<meta charset="utf8"></head><body>优化受威胁珊瑚物种的珊瑚精子冷冻保存

<ol>
	<li>Eakin, C. M., Sweatman, H. P. A., Brainard, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2014-2017+global-scale+coral+bleaching+event:+Insights+and+impacts.">The 2014-2017 global-scale coral bleaching event: Insights and impacts.</a> Coral Reefs. 38 (4), 539-545 (2019).</li><li>Anthony, K., 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=New+interventions+are+needed+to+save+coral+reefs.">New interventions are needed to save coral reefs.</a> Nature Ecology &amp; Evolution. 1 (10), 1-3 (2017).</li><li>Wildt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+resource+banking+for+wildlife+research,+management,+and+conservation.">Genome resource banking for wildlife research, management, and conservation.</a> ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 41 (4), 228-234 (2000).</li><li>Hagedorn, M., 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=Preserving+and+using+germplasm+and+dissociated+embryonic+cells+for+conserving+Caribbean+and+Pacific+coral.">Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.</a> PLoS ONE. 7 (3), e33354 (2012).</li><li>Hagedorn, M., 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=First+frozen+repository+for+the+Great+Barrier+Reef+coral+created.">First frozen repository for the Great Barrier Reef coral created.</a> Cryobiology. 65 (2), 157-158 (2012).</li><li>Zuchowicz, N., Daly, J., Lager, C., Williamson, O., Hagedorn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freezing+on+the+beach:+A+robust+coral+sperm+cryopreservation+design.">Freezing on the beach: A robust coral sperm cryopreservation design.</a> Cryobiology. 101, 135-139 (2021).</li><li>Hobbs, R. J., 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=A+decade+of+coral+biobanking+science+in+Australia+-+transitioning+into+applied+reef+restoration.">A decade of coral biobanking science in Australia - transitioning into applied reef restoration.</a> Frontiers in Marine Science. , 9 (2022).</li><li>Daly, J., 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=Cryopreservation+can+assist+gene+flow+on+the+Great+Barrier+Reef.">Cryopreservation can assist gene flow on the Great Barrier Reef.</a> Coral Reefs. 41, 455-462 (2022).</li><li>Hagedorn, M., 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=Assisted+gene+flow+using+cryopreserved+sperm+in+critically+endangered+coral.">Assisted gene flow using cryopreserved sperm in critically endangered coral.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (38), e2110559118 (2021).</li><li>Daly, J., 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=Successful+cryopreservation+of+coral+larvae+using+vitrification+and+laser+warming.">Successful cryopreservation of coral larvae using vitrification and laser warming.</a> Scientific Reports. 8, 1-10 (2018).</li><li>Daly, J., 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=The+first+proof+of+concept+demonstration+of+nanowarming+in+coral+tissue.">The first proof of concept demonstration of nanowarming in coral tissue.</a> Advanced Sustainable Systems. , (2023).</li><li>Powell-Palm, M. J., 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=Cryopreservation+and+revival+of+Hawaiian+stony+corals+via+isochoric+vitrification.">Cryopreservation and revival of Hawaiian stony corals via isochoric vitrification.</a> Nature Communications. 14, 4859 (2023).</li><li>. Reef Restoration and Adaptation Program Available from: <a target="_blank" href="https://gbrrestoration.org">https://gbrrestoration.org</a> (2023)</li><li>. Florida Coral Rescue Available from: <a target="_blank" href="https://myfwc.com/research/habitat/coral/disease/rescue">https://myfwc.com/research/habitat/coral/disease/rescue</a> (2023)</li><li>Harrison, P. 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> Coral reefs: An ecosystem in transition. , 59-85 (2011).</li><li>Hagedorn, M., Carter, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiology:+Principles,+species+conservation+and+benefits+for+coral+reefs.">Cryobiology: Principles, species conservation and benefits for coral reefs.