Special thanks to the St. Regis Mohawk Environmental Division, Welaka National Fish Hatchery, US Fish and Wildlife Service Research Unit, Southern Illinois University, US Fish and Wildlife Service Northeast Fisheries Center, Sterling Caviar, University of California-Davis, Garrison Dam National Fish Hatchery, Bears Bluff National Fish Hatchery, and US Fish and Wildlife Service Panama City Fisheries Resource Office for their help supplying sturgeon for this project.&#160;This article is Contribution 2013 of the USGS Great Lakes Science Center.

Important Note: Due to the protected status of all species of sturgeon in the United States, permits to acquire and handle fish were obtained prior to laboratory procedure. All specimens were acquired from hatchery sources post mortem due to natural causes and disposed of following standard procedure.
1. Specimen Preparation
<ol>
	<li>Before any incisions lay sturgeon carcass on its side and using a measuring tape to measure total length from the snout to the tip of the tail to the nearest 1 mm. (Figure 1A)</li>
	<li>Weigh sturgeon carcass on a tared scale to the nearest 1 g. Evaluate carcass for any external tags or marks and record.</li>
</ol>
2. Otolith Extraction
<ol>
	<li>Make a vertical incision with a large fillet knife between the base of the skull and the first dorsal scute through the body to separate the head from the body (Figure 1B). After removing the head, turn the head so the mouth is facing up (Figure 1C).</li>
	<li>Remove soft tissue of the mouth and gills by using the fillet knife to cut along the ventral side of the skull (Figure 1D). Make an incision with an electric bone saw from the tip of the snout to the base of the skull to completely bisect the head (Figure 1E). Separate the two halves by hand to expose the brain cavity (Figure 2A).</li>
	<li>Remove and discard brain matter (Red box left) with forceps to view semicircular canals (Red box right) within the brain cavity (Figure 2B).</li>
	<li>Remove semicircular canal cartilage from brain cavity with forceps (Figure 2C). Carefully puncture semicircular canal cartilage with the tip of the forceps and remove otolith (Figure 2D). Remove the otolith (hard bony structure inside the cartilage) with forceps from the semicircular canal cartilage (Figure 2E).</li>
	<li>Repeat steps 2.3 - 2.4 to remove otolith from the other half of the skull.</li>
	<li>Dip the otoliths in 92% Ethanol for 5 sec to remove any cartilage. Place otoliths in an open 25 ml scintillation vial to dry for 24 hr.</li>
</ol>

<ol>
	<li>Auer, N. A., LeBreton, G. T. O., Beamish, F. W. H., McKinley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation.">Conservation.</a> Sturgeons and Paddlefish of North America. 27, 252-276 (2004).</li><li>Campana, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy,+precision+and+quality+control+in+age+determination,+including+a+review+of+the+use+and+abuse+of+age+validation+methods).">Accuracy, precision and quality control in age determination, including a review of the use and abuse of age validation methods).</a> Journal of Fish Biology. 59, 197-242 (2001).</li><li>Bruch, R. M., Campana, S. E., Davis-Foust, S. L., Hansen, M. J., Janssen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lake+Sturgeon+age+validation+using+bomb+radiocarbon+and+known-age+fish.">Lake Sturgeon age validation using bomb radiocarbon and known-age fish.</a> Transactions of the American Fisheries Society. 138, 361-372 (2009).</li><li>Baremore, I. E., Rosati, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated,+minimally+deleterious+method+for+ageing+sturgeon.">A validated, minimally deleterious method for ageing sturgeon.</a> Fishery Bulletin. 112, 274-282 (2014).</li><li>Stevensen, J. T., Secor, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+determination+and+growth+of+Hudson+River+Atlantic+sturgeon,+Acipenser+oxyrhynchus.">Age determination and growth of Hudson River Atlantic sturgeon, Acipenser oxyrhynchus.</a> Fishery Bulletin. 97, 153-166 (1999).</li><li>Brennan, J. S., Cailliet, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+age-determination+techniques+for+white+sturgeon+in+California.">Comparative age-determination techniques for white sturgeon in California.</a> Transactions of the American Fishery Society. 118, 296-310 (1989).</li><li>Rossiter, A., Noakes, D. L. G., Beamish, F. W. H. <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+age+estimation+for+the+lake+sturgeon.">Validation of age estimation for the lake sturgeon.</a> Transactions of the American Fisheries Society. 124, 777-781 (1995).</li><li>Chalupnicki, M. A., Dittman, 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=North+American+Sturgeon+Otolith+Morphology.">North American Sturgeon Otolith Morphology.</a> Copeia. , 260-266 (2016).</li><li>Secor, D. H., Dean, J. M., Laban, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual for otolith removal and preparation for microstructural examination. , (1991).</li></ol>

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The overview of an alternative method of extracting otoliths from sturgeon carcasses has been detailed. It is important to note that special attention is needed to the lateral incision placement on the midline of skull in order to bisect the skull evenly to ensure no harm to the otoliths during dissection. If the incision is not deep enough to completely bisect the skull it will be very difficult to expose the brain cavity where the location of the otoliths is clearly visible. With experience, the overall technique is re...

