectively separate large volumes of sample17; however, the process is deleterious to the quality of the sperm collected, making confirmation of the quality groups after separation difficult20,21. PDGC, however, does have its own limitations. In the case of mammalian sperm, it should not be used for preparation of samples for in vitro fertilization, due to the possible contamination with endotoxins27. The effect of Percoll contamination with endotoxins has not been investigated in other animal classes. Sperm performance is not affected by endotoxins, meaning the PDGC is still appropriate for research purposes.
Collection of sperm by their quality using the PDGC technique will be instrumental for a wide range of applications including studies involving sperm physiologic, metabolomic, transcriptomic and proteomic research. Application of this technique will contribute to the discovery of biomarkers of sperm quality for usage in marker-assisted fertility improvement programs.

In vertebrates, male gametes undergo intense selective pressure; therefore reproductive fitness of a male is pivotal for achieving successful fertilization. Males of any given vertebrate species must be able to produce sperm cells in large quantities and of sufficient quality in order to meet the needs of fertilization. Sperm cells, having both a sperm head and a flagellum, are the most polarized cells in the body. They are also very heterogeneous in quality of sperm (live and dead, morphologically normal and abnormal, and immobile, low mobile and high mobile), which is revealed through the wide variation in reproductive efficiency of the males. The larger the proportion of high-quality sperm, the fewer the number of matings required to successfully fertilize the ovum. However, to achieve fertility, morphologically normal sperm cells rely on propulsive forces generated by their flagella to reach the site of fertilization as well as to penetrate the zona pellucida1 (ZP; in the case of mammals) or inner perivitelline layer2 (IPVL; in the case of birds and reptiles) of the ovum following natural mating or artificial insemination (AI). Determining sperm quality is necessary for use in assisted reproductive technologies3 (ART) and selection of breeding males to be used in AI programs4. On the other hand, the success of ART solely relies upon the accurate evaluation of sperm quality. A number of laboratory tests have been developed to determine the functional characteristics of sperm. The most important parameters are sperm morphology, viability, mobility, capacitation (avian sperm do not require capacitation5), acrosome reaction (AR; exocytosis and release of a proteolytic enzyme from the acrosome of the sperm head), sperm penetration of ZP or IPVL, and fertilization6,7,8,9,10,11. Measures of fertility alone do not provide an accurate evaluation of the fertilizing ability of a sperm population11. Measures of the several events leading up to fertilization of an egg allow for an appropriate representation of the performance of individual spermatocytes7.
The methodology developed for measuring sperm function is primarily species-specific. For example, in avian sperm, viability, mobility and penetration of IPVL are the most common parameters used to assess sperm quality8,11,12. The number of live sperm in the ejaculate plays a crucial role for the survival of sperm because the presence of a large number of dead sperm in the semen affects the quality of sperm. This enhances the production of reactive oxygen species in the semen and causes oxidative damage to the live sperm13. Sperm mobility, the capacity for flagellar movement of avian sperm against resistance at body temperature, is known to play a direct role in bringing about fertilization8. It is well established that mobility is positively correlated with fertility and is, therefore, a primary determinant of fertility8. However, a mobile sperm must also have the ability to undergo an AR and to penetrate the IPVL11. IPVL penetration assays take account for every sperm that participates in the process of fertilization11.
In the application of ART, ejaculate is usually processed in order to maximize the concentration of high-quality sperm and minimize concentration of low-quality sperm. After collection of semen, the proportion of high-quality sperm can be enriched through sperm separation procedures commonly used in both industry and research practices. Many of these procedures have been developed, all with respective benefits and limitations, but all utilize the heterogeneous nature of sperm to collect only the sperm with high fertilizing ability. These procedures include sperm migration methods, adherence column filtration and density gradient centrifugation (DGC)14,15,16,17,18,19,20. Among the available techniques, DGC has been found to be very simple, repeatable, cost-effective and efficient in isolating the maximum amount of high-quality sperm for use in ART with the goal of maximizing chance of fertilization14,15. In addition, DGC is not injurious to the sperm cell membrane. In contrast, sperm migration methods collect only progressively mobile sperm18,19, but the quantity of sperm collected is very low, making it inefficient in collecting large volumes of sperm18,20. Adherence column filtration is very efficient in filtering highly mobile sperm from semen17; however, it tends to be injurious to sperm membranes20,21.
In the DGC technique, the most commonly used substrate for generating the density gradient is Percoll, which consists of colloidal silica particles coated in polyvinylpryrolidone. Percoll density gradient centrifugation (PDGC) can either be continuous or discontinuous but a discontinuous gradient is most commonly used for high yield isolation of highly mobile sperm13,16,20. In a discontinuous gradient, lower density media floats above higher density media, creating a gradient that increases in density from the top to the bottom of a conical tube. This creates boundaries at the interface between the two media of differing density. The efficiency of PDGC is derived from two factors: 1) the propulsive ability of individual sperm cells and 2) the tendency of sperm cells with high structural integrity to have an increased density. Sperm with higher mobility are better able to cross from lower density media and penetrate into a higher density media. Lower mobility sperm are more likely to become trapped at the boundary created by the interface between media of differing density. Sperm cells with high structural integrity and mobility tend to have a higher density than dead, abnormal or low mobile sperm cells. When centrifugal force is applied in PDGC, this facilitates movement of sperm with different densities to their respective place in the gradient.
In general practice, after PDGC is performed, the soft pellet of sperm with high fertility potential at the bottom of the conical tube is collected, and the remainder is discarded. However, an underutilized advantage of this technique is its ability to separate sperm cells into several groups based on the quality differences. For research purposes, separation of sperm by degree of quality utilizing the PDGC technique allows for study of sperm quality as it pertains to physiologic, metabolomic and proteomic differences. Here, we aim to detail how this technique may be used to separate sperm by quality, as well as demonstrate these differences in quality, using the previously established eosin-nigrosin vital staining for viability, Accudenz assay for mobility, and sperm-IPVL interaction assay for penetrability.