</a> Reproduction, Fertility and Development. 28 (8), 1049 (2016).</li><li>Zuchowicz, N., 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=Assessing+coral+sperm+motility.">Assessing coral sperm motility.</a> Scientific Reports. 11, 61 (2021).</li><li>Thomas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+cryovial+dispensing+system+for+cell+banking.">Evaluation of an automated cryovial dispensing system for cell banking.</a> Genetic Engineering &amp; Biotechnology News. 37 (17), 22-23 (2017).</li><li>. Coral cryopreservation training course Available from: <a target="_blank" href="https://nationalzoo.si.edu/center-for-species-survival/coral-cryopreservation-training-course">https://nationalzoo.si.edu/center-for-species-survival/coral-cryopreservation-training-course</a> (2023)</li><li>Bailey, 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=Validation+of+sperm+counting+methods+using+limits+of+agreement.">Validation of sperm counting methods using limits of agreement.</a> Journal of Andrology. 28 (3), 364-373 (2007).</li><li>Hu, E., Yang, H., Tiersch, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cryopreservation+of+spermatozoa+of+blue+catfish+(Ictalurus+furcatus):+Establishment+of+an+approach+for+commercial-scale+processing.">High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing.</a> Cryobiology. 62 (1), 74-82 (2011).</li><li>Pickett, B. W., Berndtson, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+bovine+spermatozoa+by+freezing+in+straws:+A+review.">Preservation of bovine spermatozoa by freezing in straws: A review.</a> Journal of Dairy Science. 57 (11), 1287-1308 (1974).</li><li>Westbrook, C. E., Daly, J., Bowen, B., Hagedorn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+the+collector+urchin+embryo,+Tripneustes+gratilla.">Cryopreservation of the collector urchin embryo, Tripneustes gratilla.</a> Cryobiology. 15, 104865 (2024).</li><li>Maria, A. N., 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=Use+of+cryotubes+for+the+cryopreservation+of+Tambaqui+fish+semen+(Colossoma+macropomum).">Use of cryotubes for the cryopreservation of Tambaqui fish semen (Colossoma macropomum).</a> Cryobiology. 70 (2), 109-114 (2015).</li><li>Fuchs, Y. F., 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=Next-generation+biobanking:+Employing+a+robotic+system+for+automated+mononuclear+cell+isolation.">Next-generation biobanking: Employing a robotic system for automated mononuclear cell isolation.</a> Biopreservation &amp; Biobanking. 21 (1), 106-110 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Ovation ALI-Q 2 VS Pipette Controller - Aliquotting pipette</td>
			<td >Vistalab</td>
			<td >2100-1005</td>
			<td >Fluid handling &#8211; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td>
		</tr>
		<tr >
			<td >5 mL serological pipettes (bulk)</td>
			<td >Thermo Scientific&#160;</td>
			<td >Nunc 170355</td>
			<td >Fluid handling &#8211; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td>
		</tr>
		<tr >
			<td >10 mL serological pipettes (bulk)</td>
			<td >Thermo Scientific</td>
			<td >Nunc&#160; 170356</td>
			<td >Fluid handling &#8211; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td>
		</tr>
		<tr >
			<td >P2 0.2&#8211;2 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144054M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P10 1&#8211;10 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144055M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P20 2&#8211;20 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >&#160;F144056M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P200 20&#8211;200 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144058M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P1000 100&#8211;1000 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144059M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >Vacuum pump</td>
			<td >Millipore</td>
			<td >WP6122050</td>
			<td >Preparation of filtered sea water for solution preparation</td>
		</tr>
		<tr >
			<td >Reusable bottle-top filtration system</td>
			<td >Thermo Scientific</td>
