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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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatozoa+selected+by+a+discontinuous+Percoll+density+gradient+exhibit+better+motion+characteristics,+more+hyperactivation,+and+longer+survival+than+direct+swim-up.">Spermatozoa selected by a discontinuous Percoll density gradient exhibit better motion characteristics, more hyperactivation, and longer survival than direct swim-up.</a> Fertility and Sterility. 64 (1), 160-165 (1995).</li><li>Paulson, J. D., Polakoski, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glass+wool+column+procedure+for+removing+extraneous+material+from+human+ejaculate.">A glass wool column procedure for removing extraneous material from human ejaculate.</a> Fertility and Sterility. 28, 178-181 (1977).</li><li>Ahammad, M. U., Chiaki, N., Tatemoto, H., Kawamoto, Y., Nakada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+the+swim-up+migration+sedimentation+technique+to+separate+viable+and+progressively+motile+fowl+spermatozoa.">Utilization of the swim-up migration sedimentation technique to separate viable and progressively motile fowl spermatozoa.</a> World's Poultry Science Association Proceedings. , (2010).</li><li>Lucena, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+motile+sperm+using+the+migration-sedimentation+technique+in+an+in+vitro+fertilization-embryo+transfer+programme.">Recovery of motile sperm using the migration-sedimentation technique in an in vitro fertilization-embryo transfer programme.</a> Human Reproduction. 4 (2), 163-165 (1989).</li><li>Henkel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+Processing+for+IVF.">Sperm Processing for IVF.</a> Clinical Embryology: A Practical Guide. , (2013).</li><li>Sherman, J., Paulson, D., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+glass+wool+filtration+on+ultrastructure+of+human+spermatozoa.">Effect of glass wool filtration on ultrastructure of human spermatozoa.</a> Fertility and Sterility. 36, 643-647 (1981).</li><li>Freund, M., Carol, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+haemocytometer+counts+of+sperm+concentration+in+human+semen.">Factors affecting haemocytometer counts of sperm concentration in human semen.</a> Journal of Reproduction and Fertility. 8, 149-155 (1964).</li><li>Bakst, M. 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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.

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		<col />
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accudenz</td>
			<td style="width:323px;">Accurate Chemical and Scientific Corporation, Westbury, NY, USA</td>
			<td style="width:119px;">AN7050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Percoll&#160;</td>
			<td style="width:323px;">Sigma-Aldrich, Corp., St. Louis, MO, USA</td>
			<td style="width:119px;">P7828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Schiff&#8217;s reagent</td>
			<td style="width:323px;">Sigma-Aldrich, Corp., St. Louis, MO, USA</td>
			<td style="width:119px;">3952016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TES</td>
			<td style="width:323px;">Sigma-Aldrich, Corp., St. Louis, MO, USA</td>
			<td style="width:119px;">T1375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eosin Y</td>
			<td style="width:323px;">Sigma-Aldrich, Corp., St. Louis, MO, USA</td>
			<td style="width:119px;">E4009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nigrosin</td>
			<td style="width:323px;">Sigma-Aldrich, Corp., St. Louis, MO, USA</td>
			<td align="right" style="width:119px;">198285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ST 40R Centrifuge&#160;</td>
			<td style="width:323px;">Thermo Scientific, Waltham, MA, USA</td>
			<td align="right" style="width:119px;">75004524</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DU 530 Life Sciences UV/Vis Spectrophotometer</td>
			<td style="width:323px;">Beckman Coulter, Brea, CA, USA</td>
			<td style="width:119px;"></td>
			<td>No catalogue is found</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Olympus IX 71 Inverted Fluorescence and Phase Contrast Microscope</td>
			<td style="width:323px;">Olympus America Inc., PA, USA</td>
			<td style="width:119px;"></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>

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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

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 ...

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

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 ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.