The presented study was supported by Project "Pneumatic method of fish stripping - possible application, influence on the gametes' quality and quantity and the welfare of fish" (acronym: PNEUFISH) financed under "Operational Program Development of the Fisheries Sector and Coastal Areas 2007-2013" (OR-61724-OR1400001/10), funds appropriated to Institute of Animal Reproduction and Food Research Polish Academy of Sciences, Olsztyn, Poland, and support by COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE). We would like to thank, Stanley Ifeanyi Ugwu for great support in English editing. We want to acknowledge also the animation producers "Studio Filmowe Ruchome Obrazki".

Procedures were carried out in accordance with the Local Committee on the Ethics of Animal Experiments in Olsztyn, Poland.
1.Equipment Preparation
<ol>
	<li>Use a syringe and needle for injection of the gas into the fish body cavity.</li>
	<li>Set the gas pressure at 0.5 bar, and maintain this pressure to be below 1 bar throughout.</li>
	<li>Control the air flow. For the salmonid fish, 1.5 L/min is the most efficient speed of gas exhaustion. Maintain the optimum speed because higher speed might not obtain all the eggs while a slower speed might unnecessarily prolong the procedure.</li>
	<li>Connect the syringe with a screwed needle (0.8-1.2 mm diameter) to avoid needle detachment caused by the gas pressure.</li>
</ol>
2. Air Spawning
<ol>
	<li>Anesthetize the fish with 0.2% Propiscin. Place the fish into a water bath with the 0.2% Propiscin and wait until the fish becomes unresponsive.</li>
	<li>Carefully dry out before spawning by wiping with a dry cotton cloth.</li>
	<li>Place the fish on the slant bed about 35-40&#176;, on her side tail side down. With a gentle push of fish abdomen, check the egg flow from the genital tract. If the eggs are not present, the fish should not be spawned.</li>
	<li>Place a container covered with mesh below the fish papilla to collect the eggs.</li>
	<li>Quickly insert the needle below the abdominal fins.</li>
	<li>Freely remove the eggs by the gas until only singular eggs are removed from the fish body.</li>
	<li>Gently massage the abdomen to remove excess gas from the fish body.</li>
	<li>After spawning, gently transfer the fish to a tank with water flow. Fish released to tank after air spawning might initially swim on the surface of the water. After several minutes the fish releases the remaining gas while swimming and starts swimming normally. 
	NOTE: The obtained eggs are very clean and free from blood or feces contamination.</li>
</ol>

<ol>
	<li>Nelson, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fishes of the World. , (2006).</li><li>Garc&#237;a-Mar&#237;n, J. L., Sanz, N., Pla, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proportions+of+native+and+introduced+brown+trout+in+adjacent+fished+and+unfished+Spanish+rivers.">Proportions of native and introduced brown trout in adjacent fished and unfished Spanish rivers.</a> Conservation Biology. 12, 313-319 (1998).</li><li>Dietrich, G. J., Wojtczak, M., Slowinska, M., Dobosz, S., Kuzminski, H., Ciereszko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broken+eggs+decrease+pH+of+rainbow+trout+(Oncorhynchus+mykiss)+ovarian+fluid.">Broken eggs decrease pH of rainbow trout (Oncorhynchus mykiss) ovarian fluid.</a> Aquaculture. 273, 748-751 (2007).</li><li>Zurbuch, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structure+for+easy+fish+recovery+during+drainage+of+an+impoundment.">A structure for easy fish recovery during drainage of an impoundment.</a> Progressive FishCulturist. 27, 237-238 (1965).</li><li>Cejko, B. I., 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=Effects+of+different+stripping+methods+of+female+and+activation+medium+on+fertilization+success+in+northern+pike+(Esox+lucius).">Effects of different stripping methods of female and activation medium on fertilization success in northern pike (Esox lucius).</a> Czech Journal of Animal Sciences. 10, 481-486 (2016).</li><li>Morisawa, S., Morisawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+potential+for+sperm+motility+by+bicarbonate+and+pH+in+rainbow+trout+and+chum+salmon.">Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon.</a> Journal of Experimental Biology. 136, 13-22 (1988).</li><li>Billard, R., Cosson, J., Perchec, G., Linhart, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+sperm+and+artificial+reproduction+in+carp.">Biology of sperm and artificial reproduction in carp.</a> Aquaculture. 129, 95-112 (1995).</li><li>Iuchi, I., Ha, C. R., Sugiyama, H., Nomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chorion+hardening+of+eggs+of+rainbow+trout,+Oncorhynchus+mykiss.">Analysis of chorion hardening of eggs of rainbow trout, Oncorhynchus mykiss.</a> Development Growth and Differentiation. 38, 299-306 (1996).</li><li>Wojtczak, M. A., Kowalski, R. K., Dobosz, S., Goryczko, K., Ku&#378;minski, H., Glogowski, J., Ciereszko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+water+turbidity+for+evaluation+of+rainbow+trout+(Oncorhynchus+mykiss)+egg+quality.">Assessment of water turbidity for evaluation of rainbow trout (Oncorhynchus mykiss) egg quality.</a> Aquaculture. 242, 617-624 (2004).</li><li>Tabrizi, E. N., Khara, H., Nezami, S. A., Lorestani, R., Shamspour, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broken+Eggs+Influence+on+Fertilization+Capacity+and+Viability+of+Eggs,+Turbidity+and+pH+of+Ovarian+Fluid+and+Fertilization+Water+in+the+Endangered+Caspian+Brown+Trout,+Salmo+Trutta+Caspius.">Broken Eggs Influence on Fertilization Capacity and Viability of Eggs, Turbidity and pH of Ovarian Fluid and Fertilization Water in the Endangered Caspian Brown Trout, Salmo Trutta Caspius.</a> International Journal of Biology. 3 (1), 161-166 (2011).</li><li>Carl, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beware+of+the+broken+egg!+A+possible+cause+of+heavy+losses+of+salmon+eggs.">Beware of the broken egg! A possible cause of heavy losses of salmon eggs.</a> Progresive Fish-Culturist. 53, 30-31 (1941).</li></ol>

The authors have nothing to disclose.