Sturgeon (Acipenseridae) populations throughout the world have been declining for over a century due to impacts including habitat loss, population fragmentation, and overfishing such that many populations are protected by state and federal laws1,2. Management agencies throughout the USA and the world have identified sturgeon as a target species for restoration and recovery1. Population characteristics (growth, recruitment, diet) of most sturgeon populations have been studied to better understand basic biology and life history characteristics1. Due to the protected status of most sturgeon populations, age evaluation is difficult in small threatened populations where no sacrifice or harm to individuals is warranted.
The extraction of bony structures for the purpose of age estimation and growth analysis has been a common technique3.The use of fin ray sections to determine age of sturgeon has become a practical nonlethal method, but verification of the technique has been limited and age can be very difficult to decipher in older individuals (&#62; 15 years)4. Researchers evaluating the use of multiple bony structures in sturgeon for their use in validating the age of Gulf Sturgeon (Acipenser oxyrinchus desoti)5, Atlantic Sturgeon (A. o. oxyrinchus)6, White Sturgeon (A. transmontanus)7, and Lake Sturgeon (A. fluvescens)8 concluded that the first marginal pectoral fin ray provided the most accurate age but concluded this invasive procedure to be deleterious to larger individuals. &#160;Currently, there is the development of using otolith shape metrics to distinguish between North American sturgeon species with a very high rate of success9. Details on the process of removing the otolith with a dorsal transverse incision at the base of the skull is a common otolith extraction method found throughout the literature and clearly detailed by Secor et al. (1991)10. However, this method is difficult to implement on sturgeon that have very thick skulls with otoliths that are small and delicate and would tend to break. A further review of the literature shows no other detailed methodology of the otolith removal process for sturgeon. The objective of this paper was to detail an alternative method of extracting otoliths from sturgeon by entering the brain cavity from the ventral side.

<table><tbody><tr><td>Measuring Board</td><td></td><td></td><td>Metric meter stick is preferred</td></tr><tr><td>Weighing scale</td><td></td><td></td><td></td></tr><tr><td>Filleting Knife</td><td></td><td></td><td></td></tr><tr><td>Scalpel</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Sample vials</td><td></td><td></td><td>25 ml scintillation vials work well</td></tr></tbody></table>

alternative method of removing otoliths from sturgeon

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a transverse vertical incision on the top of the skull with a hand or electrical saw may damage the otolith if not performed correctly. Sturgeons (Acipenseridae) are one family in particular that have a very large and thick skull. A new laboratory method entering the brain cavity from the ventral side of the fish to expose the otoliths was easier than other otolith extraction methods found in the literature. Methods reviewed in the literature are designed for the field and are more efficient at processing large quantities of fish quickly. However, this new technique was designed to be more suited for a laboratory setting when time is not pressing and successful extraction from each specimen is critical. The success of finding and removing otoliths using this technique is very high and does not compromise the structure in any manner. This alternative technique is applicable to other similar fish species for extracting the otoliths.

The adult size of sturgeon across the world varies greatly but the location of the otoliths is consistent. The use of a sharp fillet knife was found to remove the soft tissue of the mouth easily shown in Figure 1D. An electric bone saw is the preferred tool for the lateral incision to expose the brain cavity in Figure 1E. However, attention to bisecting the midline of the skull is needed otherwise the otoliths may be crushed during this process. If this p...

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Alternative Method of Removing Otoliths from Sturgeon

The goal of the protocol is to show an effective method to extract the otoliths from sturgeon carcasses.

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a ...

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Environment

8.4K Views.  Great Lakes Science Center. The goal of the protocol is to show an effective method to extract the otoliths from sturgeon carcasses. 

Video: Alternative Method of Removing Otoliths from Sturgeon

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Necropsy-based Wild Fish Health Assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory sensitivity in high PCO2/low pH water. To assess the effects of ocean acidification, or olfactory sensitivity in marine fish in general, we propose that extracellular multi-unit recording from the olfactory nerve is the method of choice. Although invasive, it is sensitive, robust, reproducible and independent of external salinity (unlike the electro-olfactogram [EOG], for example). Furthermore, it records a primary sensory input into the CNS, prior to any central processing. We show that this method can show a reduction in olfactory sensitivity that is both temporary and odorant-dependent, using a range of amino acids to construct concentration-response curves and calculate the thresholds of detection.

Extracellular multi-unit recording from the olfactory nerve is a sensitive, robust and reproducible method for assessing olfactory sensitivity in marine fish. It records primary sensory input and is independent of external salinity.

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory ...

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.

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.

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

Developmental Biology

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

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Measuring the impacts of environmental contaminants on fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints. We describe methods including video technology to quantify swimming behavior of early life stage white sturgeon (Acipenser transmontanus) during and after 96 hr acute exposures to various concentrations of copper.

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

Neuroscience

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

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Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

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The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

Biology

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Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...