			<td >DS0320-5045</td>
			<td >Preparation of filtered sea water for solution preparation</td>
		</tr>
		<tr >
			<td >0.22-&#181;m filter discs, mixed cellulose esters&#160;</td>
			<td >Merck Millipore</td>
			<td >GSWP04700</td>
			<td >Preparation of filtered sea water for solution preparation</td>
		</tr>
		<tr >
			<td >Filtered sea water</td>
			<td >N/A</td>
			<td >&#8211;</td>
			<td >Base medium for sperm activation and cryoprotectant solutions</td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide (DMSO)</td>
			<td >Sigma-Aldrich</td>
			<td >D4540</td>
			<td >Cryoprotectant chemical used at a final concentration of 10% v/v in filtered seawater for sperm cryopreservation</td>
		</tr>
		<tr >
			<td >Caffeine</td>
			<td >Sigma-Aldrich</td>
			<td >C0750</td>
			<td >Used to activate sperm motility</td>
		</tr>
		<tr >
			<td >BSA heat shock fraction</td>
			<td >Sigma-Aldrich</td>
			<td >A9647</td>
			<td >Used to minimise sperm adherance to CASA well slides</td>
		</tr>
		<tr >
			<td >15-mL tubes - racked</td>
			<td >Thermo Scientific</td>
			<td >339651</td>
			<td >Preparation of sperm activation solution</td>
		</tr>
		<tr >
			<td >50-mL tubes racked</td>
			<td >Thermo Scientific</td>
			<td >339653</td>
			<td >For collection of gamete bundles and filtered sperm samples</td>
		</tr>
		<tr >
			<td >Transfer pipettes</td>
			<td >Thermo Scientific</td>
			<td >Samco 202PK</td>
			<td >To aid collection of gamete bundles from the water surface</td>
		</tr>
		<tr >
			<td >100-&#181;m filter baskets</td>
			<td >Fisher Scientific</td>
			<td >22363549</td>
			<td >To exclude eggs during separation of the sperm sample</td>
		</tr>
		<tr >
			<td >Eppendorf racks</td>
			<td >Interpath</td>
			<td >511029</td>
			<td>Dilution and activation of sperm for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >Eppendorf 1.5-mL tube</td>
			<td >Eppendorf</td>
			<td >30120086</td>
			<td>Dilution and activation of sperm for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >Glass coverslips 18x18 mm</td>
			<td >Brand</td>
			<td >4700 45</td>
			<td>Assessment of sperm concentration and motility using phase microscopy</td>
		</tr>
		<tr >
			<td >Plain glass slides, precleaned, 75x25 mm</td>
			<td >Corning</td>
			<td >2947</td>
			<td>Assessment of sperm concentration and motility using phase microscopy</td>
		</tr>
		<tr >
			<td >Haemocytometer</td>
			<td >Hausser Scientific</td>
			<td >1492</td>
			<td>Assessment of sperm concentration and motility using phase microscopy</td>
		</tr>
		<tr >
			<td >CASA slides (Leja 20-&#181;m 4 chamber, SC-20-01-04-B)</td>
			<td >IMV Technologies</td>
			<td >025107</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >Makler sperm counting chamber (CASA)</td>
			<td >IVFStore</td>
			<td >SM-373</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >accu-bead&#174; counting beads</td>
			<td >Hamilton-Thorne</td>
			<td >710111</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >CASA system + laptop</td>
			<td >Hamilton Thorne</td>
			<td >Ceros II</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >Safety Glasses</td>
			<td >Generic</td>
			<td >&#8211;</td>
			<td>Personal protective equi[pment for use when handling DMSO and liquid nitrogen</td>
		</tr>
		<tr >
			<td >Lab coat</td>
			<td >Long sleeve, full length</td>
			<td >&#8211;</td>
			<td>Personal protective equi[pment for use when handling DMSO and liquid nitrogen</td>
		</tr>
		<tr >
			<td >Cryogloves (pair)</td>
			<td >Tempshield</td>
			<td >Mid-Arm</td>
			<td>Personal protective equi[pment for use when handling DMSO and liquid nitrogen</td>
		</tr>
		<tr >
			<td >Medium forceps</td>
			<td >Generic</td>
			<td >&#8211;</td>
			<td >For removing cryopreserved samples from the cryo racks and manipulating samples in liquid nitrogen</td>
		</tr>
		<tr >
			<td >Barcode scanner (2D compatible)</td>
			<td >Zebra</td>
			<td >DS2278</td>
			<td >For reading 1D and 2D barcodes on cryovials for sample management</td>
		</tr>
		<tr >
			<td >2.0-mL CryoStorage Vial, external thread, pre-capped, 2D SafeCode (DataMatrix/ECC200), linear and human readable</td>
			<td >Eppendorf</td>
			<td >30079434</td>
			<td >Barcoded cryovials for cryopreservation of sperm samples</td>
		</tr>
		<tr >
			<td >Cryovial rack</td>
			<td >Simport</td>
			<td >T315</td>
			<td >Rack to hold cryovials, with locking base to allow for one hand de-capping and capping</td>
		</tr>
		<tr >
			<td >Freezing racks</td>
			<td >Custom</td>
			<td >&#8211;</td>
			<td >Cryopreservation system custom designed for coral sperm, utilising 3D-printed and readily available components. Parts list and assembly instructions are available in Zuchwicz et al., 2021 (doi:10.1016/j.cryobiol.2021.04.005)</td>
		</tr>
		<tr >
			<td >Freezing rack lid</td>
			<td >Custom</td>
			<td >&#8211;</td>
			<td >Cryopreservation system custom designed for coral sperm, utilising 3D-printed and readily available components. Parts list and assembly instructions are available in Zuchwicz et al., 2021 (doi:10.1016/j.cryobiol.2021.04.005)</td>
		</tr>
		<tr >
			<td >Freezing Thermos &#8211; 1.5 Litre 18/8 Stainless Steel Double-Wall Vacuum Food Container</td>
			<td >Isosteel</td>
			<td >&#160;VA-9683&#160;</td>
			<td >Cryopreservation system custom designed for coral sperm, utilising 3D-printed and readily available components. Parts list and assembly instructions are available in Zuchwicz et al., 2021 (doi:10.1016/j.cryobiol.2021.04.005)</td>
		</tr>
		<tr >
			<td >Lab timers</td>
			<td >Generic</td>
			<td >&#8211;</td>
			<td >For timing of cryoprotectant equilibration prior to cryopreservation</td>
		</tr>
		<tr >
			<td >Nitrogen bath 9L</td>
			<td >BelArt</td>
			<td >M16807-9104</td>
			<td >For quenching samples during cryopreservaton and holding samples during sorting and handling</td>
		</tr>
		<tr >
			<td >Thermocouple data logger- multichannel</td>
			<td >Omega</td>
			<td >HH520</td>
			<td >Temperature monitoring during cryopreservation to determine freezing rate and end point</td>
		</tr>
		<tr >
			<td >Thermocouple probe &#8211; Type K</td>
			<td >Omega</td>
			<td >5SC-TT-K-30-36</td>
			<td >Temperature monitoring during cryopreservation to determine freezing rate and end point</td>
		</tr>
		<tr >
			<td >Cryo pens/coloured permanent pen</td>
			<td >Staedtler Lumocolor</td>
			<td >318</td>
			<td >Optional for marking cryovial lids to assist with sample management</td>
		</tr>
		<tr >
			<td >Dry Shipper - charged</td>
			<td >Taylor Wharton</td>
			<td >CXR100, or CX500</td>
			<td >For transfer of cryopreserved samples from field/collection sites to the biorepository for storage</td>
		</tr>
	</tbody>
</table>

a semi-automated workflow for the cryopreservation of coral sperm to support biobanking and aquaculture

<meta charset="utf8"></head><body>作者聚焦：为全球珊瑚礁恢复开发高效的低温保存和生物样本库技术

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around ...

a-semi-automated-workflow-for-cryopreservation-coral-sperm-to-support

Research

JoVE Journal

Biology

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture

596 次观看.  Taronga Conservation Society Australia. 我们的研究计划旨在开发和应用低温保存和生物样本库技术，以帮助保护澳大利亚和全球珊瑚的遗传多样性和生物多样性。珊瑚精子的冷冻保存已经存在了十年，但珊瑚产卵仅限于每年的几个晚上，这为我们提供了一个狭窄的机会来储存受威胁物种的遗传学。我们的目标是增强这些协议，以提高珊瑚产卵事件期间的低温保存效率。我们开发的半...

视频: 作者聚焦：为全球珊瑚礁恢复开发高效的低温保存和生物样本库技术

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

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

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

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

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

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

科研

生物学

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

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.

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

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.

精子冷冻保存和恢复使用I ·冷冻包

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

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ：ImageJ的插件半自动叶形状测量

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

诱导珊瑚群落息肉获救，获得个体化微繁殖物，用于实验室实验用途

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...