<table><tbody><tr><td>Accudenz</td><td>Accurate Chemical and Scientific Corporation, Westbury, NY, USA</td><td>AN7050</td><td></td></tr><tr><td>Percoll&amp;nbsp;</td><td>Sigma-Aldrich, Corp., St. Louis, MO, USA</td><td>P7828</td><td></td></tr><tr><td>Schiff&amp;rsquo;s reagent</td><td>Sigma-Aldrich, Corp., St. Louis, MO, USA</td><td>3952016</td><td></td></tr><tr><td>TES</td><td>Sigma-Aldrich, Corp., St. Louis, MO, USA</td><td>T1375</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma-Aldrich, Corp., St. Louis, MO, USA</td><td>E4009</td><td></td></tr><tr><td>Nigrosin</td><td>Sigma-Aldrich, Corp., St. Louis, MO, USA</td><td>198285</td><td></td></tr><tr><td>ST 40R Centrifuge&amp;nbsp;</td><td>Thermo Scientific, Waltham, MA, USA</td><td>75004524</td><td></td></tr><tr><td>DU 530 Life Sciences UV/Vis Spectrophotometer</td><td>Beckman Coulter, Brea, CA, USA</td><td></td><td>No catalogue is found</td></tr><tr><td>Olympus IX 71 Inverted Fluorescence and Phase Contrast Microscope</td><td>Olympus America Inc., PA, USA</td><td></td><td>No catalogue is found</td></tr></tbody></table>

sperm collection of differential quality using density gradient centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

The PDGC technique resulted in distinct separation of three layers of sperm by degree of quality across all parameters. Sperm separates into a high-quality layer below the higher density solution, a medium-quality layer between the higher and lower density solution and a low-quality layer above the lower density solution. These differences in quality are evidenced by clear differences in viability (Figure 4), mobility (Figure 5) and penetrability (Figure 6). Sperm isolated from the high-quality layer of the PDG exhibited increases in all three parameters relative to those of the traditionally washed sample. Those layers noted as medium- and low-quality upon separation by PDGC display moderate and dramatic, respectively, decreases across all test parameters.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58833/58833fig1.jpg" /> 
Figure 1: Isolation of sperm with differential quality using PDGC. Overlay 3 mL of sperm solution on prepared PDG solution. Centrifuge at 1500 x g for 20 min. Collect three distinct groups of sperm with low-, medium- and high-quality. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58833/58833fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58833/58833fig2.jpg" /> 
Figure 2: Determination of sperm mobility using Accudenz assay. Overlay 100 &#181;L of sperm sample on a 6% assay solution in a cuvette. Incubate at 41 &#176;C for 5 min. Record the mobility of sperm as a function of absorbance at 550 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58833/58833fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58833/58833fig3.jpg" /> 
Figure 3: A model of formation of sperm penetration holes in IPVL. Incubate sperm with a small section of IPVL at 37 &#176;C for 15 min. Upon contact with IPVL, sperm cells bind with the IPVL, undergo an acrosome reaction and penetrate the IPVL, creating penetration holes. Count the number of penetration holes and measure the penetrability of sperm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58833/58833fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58833/58833fig4.jpg" /> 
Figure 4: Viability of sperm as assessed by eosin-nigrosin vital staining. Digital micrographs showing eosin-nigrosin vital staining of sperm obtained through a process of traditional wash (A) and PDGC (B, low motile sperm; C, medium motile sperm; D, high motile sperm). Scale bar = 50 &#181;m at 40 x magnification. Pink staining indicates dead sperm which have taken up eosin stain. Data were subjected to one-way ANOVA, followed by the Tukey-Kramer test. Error bars indicate the standard error of the mean (SEM). Values are presented as the mean &#177; SEM (n = 5). a-d Values without a common superscript differed (P &#60; 0.05). Sperm isolated by PDGC into low-, medium- and high-quality groups (E) reveal significant differences in percentage viability, 66.0 &#177; 4.7, 77.0 &#177; 8.9 and 92.8 &#177; 3.4, respectively. Sperm washed by traditional methods displayed an 81.6 &#177; 4.9% viability, lower than the high-quality group but higher than the medium- and low-quality groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58833/58833fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58833/58833fig5.jpg" /> 
Figure 5: Mobility of sperm as assessed by Accudenz assay. Absorbance values act as a proxy measure for sperm mobility in assessment by the Accudenz assay. Data were subjected to one-way ANOVA, followed by the Tukey-Kramer test. Error bars indicate the standard error of the mean (SEM). Values are presented as the mean &#177; SEM (n = 5). a-d Values without a common superscript differed (P &#60; 0.05). Sperm isolated into low-, medium- and high-quality groups by the PDGC technique exhibited markedly different low (0.11 &#177; 0.03), medium (0.34 &#177; 0.01) and high (0.87 &#177; 0.03) absorbance values, respectively. Sperm washed by traditional methods exhibited an absorbance of 0.71 &#177; 0.03, an absorbance lower than that of the high-quality group, but higher than that of the medium- and low-quality groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58833/58833fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58833/58833fig6.jpg" /> 
Figure 6: Penetrability of sperm as assessed by sperm-inner perivitelline layer (IPVL) interaction assay. Digital micrographs showing in vitro formation of holes on the surface of the IPVL by chicken sperm (4 x 106 cells/mL) during incubation in motility buffer at 37 &#176;C for 15 min. Micrographs represent IPVL penetrated by sperm having undergone traditional wash (A) and sperm from low- (B), medium- (C) and high- (D) quality groups from PDGC. Scale bar = 50 &#181;m at 40X magnification. Data were subjected to one-way ANOVA, followed by the Tukey-Kramer test. Error bars indicate the standard error of the mean (SEM). Values are presented as the mean &#177; SEM (n = 5). a-d Values without a common superscript differed (P &#60; 0.05). The number of penetration holes per 0.25 mm2 (E) differed between the sperm isolated from the different gradients of the density media. Sperm isolated into low-, medium- and high-quality groups by PDGC produced a low (24.40 &#177; 1.1), medium (33.20 &#177; 1.4) and high (51.20 &#177; 1.3) number of penetration holes, respectively. The sperm washed using the traditional method produced 46.40 &#177; 1.8 penetration holes, a value lower than the high-quality group but higher than the medium- and low-quality groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58833/58833fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Sperm Collection of Differential Quality Using Density Gradient Centrifugation at JoVE.com

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

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19.5K Views.  University of Georgia. In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Video: Sperm Collection of Differential Quality Using Density Gradient Centrifugation

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Developmental Biology

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

Genetics

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

U-Shaped Horizontal Swimming Technique for Preparing High-Quality Sperm with Low DNA Fragmentation Index

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and viscous seminal plasma. High-quality sperm refers to progressively motile spermatozoa with normal morphology, which often exhibit a lower DNA fragmentation index (DFI) and higher fertilization potential. Preparing high-quality sperm is a critical step in human-assisted reproductive technology. The traditional sperm preparation method, discontinuous density gradient centrifugation (DGC), is time-consuming and labor-intensive. Repeated centrifugation can damage sperm DNA, thereby affecting subsequent fertilization and embryo development. This study introduces a U-shaped horizontal swimming (UHS) method for preparing intracytoplasmic sperm injection (ICSI) sperm, which significantly eliminates the detrimental effects of&#160;centrifugation on sperm DNA.&#160;The UHS method involves creating a UHS lane using a fertilization medium within an ICSI operating dish. A 10 &#181;L fertilization medium microdroplet is placed at the starting point on the left side of the UHS lane to hold the semen. Two additional 10 &#181;L fertilization medium buffer droplets are positioned at intervals along the left middle section of the lane, with all droplets connected by fertilization medium. The dish is then covered with culture oil and incubated overnight at 37 &#176;C with 6% CO2 to equilibrate. Subsequently, 3 &#181;L of semen is added to the microdroplet at the starting point. High-quality spermatozoa swim to the right side of the UHS lane, facilitating their suction into the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities largely remain at the initial point or in the buffer droplets. We simultaneously processed 21 semen samples using both UHS and DGC techniques and compared their DFI. The results demonstrated that the DFI in the DGC group was 5.5% &#177; 3.2%, whereas the DFI in the UHS group was 1.7% &#177; 1.1%. The difference between the two groups was statistically significant (P &#60; 0.05).