The pneumatic method of egg collection from fish, although more time-consuming than the traditional method (hand stripping), can secure a high quality of matured oocytes (this work). This is related to the low risk of mechanical disruption of the eggs while applying this procedure. High pH of the ovary fluid, as well as an increase in the fertilization rate, attest to the usefulness of the pneumatic method. Overall, the air stripping method of egg collection in fish reproduction could be a promising option in terms of st...

The rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario) belong to the Salmonidae family of Salmoniformes1. The production of these two species in global aquaculture has gained rapid growth, owing to their commercial and recreational importance. In Poland, production of salmonid fish stands at around 20 000 tonnes, with rainbow trout being the dominant species. On the other hand, the brown trout represents a major source of freshwater fish resources in Europe because of its commercial value in aquaculture and importance for angling. In many aquatic ecosystems, the existence of brown trout populations is threatened. As a result, artificial reproduction has been applied to restock local populations of this species2.
Rainbow trout and brown trout attain sexual maturity usually at the age of three, with males maturing a year earlier than females1. Under artificial conditions, eggs and sperm of rainbow trout and brown trout are usually collected by gentle abdominal massage. In practice, on a small scale, a hand-stripping method of collecting eggs is cost-effective. However, on a large scale, the hand-stripping method may be labor-intensive and exhausting. As a result, this technique can result in broken eggs due to the weariness of the hatchery workers. Breakage of eggs often leads to a decrease in the pH of ovarian fluid and the release of egg yolk, both of which negatively affect the fertilization of eggs3. Furthermore, this traditional method of egg collection (repeatedly pressing of the abdomen) may also result in broodstock mortality caused by skin infection associated with the deterioration of the protective barrier of mucus. 
Pneumatic method of egg collection was first used in Australia in 19574. The method is based on the injection of gases (air, nitrogen or oxygen) into the body cavity to expel eggs using gas pressure. This fast and easy technique has been successfully applied in salmonid fish without any negative side effects on the fish4. Recently, this method was used in wild species (Northern pike, Esox lucius) to collect mature eggs in artificial conditions5. It was shown that air stripped eggs had higher fertilization and hatching rates in comparison with hand stripped eggs, regardless of the solution used for fertilization (Woynarovich solution, Billard buffer or hatchery water)5.
The principal aim of this study was to test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout in comparison with traditional hand stripping method. Efficiencies of both methods (volume of obtained eggs and time of collection, quality of gametes (ovarian fluid pH), as well as hatching rate and post-spawning mortality) were compared.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine pure P.A.</td>
			<td style="width:215px;">AVANTOR</td>
			<td align="right" style="width:119px;">527560117</td>
			<td style="width:167px;">sperm activating buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma base</td>
			<td style="width:215px;">Sigma Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td>sperm activating buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride Bioreagent</td>
			<td style="width:215px;">Sigma Aldrich</td>
			<td style="width:119px;">S5886</td>
			<td>sperm activating buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride anhydrous</td>
			<td style="width:215px;">Sigma Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td>sperm activating buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propiscin (0.2% etomidate solution)</td>
			<td style="width:215px;">IRS Olsztyn, Poland</td>
			<td style="width:119px;">not indicated</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">compressor</td>
			<td style="width:215px;">Thomas&#160; Sheboigan WI USA&#160;</td>
			<td style="width:119px;">DT/SR 070800001882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">reduction valve</td>
			<td style="width:215px;">Camozzi&#160;</td>
			<td style="width:119px;">cRJUS U7J</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">air hose</td>
			<td style="width:215px;">ZEC T.P.U.</td>
			<td style="width:119px;">&#160;SH.98&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">syrgine</td>
			<td style="width:215px;">EFD</td>
			<td align="right">7012118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Air spawning stage</td>
			<td style="width:215px;">Biopasz, Poland</td>
			<td style="width:119px;">PNEU001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Orion Ross Ultra electrode</td>
			<td style="width:215px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">8102BNUWP</td>
			<td></td>
		</tr>
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effectiveness of the air stripping in two salmonid fish, rainbow trout (oncorhynchus mykiss) and brown trout (salmo trutta morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Mature males and females of rainbow trout (age 2+, 1700 &#177; 328 g) and brown trout (age 3+, 1900 &#177; 435 g) were obtained from the Salmonid Fish Hatchery (Rutki-&#379;ukowo, Inland Fishery Institute in Olsztyn, Poland). The males and females were placed in separate tanks with a volume of about 5 m3. The water temperature was 12&#177;1 &#176;C. Before manipulation, fish were anesthetized with 0.2% Propiscin. The fish were divided into two groups. Part of them ...

Watch this Scientific Journal Video about Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario at JoVE.com

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

effectiveness-air-stripping-two-salmonid-fish-rainbow-trout

Research

JoVE Journal

Developmental Biology

7.7K Views.  Polish Academy of Science. The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

Video: Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

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Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

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Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm