Name: effectiveness of the air stripping in two salmonid fish, rainbow trout (oncorhynchus mykiss) and brown trout (salmo trutta morpha fario)
Uploaded: 2018-09-16T13:00:00
Description: 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

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 mi...

<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 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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			<td height="21" style="height:21px;width:216px;">Glycine pure P.A.</td>
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			<td>sperm activating buffer</td>
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			<td height="42" style="height:42px;width:216px;">Propiscin (0.2% etomidate solution)</td>
			<td style="width:215px;">IRS Olsztyn, Poland</td>
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			<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>
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			<td height="21" style="height:21px;width:216px;">syrgine</td>
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			<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>
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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 ...

Air stripping of fish eggs, the effectiveness of the procedure, and the impact on brood stock management in two salmonid fish, rainbow trout and brown trout. Hi, my name is Radoslaw Kowalski. We are from the Institute of Animal Reproduction and Food Research, Polish Academy of Science in Olsztyn.We would like to present the air spawning method of salmonid fish. Hand stripping is a traditional spawning procedure in fish. This procedure may cause damage to the eggs by hand resulting in lower fertilization potential of the eggs.An alternative method utilizes gas pressure to deplete eggs from the fish body. This method is called air spawning. Air spawning is a method in which fish eggs are collected without massaging the abdomen of the fish.A syringe and needle are used for injection of the gas into the fish body cavity. Air stripping of fish eggs. In this procedure gas pressure should be controlled and the best condition is maintained when its value is below one bar.Air flow should also be controlled. For salmonid fish, one and a half liters per minute is the efficient speed of gas exhaustion. And optimum speed should be maintained because higher speed may not obtain all the eggs while a slower speed may unnecessarily prolong the procedure.The syringe should be connected with a screwed needle to avoid needle detachment caused by the gas pressure. The fish should be anesthetized and carefully dried out before spawning. The fish is placed on a slant bed about 35 to 40 degrees.The needle should be quickly inserted below the abdominal fins. The eggs are freely removed by the gas until only singular eggs are being removed from the fish body. Gentle massage of the abdomen can help to remove the excess gas from the fish body.After spawning, the fish is gently transferred to the tank with a water flow. Fish release to the tank after air spawning may initially swim on the surface of the water. After several minutes the fish releases the remaining gas while swimming and starts swimming normally.The obtained eggs are very clean and free from blood or fecal contamination.Results. The duration of egg collection by the hand stripping method was shorter than air stripping in both species. The method of stripping did not influence the total volume of eggs obtained by either hand or air stripping methods.The ovarian fluid in rainbow trout after hand stripping was lower than after air stripping. The hatching rate in rainbow trout was also significantly higher after air stripping. Conclusions, air stripping was not faster than the hand stripping method.Air stripping was beneficial to rainbow trout egg quality but not to brown trout eggs. Since no special skills are required to carry out the air stripping procedure a similar quantity and quality of eggs can be obtained by both skilled and non-skilled hatchery workers. Air stripping procedure is a promising option for new hatcheries seeking an egg collection procedure characterized by a high level of reproducibility and efficiency as well as little or no experience by the operators.

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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 ...