This protocol describes a method for screening high-quality sperm with a low DNA fragmentation index by utilizing sperm motility characteristics and thigmotaxis. The method employs a U-shaped horizontal swimming lane, enabling high-quality sperm to reach the opposite side through buffer droplets, while dead sperm, cell debris, and impurities are excluded.

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and ...

Medicine

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

Density Gradient Ultracentrifugation

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact that, in suspension, particles that are more dense than the solvent will sediment, while those that are less dense will float. A high-speed ultracentrifuge is used to accelerate this process in order to separate biomolecules within a density gradient, which can be established by layering liquids of decreasing density in a centrifuge tube.
The video will cover the principles of density gradient ultracentrifugation, including a procedure that demonstrates sample preparation, creation of a sucrose gradient, ultracentrifugation, and collection of fractionated analytes. The applications section discusses isolation of multi-protein complexes, isolation of nucleic acid complexes, and separation using cesium chloride density gradients.
<div class="chapter_text" id="script_0">
Density gradient ultracentrifugation is a common approach to isolate and purify cell structures for biochemical experiments. The technique uses a high-speed, or ultra, centrifuge to nondestructively separate cellular components in a density gradient. This video describes the principles of density gradient ultracentrifugation, provides a general procedure using a sucrose gradient, and discusses some applications.
</div>
<div class="chapter_text" id="script_1">
Let&#39;s start by examining the principles of ultracentrifuges and density gradients. A suspension contains particles in a liquid solvent. Because of gravity, particles denser than the solvent sediment out while those less dense than the solvent float. The greater the difference in density between the particle and the solvent, the faster the separation.
An ultracentrifuge contains a unit called a rotor, which rotates at highly controlled speeds, simulating a strong gravitational field. Within this field, the differences in density between particles and the solvent are magnified.
The strength of the field depends on the speed of rotation. Even a small rotor at a relatively low rotational speed can create a force thousands of times stronger than the earth&#39;s gravitational field.
If a tube contains several liquids of different densities, centrifugation will keep them in separate layers in order of density, with the densest liquid closest to the base. Such a layering of multiple liquids is called a &#34;density gradient.&#34; There are two types. In step gradients, liquids of decreasing density are carefully layered on top one another. In continuous gradients, the liquids are mixed in varying proportions, so the density decreases smoothly from the base upwards.
Cellular organelles can be separated using a step gradient, through &#34;isopycnic density-gradient centrifugation.&#34; This is the simplest and most common centrifugation procedure.
This procedure is used to separate the cellular structures. The more dense the organelle, the further it descends-with mitochondria at the top and nucleic acids towards the bottom.
Now that you know the principles behind the technique, let&#39;s see it in the lab.
</div>
<div class="chapter_text" id="script_2">
Before the procedure is started, the manufacturer&#39;s speed and density ratings should be noted, and the ultracentrifuge checked for corrosion. This procedure uses a swinging-bucket rotor.
First, the cellular material is prepared by homogenizing the cells, which nondestructively releases their organelles. The homogenate may be fractionated through preliminary low-speed centrifugation, to remove low-density components. Next, the sucrose solutions are prepared.
Sucrose is added in increasing amounts so each solution is more concentrated, and therefore denser, than the preceding one. The exact densities of the solutions will depend on the components to be separated, which vary between organisms. The solutions should have densities between those of the components to be separated, with the last solution denser than the densest component of the analyte. Techniques for separating components denser than sucrose, like nucleic acids, are described in the applications.
The sucrose gradient is now created in a clean centrifuge tube. A pipette is used to draw up the most concentrated sucrose solution. With the tube held upright, the pipette tip is placed high against the wall, and the liquid dispensed steadily down. It&#39;s important that the working area is kept free of vibrations and other disturbances.
After replacing the tip, the remaining solutions are added in order of decreasing density. They are dispensed carefully to form distinct layers and avoid mixing. Finally, about half a milliliter of the cellular sample is added atop the gradient, and the tube is weighed. This is used to balance the weight distribution, the next step of the process.
</div>
<div class="chapter_text" id="script_3">
Centrifugation should begin as soon as possible. The tube is placed in the rotor, which is then balanced by placing blank solutions of equal weight in opposing slots. The rotor is placed in the ultracentrifuge and the system sealed. The temperature and rotation speed and time are set. Typical values are 4 &#176;C with a force of over 100,000 x g for 16 h.
After centrifugation, the tube is withdrawn from the rotor, taking care to keep it upright and undisturbed. The different cellular components have fractionated into discrete bands between the solution layers. The fractions can be collected with a syringe. Alternately, the bottom of the tube can be punctured with a fine, sterilized needle and the outflow collected in sterile tubes. The cellular components have now been isolated. They can be stored at -80 &#176;C.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve seen the basic procedure, let&#39;s look at some applications.
A typical application is the isolation of multi-protein complexes in plant cells. In this example, complexes responsible for cyclic electron flow are being isolated from the thylakoid, the site of the light reaction in photosynthesis. This procedure uses discrete solutions of 14 to 45% sucrose. Centrifugation occurs over 100,000 x g for 14 h at 4 &#176;C.
Because nucleic acids are denser than sucrose, isopycnic centrifugation cannot separate them from organelles nondestructively.
A different technique, known as &#34;rate-zonal centrifugation&#34; is used. It separates organelles based on their sedimentation rates, which depend not only on their densities, but also on their conformations. A continuous gradient is used to separate the components based on this property.
The procedural steps are similar to those for isopycnic cases. In this example, RNA-ribosome complexes are isolated using a continuous gradient of 5% to 20%, centrifuged at 230,000 x g. Centrifugation is interrupted after a few hours to prevent co-precipitation.
Nucleic acid strands can be separated from each other on the basis of density.
This is because strands rich in guanine and cytosine are denser than those rich in adenine and thiamine. In this case, the gradient cannot be made of sucrose, because sucrose is less dense than nucleic acids. Instead, cesium chloride gradients, typically from 1.65 to 1.75 g/mL are used, as they have sufficient density and a low viscosity.
Here we see plankton DNA being purified using a continuous cesium chloride gradient. Centrifugation occurs at over 1,000,000 x g for 18 h under vacuum.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on ultracentrifugation with a sucrose density gradient. You should now understand how a density gradient works, how to construct a step gradient, and how to load and operate an ultracentrifuge. Thanks for watching!
</div>

Density Gradient Ultracentrifugation: Principle and Applications

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact ...

JoVE Science Education

Chemistry

Biochemistry

Density Gradient Centrifugation: A Method to Isolate CLL Cells from Peripheral Blood

Source: Hay, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60426">Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking.</a> J. Vis. Exp. (2019).
This video describes the technique of isolating chronic lymphocytic leukemia (CLL) cells from peripheral blood. With this protocol, we will isolate CLL cells from human patient blood sample for subsequent efficient generation of nuclear and cytoplasmic fractions.

Wirowanie w gradiencie gęstości: metoda izolowania komórek PBL z krwi obwodowej

Source: Hay, J. et al. Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking. J. Vis. ...

JoVE Encyclopedia of Experiments

Cancer Research

Leukemia

Cell isolation & characterization

Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation

Source: Narayanan, A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61542/nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus">Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq.</a> J. Vis. Exp. (2020)
In this video, we describe a simple and efficient protocol using gradient centrifugation to isolate single nuclei. Once isolated, these nuclei can be used for sequencing and genome analysis.

Oczyszczanie jąder oparte na wirowaniu gradientowym: technika otrzymywania ultraczystej frakcji nienaruszonych jąder za pomocą wirowania w gradicynacji gęstości

Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)
In this video, ...

Cancers of the Nervous System

In vitro studies

Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

Source: Urwitz, S. N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59143/extraction-of-extracellular-vesicles-from-whole-tissue">Extraction of Extracellular Vesicles from Whole Tissue.</a> J. Vis. Exp. (2019)
This video demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

Oczyszczanie subpopulacji pęcherzyków zewnątrzkomórkowych: technika oczyszczania subpopulacji pęcherzyków zewnątrzkomórkowych za pomocą ultrawirowania w gradimencie gęstości

Source: Urwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video demonstrates the density gradient ...